cDNA ENCODING A BMP TYPE II RECEPTOR

TECHNICAL FIELD The present invention relates to the field of bone formation and development. Specifically, the present invention relates to a bone morphogenetic protein receptor, a DNA sequence coding for said receptor, and cells transfected with a DNA sequence coding for said receptor.

BACKGROUND Humans and other warm-blooded animals can be afflicted by a number of bone- related disorders. Such disorders range from bone fractures, to debilitating diseases such as osteoporosis. While in healthy individuals bone growth generally proceeds normally and fractures heal without the need for pharmacological intervention, in certain instances bones may become weakened or may fail to heal properly. For example, healing may proceed slowly in the elderly and in patients undergoing treatment with corticosteroids (e.g., transplant patients). Osteoporosis is a condition in which bone hard tissue is lost disproportionately to the development of new hard tissue. Osteoporosis can generally be defined as the reduction in the quantity of bone, or the atrophy of skeletal tissue; marrow and bone spaces become larger, fibrous binding decreases, and compact bone becomes fragile. Another bone related disorder is osteoarthritis, which is a disorder of the movable joints characterized by deterioration and abrasion of articular cartilage, as well as by formation of new bone at the joint surface.

While a variety of treatments are available for such bone-related disorders, none of the treatments provide optimum results. One of the difficulties facing individuals who treat bone-related disorders is a lack of complete understanding of bone metabolism and of the bone-related disorders. A key to such understanding is identifying and characterizing each of the components involved in bone growth. Bone morphogenetic proteins (BMPs) have been demonstrated to play a role in bone formation and development (J. M. Wozney, Molec. Reproduct. and Develop., 32: 160-167 (1992)).

Furthermore, the role of BMPs may not be limited to their role in bone The finding that the BMPs are found at significant concentrations in other tissues such as brain, kidney, stratified squamous epithelia, and hair follicle (N.A. Wall, M. Blessing, C.V.E. Wright, and B.L.M. Hogan, J. Cell Biol., 120: 493-502 (1993); E. Ozkaynak, P.N.J. Schnegelsberg, D.F. Jin, G.M. Clifford, F.D. Warren, E.A. Drier, and H.

Oppermann, J. Biol. Chem., 267: 25220-25227 (1992), K.M. Lyons, CM. Jones, and B.L.M. Hogan, Trends in Genetics, 7: 408- 12 (1991); V. Drozdoff, N.A. Wall, and W.J. Pledger, Proceedings of the National. Academy of Sciences, U.S.A., 91: 5528- 5532 (1994)) suggests that they may play additional roles in development and differentiation. In support of this, BMPs have recently been found to promote nerve cell differentiation and to affect hair follicle formation (K. Basler, T. Edlund, T.M. Jessell, and T Yamada, Cell, 73: 687-702 (1993); V.M. Paralkar, B.S. Weeks, Y.M. Yu, H.K. Kleinman, and A.H. Reddi, J. Cell Biol, 119: 1721-1728 (1992); M. Blessing, L.B. Nanney, L.E. King, CM. Jones, and B.L. Hogan, Genes Dev., 7: 204-215 (1993)). A BMP initiates its biological effect on cells by binding to a specific BMP receptor expressed on the plasma membrane of a BMP-responsive cell. A receptor is a protein, usually spanning the cell membrane, which binds to a ligand from outside the cell, and as a result of that binding sends a signal to the inside of the cell which alters cellular function. In this case, the ligand is the protein BMP, and the signal induces the cellular differentiation.

Because of the ability of a BMP receptor to specifically bind BMPs, purified BMP receptor compositions are useful in diagnostic assays for BMPs, as well as in raising antibodies to the BMP receptor for use in diagnosis and therapy. In addition, purified BMP receptor compositions may be used directly in therapy to bind or scavenge BMPs, thereby providing a means for regulating the activities of BMPs in bone and other tissues. In order to study the structural and biological characteristics of BMP receptors and the role played by BMPs in the responses of various cell populations to BMPs during tissue growth/formation stimulation, or to use a BMP receptor effectively in therapy, diagnosis, or assay, purified compositions of BMP receptor are needed. Such compositions, however, are obtainable in practical yields only by cloning and expressing genes encoding the receptors using recombinant DNA technology. Efforts to purify BMP receptors for use in biochemical analysis or to clone and express mammalian genes encoding BMP receptors have been impeded by lack of a suitable source of receptor protein or mRNA. Prior to the present invention, few cell lines were known to express high levels of high affinity BMP receptors which precluded purification of the receptor for protein sequencing or construction of genetic libraries for direct expression cloning. Availability of the BMP receptor sequence will make it possible to generate cell lines with high levels of recombinant BMP receptor for biochemical analysis and use in screening experiments. The BMPs are members of the TGF-β superfamily. Other members of the

TGF-β superfamily include TGF-β, activins, inhibins, Mϋllerian Inhibiting Substance, and the Growth and Differentiation Factors (GDFs). As expected, the receptors for various members of the TGF-β superfamily share similar structural features. Receptors

of the TGF-β ligand superfamily are typically classified into one of two sub-groups, designated as type I and type II. The type I and type II receptors are classified as such based on amino acid sequence characteristics. Both the type I and type II receptors possess a relatively small extracellular ligand binding domain, a transmembrane region, and an intracellular protein kinase domain that is predicted to have serine/threonine kinase activity (Lin and Moustakas, Cellular and Molecular Biology, 40: 337-349 (1994); L.S. Mathews, Endocrine Reviews, 15: 310-325 (1994); L. Attisano, J.L. Wrana, F. Lόpez-Casillas, and J. Massague, Biochimica et Biophysica Acta, 1222: 71- 80 (1994)). The type I receptors cloned to date belong to a distinct family whose kinase domains are highly related and share > 85% sequence similarity (B.B. Koenig et al., Molecular and Cellular Biology, 14: 5961-5974 (1994)). The intracellular juxtamembrane region of the type I receptors is characterized by an SGSGSG motif 35- 40 amino acids from the transmembrane region, and the carboxy terminus of these receptors is extremely short (B.B. Koenig et al., Molecular and Cellular Biology, 14: 5961-5974 (1994); L. Attisano, J.L. Wrana, F. Lόpez-Casillas, and J. Massague, Biochimica et Biophysica Acta, 1222: 71-80 (1994)). The extracellular domain of the type I receptors contains a characteristic cluster of cysteine residues, termed the "cysteine box", located within 25-30 amino acids of the transmembrane region, and another cluster of cysteine residues, termed the "upstream cysteine box", located after the putative signal sequence (B. B. Koenig, et al., Molecular and Cellular Biology, 14: 5961-5974 (1994); L. Attisano, et al., Biochimica et Biophysica Acta, 1222: 71-80 (1994)).

In contrast to the type I receptors, the kinase domains of the type II receptors are only distantly related to one another. The SGSGSG motif found in type I receptors is not found in type II receptors. Also, the "upstream cysteine box" of type I receptors is not present in type II receptors. Furthermore, while all of the activin type II receptors contain a proline-rich sequence motif in the intracellular juxtamembrane region, there is no characteristic sequence motif that is common to all type II receptors (L.S. Mathews, Endocrine Reviews, 15: 310-325 (1994)). The length of the carboxy terminus of the type π receptors is considerably variable, with the longest known carboxy terminus being found in the BMP type II receptor, DAF-4 (M. Estevez, L. Attisano, J.L. Wrana, P.S. Albert, J. Massague, and D.L. Riddle, Nature, 365: 644-49 (1993)), that was cloned from the nematode C. elegans. The extracellular domain of the type II receptors contains a single cysteine box located near the transmembrane region. Aside from the presence of the cysteine box, there is little sequence similarity amongst the extracellular domains of the type II receptors for TGF-β, activin, and BMPs.

Signaling by members of the TGF-β ligand superfamily requires the presence of

both type I and type II receptors on the surface of the same cell (L.S. Mathews, Endocrine Reviews, 15: 310-325 (1994), L. Attisano, J.L. Wrana, F. Lόpez-Casillas, and J. Massague, Biochimica et Biophysica Acta, 1222: 71-80 (1994)). The BMPs are members of the TGF-β ligand superfamily; given the high degree of structural similarity among these family members, it is expected that their receptors will be structurally and functionally related to the TGF-β and activin receptors. It is anticipated that, like the TGF-β and activin receptor systems (J. Massague, L. Attisano, and J.L. Wrana, Trends in Cell Biology, 4: 172-178 (1994)), both a BMP type I receptor and a BMP type II receptor will be needed in order to transduce a BMP signal within a cell or tissue. Hence, there is a need for a mammalian type II BMP receptor kinase protein in addition to the type I receptors that have already been cloned.

Three distinct mammalian type I receptors have been reported for the BMPs: BRK-1 (see U.S.S.N. 08/158,735, filed November 24, 1993 by J. S. Cook, et al.; and B.B. Koenig et al., Molecular and Cellular Biology, 14: 5961-5974 (1994)), ALK-2, and ALK-6. BRK-1 is the mouse homologue of ALK-3, which has also been demonstrated to bind BMP-4, as does ALK-6; ALK-2 binds BMP-7 (see P. ten Dijke, H. Yamashita, T.K. Sampath, A.H. Reddi, M. Estevez, D.L. Riddle, H. Ichijo, CH. Heldin, and K. Miyazono, J. Biological Chemistry, 269: 16985-16988 (1994)). It is also postulated that ALK-6 is the mouse homologue of the chicken receptor BRK-2 (also referred to as RPK-1) (S. Sumitomo, T. Saito, and T. Nohno, DNA Sequence, 3: 297-302 (1993)).

The only type II receptor for BMP-2 and BMP-4, named DAF-4, has been cloned from the nematode C. elegans (M. Estevez, L. Attisano, J.L. Wrana, P S. Albert, J. Massague, and D.L. Riddle, Nature, 365: 644-9 (1993)). Because of the large evolutionary distance between the nematode and mammals, it has not been possible to use the DAF- cDNA as a probe with which to clone the mammalian DAF-4 homologue. This implies that the DNA sequence of the mammalian type II receptor for BMPs is substantially divergent from that of DAF-4, and it is necessary to clone a mammalian type II receptor for the BMPs. Thus, the BMP receptor kinase protein of the present invention provides a mammalian type II receptor which will enable the formation of a high affinity complex that is competent for signaling a response to BMPs in concert with the mammalian type I receptor(s) for BMPs. The mammalian BMP receptor complex is therefore more relevant for the identification of novel compounds which interact with the BMP receptor, and which will be useful as therapeutic agents in humans and other mammals, than is a receptor complex that is composed of the nematode type II receptor and the mammalian type I receptor.

OBJECTS OF THE PRESENT INVENTION It is an object of the present invention to provide an isolated BMP type II receptor kinase protein.

It is also an object of the present invention to provide a DNA sequence encoding a BMP type II receptor kinase protein.

It is also an object of the present invention to provide a recombinant expression vector encoding a BMP type II receptor kinase protein.

It is also an object of the present invention to provide a host cell comprising a recombinant expression vector encoding a BMP receptor kinase protein. It is also an object of the present invention to provide a method for producing a

BMP type II receptor kinase protein, or a soluble fragment thereof.

It is also an object of the present invention to provide antibodies specific for the BMP type II receptor kinase proteins of the present invention.

It is also an object of the present invention to provide a reporter system for evaluating whether a test compound is capable of acting as an indirect agonist or antagonist of the BMP type II receptor protein kinase of the present invention.

It is also an object of the present invention to provide a method for determining whether a compound is capable of binding to a BMP receptor kinase protein of the present invention.

SUMMARY

The present invention relates to an isolated BMP type II receptor kinase protein or soluble fragment thereof, a DNA sequence coding for said BMP receptor kinase protein or said soluble fragment thereof, a recombinant expression vector comprising said DNA sequence, a host cell comprising said recombinant expression vector, a method of expressing said BMP receptor kinase protein or soluble fragment thereof, an antibody directed to a BMP type II receptor kinase protein of the present invention, a method for evaluating whether a test compound is capable of acting as an indirect agonist or antagonist to the BMP type II receptor protein kinase of the present invention, and a method for determining whether a compound is capable of binding to a

BMP receptor kinase protein of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the DNA sequence of the degenerate oligonucleotide primers used in the PCR amplification of t-BRK-3. The nucleotide bases adenine, thymine, cytosine, and guanine are represented by A, T, C and G respectively. The letter N represents the presence of an equal mixture of A, T, C, and G at that site. The primers are derived from the sequence of the TGF-β type II receptor (H.Y. Lin, X.F. Wang,

E. Ng-Eaton, R.A. Weinberg, and H.F. Lodish, Cell, 68: 775-785 (1992)).

Figure 2 shows the construct pJT4-hBRK3T, used for transient mammalian expression of t-BRK-3. CMV, cytomegalovirus early promoter/enhancer; R, the "R" element from the long terminal repeat of human T-cell leukemia virus- 1; SP, an intron splice site from the SV40 virus; T3, promoter region from the T3 bacteriophage; T7, promoter region from the T7 bacteriophage; poly A, region from the SV40 virus directing polyadenylation of the message; SV40 ORI, origin of replication from the SV40 virus; Amp, ampicillin resistance gene for selection in E. coli.

Figure 3 shows the construct pJT4-J159F, used for transient mammalian expression of BRK- 1. Abbreviations are the same as those in Figure 2.

Figure 4 shows the construct pJT3-BRK2, used for transient mammalian expression of BRK2. Abbreviations are the same as those in Figure 2.

Figure 5 shows the construct pJT4-Daf4, used for transient mamπμlian expression of the C. elegans receptor DAF-4. Abbreviations are the same as those in Figure 2.

Figure 6 shows whole cell binding of [^l]-EMP-4 to t-BRK-3 expressed in COS-7 cells, in the presence or absence of the type I receptors BRK-1 and BRK2. Bars represent specific binding of [ ¹²⁵ I]-BMP-4, normalized to cell number. Left to right, NTH3T3 embryonic fibroblasts; COS-7 cells; COS-7 cells transfected with the vector pJT-4 alone (designated "mock"); COS-7 cells transfected with BRK-1 alone, BRK-1 plus 10 or 20 μg of t-BRK-3, BRK-2 alone, BRK-2 plus 10 or 20 μg of t-BRK-3, and t- BRK-3 alone (20 μg).

Figure 7 shows crosslinking of [125τ].BMP-4 to COS-1 cells transfected with t- BRK-3, in the presence or absence of the type I receptors BRK-1 and BRK-2. Molecular weight standards are shown on the left. Labels on the right indicate the bands which migrate at the predicted molecular weights of t-BPJ -3, BRK-1, and BRK- 2 crosslinked to [ ¹²⁵ I]-BMP-4 Left to right, the lanes represent COS-1 cells transfected with BRK-1 alone; BRK-1 plus 2 μg/ml t-BRK-3; BRK-1 plus 4 μg ml t- BRK-3; BRK-2 alone; BRK-2 plus 2 μg/ml t-BRK-3; BRK-2 plus 4 μg/ml t-BRK-3; t- BRK-3 alone at 2 μg/mi; and t-BRK alone at 4 μg/ml. Volume of DNA mixture is 4 ml. In this figure, "BRK-3*" is t-BRK-3.

Figure 8 shows an immunoprecipitation of t-BRK-3 and the C. elegans type II receptor DAF-4 expressed in COS-1 cells and crosslinked to [ ¹²⁵ I]-BMP-4 in the presence or absence of the type I receptors BRK-1 or BRK-2. Molecular weight standards are shown on the left; areas shown at the right indicate labeled protein bands migrating at the predicted molecular weight of DAF-4, t-BRK-3, BRK-1, or BRK-2 crosslinked to [ ¹²⁵ I]-BMP-4. Antiserum 1379 was used for COS-1 cells transfected with BRK-1 in the presence or absence of type II receptors, and antiserum 1380 for

COS-1 cells transfected with BRK-2 in the presence or absence of type II receptors. For all others, antiserum is listed in parentheses. Left to right, NLH3T3 embryonic fibroblasts (1379), followed by COS-1 cells transfected with BRK-1 alone; BRK-1 plus DAF-4; BRK-1 plus t-BRK-3; BRK-2 alone; BRK-2 plus DAF-4; BRK-2 plus t-BRK- 3. This is followed by NLH3T3 cells (1380), followed by COS-1 cells transfected with DAF-4 alone (1379), and t-BRK-3 alone (1380). In this figure, "BRK-3*" is t-BRK-3.

Figure 9 shows an immunoprecipitation of COS- 1 cells transfected with BRK-2 and t-BRK-3 and crosslinked to [ ¹²⁵ I]-BMP-4 at a concentration of 210 pM, in the presence or absence of excess unlabeled competitors as indicated. Antiserum 1380 is used. Duplicate lanes at left show no unlabeled competitor added, followed by addition of (left to right) 10 nM BMP-4; 10 nM BMP-2; 10 nM DR-BRMP-2; and 50 nM TGF- β _j . In this figure, "BRK-3*" is t-BRK-3.

Figure 10 shows the construct pJT6-mBRK-3L, used for transient mamm.alian expression of mouse BRK-3. Abbreviations used are the same as those for Figure 2. Figure 1 1 shows the construct pJT6-mBRK-3S, used for transient mammalian expression of mouse BRK-3. In this construct, most of the untranslated 3' region has been removed. Abbreviations used are the same as those for Figure 2.

Figure 12 shows whole cell binding of [ ¹²⁵ I]-BMP-4 to mouse BRK-3 expressed in COS-1 cells, in the presence or absence of the type I receptor BRK-2. Bars represent specific binding of [^ ² ^I]-BMP-4, normalized to cell number. Constructs used for mouse BRK-3 are pJT6-mBRK-3L and pJT6-mBRK-3S; for BRK- 2, the construct is pJT3-BRK-2. Both constructs contain the complete coding region of mouse BRK-3. In pJT6-mBRK-3S,an A-T rich region in the 3' untranslated region has been deleted. Left to right, COS-1 cells transfected with the vector pJT-6 alone (designated "mock"); pJT3-BRK-2 alone; the construct pJT6-mBRK-3S alone; pJT6- mBRK-3L alone; pJT3-BRK-2 plus pJT6-BRK-3S; and pJT3-BRK-2 plus pJT6-BRK- 3L.

Figure 13 shows crosslinking of [ 5rj-BMP-4 to m-BRK-3 in the presence and absence of type I BMP receptors. COS-1 cells are transfected with the cDNA for BRK-3 using the construct pJT6-mBRK-3S, and/or with cDNAs for BRK-1 (using pJT4-J159F) or BRK-2 (using pJT3-BRK-2). The cells are then allowed to bind crosslinked with disuccinimidyl suberate, and subjected to SDS gel electrophoresis. Position of molecular weight standards is indicated on the left. Left to right: COS-1 cells transfected with BRK-1 alone; BRK-1 plus m-BRK-3; m-BRK-3 alone; BRK-2 plus m-BRK-3; BRK-2 alone; and vector alone. Bands identified with BRK-1, BRK-2, and BRK-3 are indicated on the right.

Figure 14 shows immunoprecipitation of m-BRK-3 in the presence and absence of type I BMP receptors. COS-1 cells are transfected with the cDNA for m-

BRK-3 using the construct pJT6-mBRK-3S, and/or with cDNAs for BRK-1 (using ρJT4-J159F) or BRK-2 (using pJT3-BRK-2). The cells are then allowed to bind [125r]_BMP-4, crosslinked with disuccinimidyl suberate, immunoprecipitated with antibodies to BRK-1 or BRK-2, and subjected to SDS gel electrophoresis. Antisera used are indicated below the lanes: PI, preimmune; 1379, for cells transfected with cDNA for BRK-1; 1380, for cells transfected with cDNA for BRK-2. Position of molecular weight standards is indicated on the left. Left to right, COS-1 cells transfected with BRK-1 plus m-BRK-3 (preimmune serum); BRK-1 alone; BRK-1 plus m-BRK-3; BRK-2 plus m-BRK-3; BRK-2 alone; and BRK-2 plus m-BRK-3 (preimmune serum).

Figure 15 shows a map of the insert of pHSK1040. This construct contains the complete coding region of human BRK-3 in BLUESCRIPT II SK (-).

DESCRIPTION The present invention answers the need for a mammalian BMP type II receptor by providing an isolated BMP receptor kinase protein; a DNA sequence coding for said protein; a recombinant expression vector comprising said DNA sequence; a host cell comprising said recombinant expression vector, and a method of expressing said BMP receptor kinase protein. The BMP type II receptor of the present invention will also reconstitute the high affinity BMP receptor complex thought to be necessary for signaling in concert with the BMP type I receptors.

As used herein, "human BMP receptor kinase protein-3" or "h-BRK-3" means a protein having the amino acid sequence SEQ ID NO:2, as well as proteins having amino acid sequences substantially similar to SEQ LD NO:2, and which are biologically active in that they are capable of binding a BMP molecule (including, but not limited to BMP- 2, DR-BMP-2, BMP-4, and/or BMP-7), or transducing a biological signal initiated by a BMP molecule binding to a cell, or crossreacting with antibodies raised against h-BRK- 3 protein, or peptides derived from the protein sequence of h-BRK-3 or m-BRK-3 (see below), or forming a complex with a BMP type I receptor, or co-immunoprecipitating with a BMP type I receptor when antibodies specific for either h-BRK-3 or a BMP type I receptor are used.

As used herein, "truncated human BMP receptor kinase protein" or "t-BRK-3" means a protein having amino acid sequence SEQ LD NO:4, or a sequence having the properties described above for BRK-3. As used herein, "mouse BMP receptor kinase protein" or "m-BRK-3" means a protein having amino acid sequence SEQ LD NO: 8, or a sequence having the properties described above for BRK-3.

As used herein, "BRK-3" refers generally to h-BRK-3, t-BRK-3 and m-BRK-3,

or a substantially similar BMP receptor kinase protein.

As used herein, "substantially similar" when used to define either amino acid or nucleic acid sequences, means that a particular subject sequence, for example, a sequence altered by mutagenesis, varies from a reference sequence by one or more substitutions, deletions, or additions, the net effect of which is to retain biological activity of the BRK-3 protein. Alternatively, nucleic acid sequences and analogs are "substantially similar" to the specific DNA sequence disclosed herein if the DNA sequences, as a result of degeneracy in the genetic code, encode an amino acid sequence substantially similar to the reference amino acid sequence. In addition, "substantially similar" means a receptor protein that will react with antibodies generated against the BRK-3 protein or peptides derived from the protein sequence of BRK-3.

As used herein, "biologically active" means that a particular molecule shares sufficient amino acid sequence similarity with the embodiments of the present invention disclosed herein to be capable of binding detectable quantities of BMP-2 or BMP -4, or transmitting a BMP-2 or BMP -4 stimulus to a cell, for example, as a component of a hybrid receptor construct. Preferably, biologically active BRK-3 within the scope of the present inve tion means the receptor protein is capable of binding [^ ² ^I]-BMP-4 with nanomolar or subnanomolar affinity (Krj approximately equal to 10'^M). Preferably, the affinity is from about lxlO'l^M to lxlO'^M, with a proportion of binding sites exhibiting a Kj less than 10" 1 °M.

As used herein, "soluble fragment" refers to an amino acid sequence corresponding to the extracellular region of BRK-3 which is capable of binding BMPs. Soluble fragments include truncated proteins wherein regions of the receptor molecule not required for BMP binding have been deleted. Examples of such soluble fragments of the present invention include, but are not limited to, polypeptides having the amino acid sequences substantially similar to SEQ LD NO:6; SEQ LD NO: 10; amino acid residues 1-150 depicted in SEQ LD NO:2; amino acid residues 1-150 depicted in SEQ ID NO: 8; or polypeptides encoded by nucleic acid residues substantially similar to SEQ ID NO:5; SEQ LD NO:9; nucleic acid residues 409-858 depicted in SEQ LD NO: l, or nucleic acid residues 17-466 depicted in SEQ LD NO:7.

As used herein, "digit-removed BMP-2" and "DR-BMP-2" refer to a fragment of BMP-2 protein wherein the amino terminus of mature BMP-2 has been removed by mild trypsin digestion (B.B. Koenig et al., Molecular and Cellular Biology, 14: 5961-5974 (1994)). As used herein, "isolated", in reference to the receptor protein of the present invention or DNA sequences encoding said protein, means that the protein or DNA sequence is removed from the complex cellular milieu in which it naturally occurs, and said protein is expressible from said DNA sequence in a cell that does not naturally

express it when operably linked to the appropriate regulatory sequences.

As used herein, "operably linked" refers to a condition in which portions of a linear DNA sequence are capable of influencing the activity of other portions of the same linear DNA sequence. For example, DNA for a signal peptide (secretory leader) is operably linked to DNA for a polypeptide if it is expressed as a precursor which participates in the secretion of the polypeptide; a promoter is operably linked to a coding sequence if it controls the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation. Generally, operably linked means contiguous and, in the case of secretory leaders, contiguous in reading frame.

As used herein, "ATCC" means American Type Culture Collection, Rockville, Maryland.

As used herein, "bone morphogenetic protein 2" or "BMP-2" means a peptide encoded by a DNA sequence contained in ATCC No. 40345 (see ATCC NIH REPOSITORY CATALOGUE OF HUMAN AND MOUSE DNA PROBES AND LIBRARIES, sixth Edition, 1992, p. 57, hereinafter "ATCC/NTH REPOSITORY CATALOGUE"). Isolation of BMP-2 is disclosed in U.S. Patent No. 5,013,649, Wang, Wozney and Rosen, issued May 7, 1991; U.S. Patent No. 5,166,058, Wang, Wozney and Rosen, issued November 24, 1992; and U.S. Patent No. 5,168,050, Hammonds and Mason, issued December 1, 1992; each of which is incorporated herein by reference.

As used herein, "bone morphogenetic protein 4" or "BMP-4" means a peptide encoded by a DNA sequence contained in ATCC No. 40342 (see ATCC/NLH REPOSITORY CATALOGUE). Isolation of BMP-4 is disclosed in U.S. Patent No. 5,013,649, Wang, Wozney and Rosen, issued May 7, 1991, incorporated herein by reference.

As used herein, "bone morphogenetic protein 7" or "BMP-7" means a peptide encoded by a DNA sequence contained in ATCC No. 68020 and ATT 68182 (see ATCC NTH Repository Catalogue), where the cDNA in ATCC 68182 is claimed to contain all of the nucleotide sequences necessary to encode BMP-7 proteins. Isolation of BMP-7 is disclosed in U.S. Patent 5,141,905, issued August 25, 1992, to Rosen, et al., which is incorporated herein by reference.

As used herein, a "BMP Type I Receptor Kinase" is a protein capable of binding BMP-2, BMP-4 and/or other known BMPs, and bears sequence characteristics of a type I receptor including, but not limited to, an extracellular ligand binding domain containing a cysteine box and an upstream cysteine box, an SGSGSG motif, designated the GS domain, in the intracellular juxtamembrane region, an intracellular kinase domain that is > about 85% similar to other type I receptors for other ligands in the TGF-β superfamily, and/or a relatively short carboxy terminus. As used herein, "BMP Type I

Receptor Kinase" also includes receptor proteins having the characteristics of a BMP type I receptor as described in the literature, such as in: B.B. Koenig et al., Molecular and Cellular Biology, 14: 5961-5974 (1994); L. Attisano, et al., Biochimica et Biophysica Acta, 1222: 71-80 (1994); J. Massague, L. Attisano, and J. L Wrana, Trends in Cell Biology, 4: 172-178 ( 1994); and ten Dijke, et al., J. Biological Chemistry, 269: 16985-16988 (1994).

Examples of BMP type I receptors include, but are not limited to: BRK-1 (B.B. Koenig et al., Molecular and Cellular Biology, 14: 5961-5974 (1994)); BRK-2, also referred to as RPK-1 (S. Sumitomo, T. Saito, and T. Nohno, DNA Sequence, 3: 297- 302 (1993); ALK-2, which has been shown to be a receptor for BMP-7 (ten Dijke et al., J. Biological Chemistry, 269: 16985-16988 (1994)); the Xenopus BMP type I receptor that binds BMP-2 and BMP-4 and which is involved in mesoderm induction (J.M. Graff, R.S. Thies, J.J. Song, A.J. Celeste, and D A. Melton, Cell, 79: 169-179 (1994)) _% and type I receptors from Drosophila that bind the decapentaplegic peptide, which is the Drosophila homologue of BMP-2 and BMP-4. These Drosophila receptors are designated 25D1, 25D2, and 43E (T. Xie, A.L. Finelli, and R.W. Padgett, Science, 263: 1756-1759 (1994); A. Penton, Y. Chen, K. Staehling-Hampton, J.L. Wrana, L. Attisano, J. Szidonya, A. Cassill, J. Massague, and F.M. Hoffmann, Cell, 78: 239-250 (1994); and T. Brummel, V. Twombly, G. Marques, J. Wrana, S. Newfeld, L. Attisano, J. Massague, M. O'Connor, and W. Gelbart, Cell, 78: 251-261 (1994)).

As used herein, "DNA sequence" refers to a DNA polymer, in the form of a separate fragment or as a component of a larger DNA construct, which has been derived from DNA isolated at least once in substantially pure form, i.e., free of contaminating endogenous materials and in a quantity or concentration enabling identification, manipulation, and recovery of the sequence and its component nucleotide sequences by standard biochemical methods, for example, using a cloning vector. Such sequences are preferably provided in the form of an open reading frame uninterrupted by internal nontranslated sequences (introns) which are typically present in eukaryotic genes. Genomic DNA containing the relevant sequences could also be used. Sequences of non-translated DNA may be present 5' or 3' from the open reading frame, where the same do not interfere with manipulation or expression of the coding regions. DNA sequences encoding the proteins provided by this invention can be assembled from cDNA fragments and short oligonucleotide linkers, or from a series of oligonucleotides, to provide a synthetic gene which is capable of being expressed in a recombinant transcriptional unit.

As used herein, "recombinant" means that a protein is derived from a DNA sequence which has been manipulated in vitro and introduced into a host organism.

As used herein, "microbial" refers to recombinant proteins made in bacterial,

fungal (e.g., yeast), or insect expression systems.

As used herein, "recombinant expression vector" refers to a DNA construct used to express DNA which encodes a desired protein (for example, BRK-3) and which includes a transcriptional subunit comprising an assembly of 1) genetic elements having a regulatory role in gene expression, for example, promoters and enhancers, 2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and 3) appropriate transcription and translation initiation and termination sequences. Using methodology well known in the art, recombinant expression vectors of the present invention can be constructed. Possible vectors for use in the present invention include, but are not limited to: for mammalian cells, pJT4 (discussed further below), pcDNA-1 (Invitrogen, San Diego, Ca) and pSV-SPORT 1 (Gibco-BRL, Gaithersburg, MD); for insect cells, pBlueBac III or pBlueBacHis baculovirυs vectors (Invitrogen, San Diego, CA); and for bacterial cells, pET-3 (Novagen, Madison, WI). The DNA sequence coding for a BRK-3 protein receptor kinase of the present invention can be present in the vector operably linked to regulatory elements.

In one embodiment of the present invention, mammalian host cells are preferably transfected with the plasmid construct pJT6-mBRK-3L, thereby resulting in expression of m-BRK-3. In another embodiment of the present invention, mammalian host cells are preferably transfected with the plasmid construct, pJT4-hBRK3T, thereby resulting in expression of t-BRK-3. Transfection with the recombinant molecules can be effected using methods well known in the art.

As used herein, "host cell" means a cell comprising a recombinant expression vector of the present invention. Host cells may be stably transfected or transiently transfected within a recombinant expression plasmid or infected by a recombinant virus vector. The host ceils include prokaryotic cells, such as Escherichia coli, fungal systems such as Saccharomyces cerevisiae, permanent cell lines derived from insects such as Sf-9 and Sf-21, and permanent mammalian cell lines such as Chinese hamster ovary (CHO) and SV40-transformed African green monkey kidney cells (COS).

In one embodiment, the present invention relates to a type II BMP receptor kinase protein, or soluble fragment thereof. Preferably, the BMP receptor kinase protein is h-BRK-3, having an amino acid sequence SEQ LD NO: 2, or the soluble fragment thereof having an amino acid sequence SEQ LD NO: 6. Also preferred is the BMP receptor kinase protein m-BRK-3 having an amino acid sequence SEQ LD NO: 8, or the soluble fragment thereof having an amino acid sequence SEQ LD NO: 10. Also preferred is the BMP receptor kinase protein t-BRK-3 having an amino acid sequence SEQ LD NO: 4.

In another embodiment, the present invention relates to a DNA sequence coding for the h-BRK-3 receptor protein, or a soluble fragment thereof. (The DNA can be

genomic or cDNA.) Preferably the h-BRK-3 protein is coded for by the nucleic acid sequence SEQ LD NO: 1; the soluble fragment thereof is preferably coded for by the nucleic acid sequence SEQ LD NO: 5.

In another embodiment, the present invention relates to a DNA sequence coding for the t-BRK-3 protein. (The DNA sequence can be genomic DNA or cDNA.) Preferably the DNA sequence is SEQ LD NO:3.

In another embodiment, the present invention relates to a DNA sequence coding for the m-BRK-3 protein, or a soluble fragment thereof. (The DNA sequence can be genomic DNA or cDNA.) Preferably the m-BRK-3 protein is coded for by the DNA sequence SEQ LD NO: 7; the soluble fragment is preferably coded for by the DNA sequence SEQ LD NO: 9.

In another embodiment, the present invention relates to a recombinant expression vector comprising a DNA sequence coding for the m-BRK-3 prqtein. Preferably the recombinant expression vector is a plasmid having all of the identifying characteristics of the pJT6-mBRK-3S or pJT6-mBRK-3L plasmid constructs contained in ATCC No. 69694 and ATCC No. 69695, respectively. In another embodiment, the present invention relates to a host cell comprising the above described recombinant expression vector. Preferably the host cell is a mammalian cell; more preferably a CHO cell or COS cell, or a mink lung epithelial cell. In another embodiment, the present invention relates to a recombinant expression vector comprising a DNA sequence coding for t-BRK-3. Preferably the recombinant expression vector is a plasmid having all of the identifying characteristics of the pJT4-hBRK3T plasmid construct contained in ATCC No. 69676. In another embodiment, the present invention relates to a host cell comprising the recombinant expression vector comprising a DNA sequence that codes for t-BRK-3. Preferably the host cell is a mammalian cell; more preferably a CHO ceil or COS cell.

In another embodiment, the present invention relates to a recombinant expression vector comprising a DNA sequence coding for h-BRK-3 In another embodiment, the present invention relates to a host cell comprising the recombinant expression vector comprising a DNA sequence that codes for h-BRK-3. Preferably the host cell is a mammalian cell; more preferably a CHO cell or COS cell.

In another embodiment, the present invention relates to a method for producing BRK-3, t-BRK-3, or m-BRK-3 comprising isolating BRK-3, t-BRK-3, or m-BRK-3 from the host cell described above. The BMP type II receptor of the present invention is useful for identifying compounds (e.g., BMP (preferably BMP-2, BMP-4, or BMP-7), or other as yet to be discovered compounds) capable of binding to a BMP receptor kinase protein, the method comprising introducing a sample comprising the compound to the BMP type II

receptor kinase protein of the present invention that is expressed in a cell, and allowing the compound to bind to the receptor kinase protein. Preferably the type II receptor kinase protein has amino acid sequence SEQ LD NO:2 (h-BRK-3) or a soluble fragment thereof, or SEQ LD NO:8 (m-BRK-3) or SEQ LD NO:4 (t-BRK-3) or soluble fragment thereof. Such a method is also useful for determining the amount of BMP or other receptor binding compound present in the sample.

For example, BMP concentration in a sample can be determined by radioreceptor assay, in which unlabeled BMP in the sample competes with labeled tracer BMP for binding to the BRK-3 receptor. As described in co-pending application U.S.S.N. , filed by Rosenbaum on November 4, 1994, the BRK-3 receptor of the present invention may be complexed to a BMP type I receptor. As the amount of BMP in the sample increases, it reduces the amount of labeled BMP which is able to bind to BRK-3 or a receptor protein complex comprising BRK-3. Comparison with a standard curve prepared with known concentrations of unlabeled BMP allows accurate quantitation of BMP concentration in the sample. Labeling of tracer BMP is preferably done by iodination with BRK-3 can be expressed in the outer membrane of a stable cell line, or supplied as a soluble fragment, or as a soluble fragment covalently attached to a solid support. To perform the assay, unlabeled BMP from the sample and labeled tracer BMP compete for binding to the receptor until equilibrium is reached. The receptor-BMP complex is then isolated from free ligand, for example by washing (in the case of an adherent cell line), rapid filtration or centrifugation (in the case of a nonadherent cell line or receptor bound to a solid support), or precipitation of the receptor-ligand complex with antibodies, polyethylene glycol, or other precipitating agent followed by filtration or centrifugation (in the case of a soluble receptor). The amount of labeled BMP in the complex is then quantitated, typically by gamma counting, and compared to known standards. These methods have been described in the literature using other receptors (M. Williams, Med Res. Rev., 1 1 : 147-184 (1991); M. Higuchi and B.B. Aggarwal, Anal. Biochem., 204: 53-58 (1992); M.J. Cain, R.K. Garlick and P.M. Sweetman, J. Cardiovasc. Pharm., 17: S 150-S151 (1991), each of which are incorporated herein by reference), and are readily adapted to the BRK-3 receptor/BMP system. Such a radioreceptor assay can be used for diagnostic purposes for quantitation of BMP in clinical samples, where such quantitation is necessary.

The BMP type II receptor protein of the present invention is also useful in high- throughput screens to identify compounds capable of binding to BRK-3, or a homologous receptor protein. In such a method, the higher the affinity of the compound for BRK-3, the more efficiently it will compete with the tracer for binding to the receptor, and the lower the counts in the receptor-ligand complex. In this case, one compares a series of compounds at the same concentration range to see which competed

for receptor binding with the highest affinity.

This invention is useful for determining whether a ligand, such as a known or putative drug, is capable of binding to and/or activating the receptors encoded by the DNA molecules of the present invention. Transfection of said DNA sequence into the cell systems described herein provides an assay system for the ability of ligands to bind to and or activate the receptor encoded by the isolated DNA molecule. Recombinant cell lines, such as those described herein, are useful as living cell cultures for competitive binding assays between known or candidate drugs and ligands which bind to the receptor and which are labeled by radioactive, spectroscopic or other reagents. Membrane preparations containing the receptor isolated from transfected cells are also useful for competitive binding assays. Soluble receptors derived from the ligand binding domain of the receptor can also be employed in high throughput screening of drug candidates. Functional assays of intracellular signaling can act as assays for binding affinity and efficacy in the activation of receptor function. In addition, the recombinant cell lines may be modified to include a reporter gene operably linked to a response element such that a signal sent by the receptor turns on the reporter gene. Such a system is especially useful in high throughput screens directed at identification of receptor agonists. These recombinant cell lines constitute "drug discovery systems", useful for the identification of natural or synthetic compounds with potential for drug development. Such identified compounds could be further modified or used directly as therapeutic compounds to activate or inhibit the natural functions of the receptor encoded by the isolated DNA molecule.

The present invention relates to a receptor-reporter system to identify compounds which will alter transcription of the gene for the BMP type II receptor BRK-3, thereby acting as indirect BRK-3 receptor agonists or antagonists. The reporter system for evaluating whether test compounds are capable of acting as agonists of the BMP type LI receptor protein kinase BRK-3, or functionally modified forms thereof, comprises:

(a) culturing cells containing: (i) DNA encoding BRK-3 protein, or functionally modified forms thereof, and

(ii) DNA encoding a hormone response element operatively linked to a reporter gene, wherein the culturing is carried out in the presence of at least one test compound whose ability to induce the transcriptional activity of BRK-3 protein is sought to be determined, and thereafter

(b) monitoring the cells for expression of the reporter gene.

The reporter system for evaluating whether test compounds are capable of acting

as antagonists of the BMP type II receptor protein kinase BRK-3, or functionally modified forms thereof, comprises:

(a) culturing cells containing:

(i) DNA encoding BRK-3 protein, or functionally modified forms thereof, and

(ii) DNA encoding a hormone response element operatively linked to a reporter gene, wherein the culturing is carried out in the presence of: a fixed concentration of at least one agonist for transcription of BRK-3 or, functionally modified forms thereof, and increasing concentrations of at least one test compound whose ability inhibit transcriptional activation of the BRK-3 receptor protein is sought to be determined; and thereafter

(b) monitoring in the cells the level of expression of the product of the reporter gene as a function of the concentration of the test compound, thereby indicating the ability of the test compound to inhibit activation of transcription.

Cell lines expressing a high number of the BMP type II receptor proteins, or a soluble form thereof, of the present invention are also useful as a source of protein for receptor purification. The purified receptor or its soluble form can then be used for high-throughput screening assays for the purposes described above. The purified receptor or its soluble form can also be used for determination of the structure of the BMP:BRK-3 complex, using X-ray crystallography or NMR techniques, which can then be used in rational design of BMP agonists or antagonists. In addition, the purified receptor or its soluble form can be used in combination with a type I receptor or its soluble form for determination of the structure of a BMP:BRK-3:type I receptor complex. The soluble receptor proteins can also be used therapeutically as an agonist or antagonist of BMP function in vivo.

The present invention also relates to antibodies generated against the BMP type JJ receptor kinase proteins of the present invention. Such antibodies can be prepared by employing standard techniques as are well known to those skilled in the art, using the BMP type JJ receptor kinase protein of the present invention as antigens for antibody production. These antibodies can be employed for diagnostic applications, therapeutic applications, and the like. Preferably for therapeutic applications, the antibodies will be monoclonal antibodies.

The soluble receptor proteins of the present invention and the antibodies of the invention can be administered in a clinical setting using methods such as by intraperitoneal, intramuscular, intravenous, or subcutaneous injection, implant or transdermal modes of administration, and the like. Such administration can be expected to provide therapeutic alteration of the activity of the BMPs.

The nucleotide sequences disclosed herein, SEQ LD NO:3 and SEQ LD NO: l, represent the sequence of the DNA that codes for t-BRK-3 and h-BRK-3, respectively, isolated from human skin fibroblasts. SEQ ID NO: 7 represents the DNA sequence coding for m-BRK-3 receptor protein from mouse NLH3T3 cells. These sequences could be readily used to obtain the cDNA for BRK-3 from other species, including, but not limited to, rat, rabbit, Drosophila, and Xenopus. These cDNA sequences can also be readily used to isolate the genomic DNA for BRK-3 This would permit analysis of the regulatory elements controlling receptor gene expression, which may offer new opportunities for therapeutic intervention and disease diagnosis. The nucleotide sequences are also useful to determine the distribution of the BRK-3 receptor in normal tissues and in disease states, which allows an assessment of its physiological role in vivo. For purposes of illustrating a preferred embodiment of the present invention, the following non-limiting examples are discussed in detail.

Example 1 Generation of PCR Fragments

In order to generate a PCR fragment of type II receptors related to the TGF-β type π receptor, primers shown in Figure 1 are designed from the kinase domains of the TGF- ^β type II receptor. For the first round of PCR, the primers are TSK-1, derived from kinase domain II, and TSK-2, derived from kinase domain VIII. The template DNA consists of cDNA prepared from mRNA isolated from human skin fibroblasts from a 9 month old male. The PCR reaction, carried out in a total volume of 50 μl, contains approximately 0.2 μg of this cDNA, primers TSK-1 and TSK-2 at a concentration of 15 μM, stocks of all four deoxynucleotides at a concentration of 0.2 mM each, 1.5 unit of DNA polymerase from Thermus thermophilus (hereafter, Tth polymerase) (Toyobo, Osaka, Japan) and reaction buffer for the Tth polymerase (Toyobo, Osaka, Japan). After an initial melting period of 1 min at 94°C, the temperature cycle is carried out as follows for 35 cycles: melting, 92°C for 40 sec; annealing, 48 ^e C for 40 sec; extension, 75°C for 90 sec. After the 35th cycle, the reaction is held at 75°C for an additional 5 min to complete the extension. Several bands are amplified, including some in the area of 470 base pairs (bp) corresponding to the predicted sequence length of a type II receptor homologous to the TGF-β type II receptor. Accordingly, fragments in this size range are recovered from an agarose gel using QIAEX (Qiagen, Chatsworth, CA, a kit for gel purification of DNA fragments, including activated silica spheres and buffers) according to the manufacturer's instructions, then resuspended in 10 mM Tris, pH 8.0, 1 mM EDTA (TE) in a volume of 20 μl.

To reduce the background from fragments amplified from cDNAs not related to the TGF-β type II receptor, a second round of PCR is carried out using "nested"

primers based on conserved regions of the TGF- ^β type II receptor located within the 470 bp region amplified in the first round. The nested primers are AVR-5, derived from kinase domain IV of the TGF- ^β type II receptor, and TSK-4, derived from kinase domain VLB (Figure 1). The template consists of an aliquot (0.5 μl) of the PCR fragments isolated from the first round of PCR. To this is added the primers AVR-5 (5 μM) and TSK-4 (15 μM), all four deoxynucleotides (0.2 mM each), 1.5 units of Tth DNA polymerase, and reaction buffer for the Tth DNA polymerase, in a total volume of 50 μl. The temperature cycle program is executed exactly as described above for the first round of PCR. Agarose gel electrophoresis of the PCR reaction products shows amplification of a band in the range of 300 bp, as expected. This fragment is isolated using QIAEX.

In order to subclone the PCR product of the second PCR reaction, the purified fragment is phosphorylated using polynucleotide kinase and ligated to the cloning vector pGEM7Zf (+) (Promega, Madison, WI) which has previously been cut with Sma I and dephosphoryiated. The ligation mix is used to transform E. coli XL 1 -Blue (Stratagene, La Jolla, CA). When the transformation mix is plated on agar containing isopropyl-β-D- thiogalactoside (LPTG) and 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal), colonies are obtained which lack blue color, indicating the presence of an insert. Plasmid DNA is prepared from a selection of these colonies. Three of the candidate plasmids, designated HSK7-1, HSK7-2, and HSK7-4 are found to have inserts of the expected size (300 bp). Upon sequencing of the inserts, the 300 bp insert from HSK7-2 is found to encode a portion of a novel kinase that is predicted to be a novel member of the TGF-β receptor superfamily. Accordingly, the HSK7-2 PCR fragment is used as a probe to isolate the full-length receptor clone.

Example 2 Isolation of human t-BRK-3 cDNA In order to locate the cDNA corresponding to the 300 bp insert in HSK7-2, a cDNA library is constructed from the same mRNA used to isolate the PCR fragment. This is accomplished using the SUPERSCRIPT Choice System (Life Technologies, Gaithersburg, MD; a kit for cDNA synthesis, including primers, adapters, SUPERSCRIPT II RNAse H" Reverse Transcriptase (Life Technologies, Gaithersburg MD; a modified form of reverse transcriptase from Moloney murine leukemia virus), enzymes, nucleotides, buffers, and gel filtration columns) according to the manufacturer's instructions, except that 180 units of RNase inhibitor (Takara, Kyoto, Japan) is added to the first strand synthesis. The template is mRNA (4 μg) from human skin fibroblasts from a 9 month old male. A total of 4 μg of cDNA is obtained after first and second strand synthesis. This is followed by the addition of Eco RI adapters

(supplied with the kit) which contain internal Not I and Sal I sites. The Eco Rl-adapted cDNA is then phosphorylated and subjected to size fractionation according to the manufacturer's instructions, using gel filtration columns provided with the kit.

The size fractionated cDNA is ligated into the Eco RI site of the phage λgtlO, and packaged in vitro with GIGAPACK II Gold Packaging Extract (Stratagene, La

Jolla, CA; a restriction-minus in vitro packaging extract for high-efficiency construction of cDNA libraries in λ phage) according to the manufacturer's instructions. A total of

8.1 x 10^ phages are obtained.

The library is screened on ten HYBOND Nylon membranes (Amersham, Arlington Heights, LL; nylon membranes optimized for immobilization of nucleic acids), at a density of 1 x 10 ⁵ plaques filter. The insert from HSK7-2 is labeled with the

MULTLPRLME DNA Labeling System (Amersham, Arlington Heights, LL; a kit for random primer labeling of DNA, including Klenow DNA polymerase, primers, and buffers) according to the manufacturer's instructions. The labeled probe is allowed to hybridize to the library filters in 50% formamide, 6X SSPE ( lx SSPE = 0.14 M NaCI, 8 mM sodium phosphate, 0.08 mM EDTA, pH 7.7), 5X Denhardt's solution (IX

Denhardt's = 0.02% Ficoll type 400, 0.02% polyvinylpyrrolidone, 0.02% BSA), 0.5% sodium dodecyl sulfate (SDS), and 100 μg/ml denatured salmon sperm DNA at 42°C for 12 hr. The blot is then washed in 2X SSPE, 0.1% SDS three times at room temperature (15 minutes each), followed by a 1 hr wash at 42°C

After three rounds of screening, 3 independent clones are obtained. One of the clones, designated HSK723, is found to encode the same sequence as the HSK7-2 insert. Complete DNA sequence is obtained for this clone. The cDNA from this clone is designated t-BRK-3.

Example 3 t-BRK-3 Sequence Analysis The DNA sequence of this t- BRK-3 clone is shown in SEQ LD NO: 3, and the deduced protein sequence of t-BRK-3 in SEQ LD NO: 4. The t-BRK-3 open reading frame derived from clone HSK723 encodes a protein of at least 583 amino acids. No stop codon is observed to be located in-frame in the 3' region of the HSK723 cDNA, indicating that this clone is incomplete at the 3' end. It is thus designated t-BRK-3.

The deduced protein sequence of t-BRK-3 shown in SEQ LD NO: 4 is searched against all translated protein sequences in GenBank Release 84.0, dated August 15, 1994, using a standard Needleman-Wunsch algorithm (S B. Needleman and CD.

Wunsch, J. Mol. Biol. 48: 443-453 (1970)), and is found to represent a novel sequence.

Analysis of the predicted protein sequence reveals a predicted structure of a

TGF-β type II superfamily member transmembrane serine threonine kinase. The

predicted single transmembrane region encompasses residues 151-172 in SEQ LD NO:4. Three potential N-linked glycosylation sites are located at amino acid residues 55, 1 10, and 126 in the predicted extracellular domain. Amino acids 1 16-123 in SEQ LD NO:4 contain the cluster of cysteine residues called the "cysteine box" that is a characteristic of receptors for ligands of the TGF-β superfamily. The cysteine box of t-BRK-3 is identical in 6 of 8 amino acid residues to the cysteine box of the DAF-4 type II receptor for BMP-2 and BMP-4. However, the overall sequence identity of t-BRK-3 to DAF-4 in the extracellular domain is only 7.1%.

Amino acids 200-504 (in SEQ LD NO: 4) in the predicted cytoplasmic region of t-BRK-3 contains all of the consensus sequences that characterize a protein kinase domain with predicted specificity for serine threonine residues (S. K. Hanks, A.M. Quinn, and T. Hunter, Science, 241 : 42-52 (1988)).

Example 4 Construction of expression vectors for t-BRK-3.

BRK-1. BRK-2. and DAF-4 In order to express t-BRK-3 in mammalian cells, it is subcloned into the vector pJT4, designed for transient expression. The pJT4 vector, optimized for transient expression in COS cells, includes the cytomegalovirus early promoter and enhancer, which gives very efficient transcription of message; an "R" element from the long terminal repeat of the human T-cell leukemia virus- 1, which has been shown to increase expression levels further; an intron splice site from SV40, which is believed to enhance message stability; a multiple cloning site; a polyadenylation signal derived from SV40, which directs the addition of a poly A tail to the message, as is required for most eukaryotic mRNA; and the SV40 origin of replication, which permits the replication of the plasmid to extremely high copy number in cells which contain the SV40 large T antigen, such as COS cells. In addition, for manipulation and amplification of the vector in bacteria, the vector contains an £1 coli origin of replication and an ampicillin resistance gene. Insertion of the truncated human BRK-3 cDNA into pJT4 is accomplished as follows.

Since no stop codon had been identified in the 3' region of the kinase domain, PCR is performed to insert a stop codon to permit translation of the protein. Accordingly, a PCR primer is designed to insert two stop codons after nucleotide 2028 in SEQ LD NO: 3, thus terminating the kinase after He 540 in SEQ LD NO: 4. This is chosen to correspond to the length of the activin type II receptor (L.S. Mathews and W.V Vale, Cell, 65: 973-982 (1991)), so that it should be sufficient for proper folding of the kinase domain. The stop codons are followed by a Kpn I site. The complete sequence of the primer (which includes the reverse complement of nucleotides 2013-

2028 in SEQ LD NO:3) is 5' ACG CGG TAC CTC ACT AAA TTT TTG GCA CAC GC 3'. A second primer is designed as an exact match to the t-BRK-3 sequence in the area of the Afl III site (nucleotides 1618-1637 in SEQ LD NO:3), having the sequence 5' GTA GAC ATG TAT GCT CTT GG 3'. The template for the reaction is clone HSK723, described in example 2, which contains the cDNA for t-BRK-3 in BLUESCRLPT II SK (+) (Stratagene, La Jolla, CA; a 2.96 kb colony-producing phagemid derived from pUC 19).

PCR is carried out using the GENE AMP PCR Kit with AMPLITAQ DNA Polymerase (Perkin Elmer, Norwalk, CT; a kit containing components necessary for amplification of DNA using the polymerase chain reaction, including AMPLITAQ, a recombinant modified form of the DNA polymerase from Ther us aquaticus (Perkin- Elmer, Norwalk CT), nucleotides, and buffers), according to the manufacturer's instructions, using a GENE AMP PCR System 9600 Thermocycler (Perkin Elmer, Norwalk, CT). An initial melting at 95°C for 5 min is followed by 20 cycles of the following program, melting at 95°C for 1 min, annealing at 50°C for 1 min, and extension at 72°C for 1 min. After the last cycle, the temperature is held at 72°C for an additional 2 min to complete extension.

The resulting amplified band, at the expected size of 400 bp, is isolated from an agarose gel and digested with Afl III and Kpn I. Meanwhile, the cDNA for t-BRK-3 is digested with Eco RV and Afl III, and the vector pJT4 is digested with Eco RV and Kpn I. These three isolated fragments are ligated in a single step to give the construct pJT4-hBRK3T, shown in Figure 2. To confirm that no errors are introduced during PCR, the region from the Afl III site to the Kpnl site at the 3' end is sequenced using the TAQ DYE DEOXY Terminator Cycle Sequencing Kit (Applied Biosystems, Foster, CA; kit containing components for automated DNA sequencing using the dideoxy terminator method, including AMPLITAQ, nucleotide mix, dye-labeled dideoxy nucleotide terminators, and buffers) and an Applied Biosystems Model 373A Automated DNA Sequencer. No errors are found.

To determine the effects of co-expression of t-BRK-3 with type I BMP receptors, it is necessary to co-express the cDNA for t-BRK-3 with the cDNA for BRK- 1 or the cDNA for BRK-2. The DNA sequence for mouse BRK-1 is shown in SEQ LD NO: 11, and the deduced amino acid sequence for mouse BRK-1 is shown in SEQ LD NO: 12. The DNA sequence for chicken BRK-2 is shown in SEQ LD NO: 13, and the deduced protein sequence shown for chicken BRK-2 is shown in SEQ LD NO: 14. For mammalian expression of BRK-1, the plasmid pJT4-J159F is used.

Construction of this plasmid is described in U.S.S.N. 08/158,735, filed November 24, 1993 by Cook, et al. and B.B. Koenig et al., Molecular and Cellular Biology 14: 5961- 5974 (1994); ATCC 69457. Briefly, the construct containing the BRK-1 cDNA

subcloned in BLUESCRLPT SK (-) is linearized with the restriction endonuclease Alf III, and the overhanging end filled in using DNA Polymerase I Klenow fragment. The linearized plasmid is then digested with Not I, liberating the insert from the plasmid. The insert is then subcloned into the ρJT4 expression vector at the Not I and EcoRV sites. The blunt end generated by the Klenow reaction is compatible with the EcoRV site, which is also a blunt end; ligation eliminates the Eco RV site. The construct pJT4- J159F is shown in Figure 3.

For mammalian expression of BRK-2, its cDNA is subcloned into the vector pJT3. This vector is identical to pJT4, described in this example, except that the multiple cloning site is in the opposite orientation, and an additional Not I site is present at the 5' end of the multiple cloning site. The cDNA for BRK-2 (see S. Sumitomo, et al., DNA Sequence, 3: 297-302 (1993)), originally obtained in the vector pRc CMV (Invitrogen, San Diego, CA; a mammalian expression vector), is excised by digestion with Kpn I and Xho I. It is subcloned into pJT3 at the Kpn I and Sal I sites. This regenerates a Kpn I site at the 5' end of BRK-2, while the Xho I and Sal I sites are destroyed. The resulting construct is designated pJT3-BRK-2 and is shown in Figure 4.

For mammalian expression of DAF-4, the type II BMP receptor from Caenorhabditis elegans (M. Estevez, L. Attisano, J.L. Wrana, P S. Albert, J. Massague, and D.L. Riddle, Nature, 365: 644-9 (1993), the cDNA is obtained in BLUESCRLPT II and subcloned into pJT4 as follows. A 2.4 kb fragment containing the daf-4 cDNA is excised by digestion with Dra I and Apa I. This fragment is subcloned into pJT4 at the Sma I and Apa I site. The Apa I site is regenerated, while the Dra I and Sma I sites are destroyed. This construct is designated pJT4-Daf4, and is shown in Figure 5. For mammalian expression of m-BRK-3, see Example 10, below.

Example 5 Mammalian expression of t-BRK-3. BRK-1. BRK-2. and DAF-4 Transient expression of BRK-3 in mammalian cells using pJT4-hBRK3T is carried out in COS-7 cells (ATCC CRL 1651) using electroporation or COS-1 cells (ATCC CRL 1650) using DEAE Dextran (Pharmacia Biotech, Piscataway, NJ).

COS-7 cells are grown to confluence in Dulbecco's Modified Eagle (DME) high glucose media supplemented with 10% fetal bovine serum (Hyclone, Logan, Utah), nonessential amino acids (GLBCO, Gaithersburg, MD), and glutamine, then trypsinized to release cells from the plate. The detached COS-7 cells are pelleted in a tabletop centrifuge, then resuspended in fresh media at a concentration of 6.25 x 10^ cells ml. The cell suspension (5 x 10 ⁶ cells, 0.8 ml) is transferred to the cuvette of a BioRad GENE PULSER electroporation system (BioRad, Hercules, CA). The purified plasmid containing the receptor DNA of interest (10 μg for pJT4-J159F and pJT3-BRK2 and/or

20 μg for pJT4-hBRK3T) is added to the cuvette, and the cells subjected to electoporation at 4.0 kV/cm, with a capacitance of 25 μFd. Cells are then plated (400,000 cells per well for 12 well plates and 5 x 10 ⁶ cells for 100 mm plates) and allowed to recover. Fresh media is supplied after 24 hr. At 48 hr, cells are ready to be tested for binding of BMP-4.

For transient expression of BMP receptors in COS-1 cells, the cells are grown to approximately 50%-80% confluence in DME high glucose media supplemented with

10% fetal bovine serum (HyClone, Logan, Utah), nonessential amino acids, and glutamine in 100 mm plates. The cells are washed twice with 37°C serum-free DME media, after which 4 ml of DNA mixture is added to each 100 mm plate. The DNA mixture contains DME, 10% Nu-Serum (Collaborative Biomedical Products, Bedford,

MA), 400 μg ml DEAE-Dextran (Pharmacia, Piscataway, NJ), 0.1 mM chloroquine

(Sigma, St. Louis. MO), and the cDNAs of interest: for t-BRK-3, 16 μg pJT4-hBRK3T; fo; RK-1, 8 μg pJT4-J159F; for BRK-2, 8 μg pJT3-BRK2; for DAF- 4, 16 μg pJT4-Daf4. The cells are then incubated at 37°C with the DNA mixture for 3 hr. The solution is aspirated and the cells are incubated with 4 ml of a solution containing 10% dimethylsulfoxide (DM! ⁾ ) in Dulbecco's phosphate buffered saline without calcium or magnesium (PBS; Lite Technologies, Gaithersburg, MD). After 2 min, the DMSO solution is aspirated, the cells are washed with the growth media described above, and fresh media is returned to the plates. The transfeαed cells are split into 12 well plates 24 hr post transfection for whole cell binding or cross linking. After

48 to 68 hr the cells are suitable for binding analysis.

Example 6 Generation of the Radiolabeled BMP-4 Ligand

[ ¹²⁵ I]-BMP-4 is prepared using IODOBEADS (Pierce, Rockford, LL; immobilized chloramine-T on nonporc s polystyrene beads). Lyophilized BMP-4 (2 μg) is taken up in 50 μl of 10 mM acetic acid and added to 450 μl of phosphate-buffered saline (PBS) (Sigma, St. Louis, MO) on ice. To the tube is added 500 μCurie of ¹²⁵ I (Amersham, Arlington Heights, LL) (2200Ci/mmol) in 5 μl, and one lODOBEAD. The reaction is incubated on ice for 10 min with occasional shaking. The reaction is then terminated by removal of the reaction from the lODOBEAD. To remove unreacted l^L ^e mixture is applied to a PD-10 gel filtration column (Pharmacia, Piscataway, NJ) previously equilibrated in 10 mM acetic acid, 0.1 M NaCI, 0.25% gelatin. The resulting labeled protein is >95% precipitable by trichloroacetic acid, indicating that all 1 ² ^I is protein bound, and has a typical specific activity of 4000 to 9000 Ci/mmol.

Alternatively, BMP-4 is labeled with ^ ² ^I by the chloramine-T method (CA. Frolik, L.M. Waksfield, D.M. Smith, and M.B. Sporn, J. Biol. Chem., 259: 10995-

1 1000 (1984)). BMP-4 (2 μg) is taken up in 5 μl of 30% acetonitrile, 0.1% trifluoracetic acid (TFA) plus an additional 5 μl of 1.5 M sodium phosphate, pH 7.4. Carrier free ^I25 I (1 mCi, 9 μl) is added, together with 2 μl of a chJoramine T solution (100 μg/ml). An additional 2 μl of the chJoramine T solution is added at 2.0 min and at 3.5 min. After 4.5 minutes, the reaction is stopped by the addition of 10 μl of 50 mM N-acetyl tyrosine, 100 μl of 60 mM potassium iodide, and 100 μl of 1 1M urea, 1 M acetic acid. After a 3.5 minute incubation, unreacted iodine is removed on a PD-10 gel filtration column (Pharmacia, Piscataway, NJ) run in 4 mM HCI, 75 mM NaCI, 1 mg/ml bovine serum albumin (BSA). The resulting labeled protein is >95% precipitable by trichloroacetic acid, indicating that all ¹² ^I is protein bound, and has a typical specific activity of 3000-8000 Ci mmol.

Example 7 Characterization of BMP-4 Binding to t-BRK-3 Binding of BMP-4 to t-BRK-3 can be demonstrated by whole cell binding of radiolabeled BMP -4, and by covalent crosslinking of radiolabeled BMP-4 to the receptor. These two methods are described in detail below, a. Whole Cell Binding:

COS-7 or COS-1 cells are transfected with pJT4-hBRK3T as described Si example 5. After transfection, cells are seeded into 12 well plates and the binding experiments are carried out at 48 to 68 hr. At that time, cells are washed once with binding buffer (50 mM HEPES, pH 7.4, 128 mM NaCI, 5 mM KC1, 5 mM MgSO4, 1.2 mM CaCl2, 2 mg/ml BSA), then equilibrated in the same buffer at 4°C for 30 - 60 min with gentle shaking. The buffer is then aspirated, and to each well is added 500 μl of binding buffer (4 ^β C), containing [ ¹²⁵ I]-BMP-4 tracer (100 - 400 pM), as well as varying concentrations of unlabeled BMP-2, BMP -4, or other unlabeled ligand, depending on the assay. For determination of nonspecific binding, BMP-4 is added to the binding buffer at a final concentration of 10 to 50 nM. To prevent degradation of ligand during the incubation, a protease inhibitor cocktail is also added, to give a final concentration of 10 μg/ml leupeptin, 10 μg/ml antipain, 50 μg/ml aprotinin, 100 μg/ml benzamidine, 100 μg/ml soybean trypsin inhibitor, 10 μg ml bestatin, 10 μg/ml pepstatin, and 300 μM phenylmethylsulfonyl fluoride (PMSF). The cells are incubated for 4 hr at 4°C with gentle shaking. At the end of the incubation period, the buffer is aspirated, and the cells are rinsed 4 times with 1 ml washing buffer (50 mM HEPES, pH 7.4, 128 mM NaCI, 5 mM KCI, 5 mM MgSO4, 1 2 mM CaCl2, 0.5 mg ml BSA). After the final wash is aspirated, 200 μl of solubilization buffer (10 mM Tris Cl, pH 7.4, 1 mM EDTA, 1% (v/v) Triton X-100) is added to each well and incubated at room temperature for 15 - 30 min. The solubilized cells are then transferred to fresh tubes and

counted in a Packard Model 5005 COBRA Gamma Counter (Packard Instruments, Meriden, CT).

Results are shown in Figure 6, which shows specific binding of [^ ² ^I]-BMP-4 to NLH3T3 cells (ATCC CRL 1658), which display significant endogenous binding of BMP-4, and COS 7 cells transfected with the cDNA for t-BRK-3 in the presence and absence of BRK-1 and BRK-2. ^' t-BRK-3 is capable of binding [ ^{1 5} I]-BMP-4 when expressed alone (bar on far right), at a level similar to that seen for BRK-1 and BRK-2 expressed alone. Binding of [^ ² ^I]-BMP-4 is increased by co-expression of t-BRK-3 with BRK-1, and to a greater extent by co-expression of t-BRK-3 with BRK-2. b. Covalent Crosslinking:

Bifiinctional crosslinking reagent disuccinimidyl glutarate (DSG) (Pierce, Rockford, LL) is used to covalently crosslink bound radiolabeled ligand to its receptor by reaction with free amino groups on lysine residues in the two proteins. Following the crosslinking, cellular proteins are separated by gel electrophoresis, and radioactive bands visualized. The labeled bands represent the receptor selectively "tagged" with the radiolabeled ligand. In this procedure, cells are transfected with the cDNA for BRK-3, and/or BRK-1 or BRK-2, as described in example 5, then seeded into 12 well plates. At 48 - 68 hr after transfection, the cells are washed, equilibrated, and incubated with [125η_BMp_4 and competing unlabeled ligands as described in this example for whole cell binding studies. After completion of the 4 hr incubation with ligand, the cells are washed two to three times at 4°C with 2 ml of binding buffer having the same composition as described above, except that no BSA is added. To each well is then added 1 ml of fresh BSA-free binding buffer, followed by freshly prepared DSG to a final concentration of 135 μM. After swirling gently to mix the DSG, the plates are incubated for exactly 15 minutes at 4°C with gentle shaking. At this point the media is aspirated and the cells washed with 3 ml detachment buffer (10 mM Tris base, 0.25 M sucrose, 1 mM EDTA, 0.3 mM PMSF) or PBS. Solubilization buffer (50 μl) is then added to each well and the cells are allowed to solubilize for 30 - 45 minutes at 4°C with shaking. An aliquot of the sample (20 μl) is transferred to a fresh tube and 5 μl of 5X sample loading buffer (0.25 M TrisCl, pH 6.8, 10% SDS, 0.5 M DTT, 0.5% bromophenol blue, 50% glycerol; purchased from Five Prime Three Prime, Boulder, CO) is added. The samples are boiled for 5 min and centrifuged (13,0000 x g, 5 min). The supernatants are loaded onto 7.5% SDS-polyacrylamide gels (Integrated Separation Systems, Natick, MA) and subjected to electrophoresis. The gels are stained in 0.12% Coomassie Blue R250, 5% methanol, 7.5% acetic acid; destained in 5% methanol, 7.5% acetic acid; then dried. Radioactivity on the dried gel is visualized and quantitated on a PHOSPHORLMAGER (Molecular Devices, Sunnyvale, CA. a device for quantitation of radioactivity using stable phosphor screens), or subjected to autoradiography using

Kodak X-OMAT AR autoradiography film (Kodak, Rochester, NY).

Results are shown in Figure 7. When t-BRK-3 is expressed alone in COS-1 cells, no crosslinked band is seen. Expression of BRK-1 alone results in a crosslinked band at a molecular weight of 78 kD, corresponding to the predicted molecular weight of BRK-1 plus the monomer molecular weight of BMP-4 Co-expression of t-BRK-3 and BRK-1 results in the appearance of a band of similar size to that for BRK-1, as well as a new crosslinked band at 94 kD, corresponding to the predicted molecular weight of t-BRK-3 plus the monomer molecular weight of crosslinked BMP -4. Similarly, expression of BRK-2 alone yields a single crosslinked band at 75 kD, corresponding to the predicted molecular weight of BRK-2 plus the crosslinked BMP-4 monomer. Co- expression of t-BRK- 3 with BRK-2 yields a crosslinked band corresponding to that seen for BRK-2 alone, as well as a new crosslinked band at 94 kD, again corresponding to the predicted molecular weight of t-BRK-3 plus the monomer molecular weight of crosslinked BMP-4. Thus, crosslinking of [ ^l25 I]-BMP-4 to t-BRK-3 is observed only in the presence of a co-expressed type I BMP receptor.

Example 8 Demonstration of Complex Formation with Type I BMP Receptors Receptors of the TGF-B receptor family have been shown to form complexes involving a type I and a type II receptor (L. Attisano, J.L. Wrana, F. Lopez-Casillas, and J. Massague, J. Biochim Biophys. Acta, 1222: 71-80 (1994)). In order to demonstrate that the type LI BMP receptor t-BRK-3 can form a complex with the type I BMP receptors BRK-1 and BRK-2, COS-1 cells are co-transfected with the cDNA for t- BRK-3 and BRK-1, or t-BRK-3 and BRK-2, as described in Example 5. The receptors are crosslinked to [^ ^I]-BMP-4, then subjected to immunoprecipitation with antibodies specific for the type I receptors BRK-1 and BRK-2. If antibodies specific for a type I receptor precipitate not only the type I receptor crosslinked to [^ ² ^I]-BMP-4, but also BRK-3 crosslinked to [^ ² ^I]-BMP-4, this indicates that the two receptors must be forming a complex, as expected for type I and type II receptors having the same ligand- binding specificity.

Antibodies specific for the type I receptors BRK-1 and BRK-2 are generated using as antigen the peptide LNTRVGTKRYMAPEVLDESLNKNC (B.B. Koenig, et al., Molec. Cell. Biol., 14: 5961-5974 (1994)). This peptide is based on the amino acid sequence of BRK-1 in the intracellular kinase domain, amino acids 398-420 in SEQ LD NO: 12, with the addition of a cysteine at the C terminus to permit conjugation of the peptide. Comparison of the amino acid sequence of the kinase domain of BRK- 1 with the kinase domain of the Raf protein suggests that this region of BRK- 1 corresponds to a region of the Raf kinase which was used to make highly specific antibodies (W. Kolch,

E. Weissinger, H. Mischak, J. Troppmair, S.D. Showalter, P. Lloyd, G. Heidecker, and U.R. Rapp, Oncogene, 5. 713-720 (1990)). This peptide is conjugated by standard methods to keyhole limpet hemocynanin, and used to immunize three New Zealand White rabbits (Hazleton Washington, Vienna, VA). The resulting antisera are evaluated for their ability to recognize the original peptide coated on plastic, using an antibody capture ELISA. The antisera are designated 1378, 1379, and 1380. These antibodies are shown to immunoprecipitate BRK-1 from COS-7 cells transfected with the cDNA for BRK-1, using the procedure detailed in this example (B.B. Koenig, et al., Mol. Cell. Biol., 14: 5961-5974 (1994)). Because the sequence of BRK-2 is nearly identical to that of BRK- 1 in this region, these antibodies are subsequently tested for their ability to immunoprecipitate BRK-2 as well, and are found to be effective for this purpose. Antibody 1379 gives superior results for immunoprecipitation of BRK- 1, and antibody 1380 is preferred for immunoprecipitation of BRK-2.

In the immunoprecipitation procedure, COS-7 or COS-1 cells are transfected with the cDNA for t-BRK-3 and/or BRK-1, BRK-2, or DAF-4 as described in Example 5, and plated into 100 mm dishes. They are then crosslinked to [ ¹²⁵ I]-BMP-4 as described in example 7, except that the incubation with [^ ² ^I]-BMP-4 and unlabeled ligand is carried out in a total of 4 ml, instead of 500 μl, and all other volumes are increased accordingly. Following the crosslinking, cells are washed three times with ice- cold PBS, then lysed with 1 ml of RIP buffer (20 mM TrisCl, pH 8.0, 100 mM NaCI, 1 mM Na2EDTA, 0.5% Nonidet P-40, 0.5% sodium deoxycholate, 10 mM sodium iodide, and 1% bovine serum albumin) for 10 min. The lysate is centrifuged in a microcentrifuge at 13,000 rpm for 10 min at 4°C. The supernatant is transferred to a fresh tube and made 0 1% in SDS. To remove any existing antibody present in the lysate, 50 μl of PANSORBIN (Calbiochem, La Jolla, CA, a 10% solution of Staphylococcus aureus) is added. After a 30 minute incubation at 4°C, the lysate is centrifuged as before, and the supernatant again transferred to a fresh tube.

The primary antibody — 1379 when cells are transfected with t-BRK-3 and BRK- 1; 1380 when cells are transfected with t-BRK-3 and BRK-2 — is then added to the tube at a final dil on of 1: 100, and incubated for 2 hr on ice or overnight at 4°C To precipitate the complex of antigen: primary antibody, 25-50 μl of PANSORBIN is then added and incubated 30 min on ice. The complex is pelleted at 13,000 rpm for 10 min in a microcentrifuge and the supernatant discarded. The pellet is washed twice in RLP buffer containing 0.1% SDS, and once in TNEN buffer (20 mM Tris, pH 8.0, 100 mM NaCI, 1 mM EDTA, 0.5% NP-40). The pellet is resuspended in 25 μl of IX sample loading buffer. (Alternatively, the pellet may be washed twice with TNEN buffer, with similar results.) The sample is boiled for 5 min, centrifuged for 5 min, and subjected to gel electrophoresis after loading of the samples onto a 7.5% SDS-polyacrylamide gel.

Results of this experiment are shown in Figure 8, which shows the results of immunoprecipitations on COS-1 cells transfected with t-BRK-3 in the presence or absence of BRK- 1 or BRK-2. Cells transfected with t-BRK-3 alone, crosslinked to [ ¹² 5l]-BMP-4, and immunoprecipitated with antibody 1380 show no radiolabel in the immunoprecipitate, as expected since t-BRK-3 does not crossreact with this antibody. Cells transfected with BRK-1, crosslinked, and immunoprecipitated with antibody 1379 show a single labeled band at 78 kD, consistent with the predicted molecular weight of BRK-1 plus the cross-linked monomer of BMP -4 Immunoprecipitation of cells co- transfected with BRK-1 and t-BRK-3 yields the same band seen with BRK-1 alone, plus an additional labeled band at 94 kD, consistent with the predicted molecular weight of t- BRK-3 plus the crosslinked BMP-4 monomer. (A less intense band at 120 kD is also observed.) The fact that antibodies to BRK-1 precipitate not only BRK-1, but t-BRK-3 as well in these cells indicates complex formation between BRK-1 and t-BRK-3. Similarly, cells transfected with BRK-2, crosslinked to [^ ² 5l]-BMP-4, and subjected to immunoprecipitation with antibody 1380 show a labeled band at 75 kD, consistent with the predicted molecular weight of BRK-2 plus the crosslinked monomer of BMP-4 Immunoprecipitation of cells co-transfected with BRK-2 and t-BRK-3 yields the same band seen with BRK-2 alone, plus a strongly labeled band at 94 kD, consistent with the predicted molecular weight of t-BRK-3 plus the crosslinked monomer of BMP-4. As expected, this band co-migrates with the larger labeled band in cells co-transfected with BRK-1 and t-BRK-3. (A less intense band at 120 kD is also observed.) Again, the fact that an antibody to BRK-2 precipitates not only BRK-2 but t-BRK-3 as well in these cells strongly indicates that BRK-2 and t-BRK-3 form a complex. Thus, t-BRK-3 forms a complex with two different type I BMP receptors, as expected for a type II BMP receptor.

A second immunoprecipitation experiment is carried out to test the ligand specificity of the t-BRK-3 receptor complex for BMP-2, BMP-4, and TGF-β _j . A derivative of BMP-2 designated "digit -removed" BMP-2 (DR-BMP-2) is also tested; DR-BMP-2 is prepared by mild trypsin digestion of BMP-2 to remove the amino terminus, and shows significantly reduced nonspecific binding to whole cells (B.B. Koenig, et al., Molec. Cell. Biol., 14: 5961-5974 (1994)).

COS-1 cells are co-transfected with the cDNA for BRK-2 and t-BRK-3 as described in Example 5, crosslinked to [ ¹²⁵ I]-BMP-4, and subjected to immunoprecipitation with antibody 1380 as described in this example, except that an excess of unlabeled ligand (10 nM BMP-4, 10 nM BMP-2, 10 nM DR-BMP-2, or 50 nM TGF-β _j ) is added to the incubation at the same time as the [ ¹²⁵ I]-BMP-4. The results are shown in Figure 9. When no competing unlabeled ligands are present, two labeled bands are observed, at 75 kD and 94 kD, consistent with crosslinked BRK-2 and

BRK-3 respectively, as seen in Figure 8. In the presence of excess unlabeled BMP-4, BMP-2, or DR-BMP-2, however, these bands are completely abolished, demonstrating that these ligands compete effectively with [ ^ ² 5l]-BMP-4 to bind to the complex, and that all these ligands show specific binding to the BRK-2 and BRK-3 receptor complex. However, the presence of 50 nM TGF- ^β \ has no effect on the labeled bands, indicating that TGF-βj does not bind to the same site as [^ ² ^I]-BMP-4. This shows that the BRK-2Λ-BRK-3 complex binds specifically to BMP-2 and BMP-4 and does not bind TGF-β.

Example 9 Isolation of Mouse BRK-3

In order to isolate the full-length mouse homologue of BRK-3, a cDNA library is constructed from NLH3T3 mouse embryonic fibroblasts (ATCC CRL 1658). Total RNA (1.26 mg) is isolated from the cells using a Total RNA Separator Kit (Clontech, Palo Alto, CA). Messenger RNA (81 μg) is isolated from this total RNA (1 mg) using the mRNA Separator Kit (Clontech, Palo Alto, C A). An aliquot of the mRNA (4 μg) is used to make cDNA library using the SUPER SCRIPT Plasmid System for cDNA Synthesis and Plasmid Cloning (Life Technologies, Gaithersburg, MD) according to the manufacturer's instructions. The resulting library contained approximately 4.9 x 10 ⁵ primary colonies, and is divided into 98 pools, each containing 5000 colonies. The initial screen of the library is accomplished by Southern blotting. Plasmids are purified from each of the 98 pools, using QIAGEN columns (Qiagen, Chatsworth, CA). DNA from each pool (approximately 5 μg) is digested with Mlu I to release the cDNA insert, then run on a 1% agarose gel. The gel is denatured for 30 min in 0.6 M NaCI, 0.4 N NaOH, then neutralized 30 min in 1.5 M NaCI, 0.5 M Tris, pH 7.5. The DNA is then transferred overnight to a HYBOND Nylon membrane (Amersham, Arlington Heights, LL) using 10XSSC as the transfer buffer (1XSSC = 0.15 M NaCI, 0.015 M sodium citrate, pH 7.0).

Human t-BRK-3 is cut with EcoRV and Afl III to give a 1.5 kb fragment. The fragment is randomly labeled with alpha[^ ² P]-dCTP having a specific activity of 3000 Ci mmol (NEN Research Products, Boston, MA), using a PRLME-IT II Random Primer Labeling Kit (Stratagene, La Jolla, CA; a kit for random primer labeling of DNA, including Klenow DNA polymerase, primers, and buffers). The labeled probe is allowed to hybridize to the Southern blot for 18 hr at 42°C in hybridization buffer (Sigma, St. Louis, MO) consisting of 50% deionized formamide, 5 X SSPE (lx SSPE = 0.14 M NaCI, 8 mM sodium phosphate, 0.08 mM EDTA, pH 7.7), IX Denhardt's solutions, and 100 μg ml of denatured salmon testis DNA. The blot is then washed in 0.25X SSPE, 0.5% sodium dodecyl sulfate (SDS), two times at 42°C for 15 min each, then two times at 65 ^e C for 20 min each. The blot is then exposed to Kodak X-OMAT

AR autoradiography film for 18 hr at -80°C. Development of the film shows five positive pools, as judged by the presence of a labeled band of approximately 2.5 kb.

For secondary screening, plates are streaked with the E. coli stocks from the five positive pools (5000 colonies plate). A HYBOND nylon membrane is placed on top of the plate so that the bacterial colonies are transferred to the filter. The colonies are then allowed to recover at 37°C for 2-3 hr. The filter is soaked in 10% SDS for 3 min, then transferred to 1.5 M NaCI, 0.5 M NaOH for 5 min, neutralized in 1.5 M NaCI, 1.5 M Tris, pH 7.5 for 5 min, and washed in 2X SSC. To remove proteins, the blots are then shaken with 50 μg/ml of proteinase K (Boehringer Mannheim, Indianapolis, IN) in 0.1 M Tris, pH 7.6, 10 mM EDTA, 0.15 M NaCI, 0.02% SDS at 55°C for 1 hr. The human BRK-3 fragment (Eco RV-Afl III) is labeled and the blots hybridized, washed, and subjected to autoradiography exactly as described above for the primary screening.

Colonies which corresponded to labeled spots on the autoradiograph are streaked on plates for tertiary screening, which is performed exactly as described above for secondary screening. Four positive clones are isolated. One clone, pSPORTl/N89- 5, is found to have the largest insert size, 2.9 kb.

The inserts from the four positive clones are sequenced using the TAQ DYE DEOXY Terminator Cycle Sequencing Kit and an Applied Biosystems Model 373 A Automated DNA Sequencer. Comparison of the four sequences shows that three of the four are identical at the 3' end, and all four align with the coding region of human BRK- 3 at the 5' end. The longest clone, pSPORTl/N89-5, aligns with the human BRK-3 sequence approximately 600 pairs from the beginning of the coding region.

To generate more sequence information, the insert from pSPORTl/N89-5 is digested with EcoRI and Sea I, and the resulting 1.4 kb fragment is subcloned into BLUESCRIPT II SK(-) at the Eco RI and Hinc II sites. pSPORTl/N89-5 is also digested with Eco RI and Eco RV and the resulting 2.1 kb insert subcloned into the same vector at the same sites. Finally, the plasmid is digested with Sea I and Not I, and subcloned into the same vector at the Hinc II and Not I sites. Sequencing of these three constructs yields the complete sequence of the insert from pSPORTl/N89-5. The missing 600 base pairs at the 5' end of the coding region is cloned using the

5' RACE System for Rapid Amplification of cDNA Ends (Life Technologies, Gaithersburg, MD). An antisense primer is designed corresponding to the known sequence of pSPORTl/N89-5, having the sequence 5'CTG TGT GAA GAT AAG CCA GTC 3' (the reverse complement of nucleotides 968-948 in SEQ LD NO:7). After first strand synthesis of cDNA from 1 μg of NLH3T3 mRNA, a poly C tail is added to the newly synthesized cDNA using terminal deoxynucleotidyi transferase, according to the manufacturer's instructions. The primer above is used to amplify the 5' end of the BRK- 3 cDNA, together with the Anchor Primer supplied with the kit, having the sequence 5'

(CUA)4 GGC CAC GCG TCG ACT AGT ACG GGI IGG GII GGG IIG 3' (where I = inosine and U=uracil). PCR was performed using the GENE-AMP PCR Kit with AMPLITAQ DNA Polymerase. An initial melting period at 95°C for 5 min was followed by 35 cycles of the following program: melting at 95°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 2 min. After the last cycle, the reaction was held at 72°C for 5 min to complete extension. To reduce background from nonspecific primer binding, a second round of PCR is performed using the nested primer 5' CAA GAG CTT ACC CAA TCA CTT G 3', again derived from the known sequence of the insert from pSPORTl/N89-5 (the reverse complement of nucleotides 921-900 in SEQ LD NO: 7), together with same 5' anchor primer used in the first round of PCR.

The amplified products of the second PCR reaction in the size range of 600-1000 bp are digested with Eel XI and Sal I and subcloned into BLUESCRIPT II SK(-) at the Eel XI and Sal I sites. The inserts are then sequenced, yielding an additional 600 bp of sequence which align with the coding region of human t-BRK-3. Three separate clones, designated R6-8B2, R6-11-1, and R6-11-2, are sequenced with identical results.

In order to assemble a full length clone of mouse BRK-3, a Sal I site is first placed at the 5' end of clone R 6-11-1 as follows. A primer is synthesized which contains a Sal I site followed by nucleotides 1-20 of the sequence of R6-11-1; the sequence of the primer is 5 ^* CAC ACG CGT CGA CCA TGA CTT CCT CGC TGC ATC G 3'. This is used together with the M13 reverse primer, 5' AAC AGC TAT G "AT G 3', in order to amplify a DNA fragment using plasmid DNA from clone R6- -1 as the template. PCR was performed using the GENE- AMP PCR Kit with AMPLITAQ DNA Polymerase. An initial melting period at 95°C for 5 min was followed by 35 cycles of the following program: melting at 95°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 2 min. After the last cycle, the reaction was held at 72°C for 5 min to complete extension. The fragment amplified from R6-11-1, together with the insert from pSPORTl/N89-5 (230 ng), is then subcloned in to BLUESCRIPT LI SK(-) as follows. The amplified fragment from R6-11-1 is digested with Sal I : A Eel XI . The insert from pSPORTl N89-5 is digested with Eel XI and Pst I. The vector BLUESCRIPT II SK(-) is digested ,ith Sal I and Pst I The three fragments are combined in a three-way ligation using T4 DNA ligase (3 hr, 25°C) and used to transform electrocompetent E. coli, strain DH5-a, using a BIO-RAD Gene PULSER (BIO-RAD, Hercules, CA) according to the manufacturer's instructions. A positive colony is selected and is designated pBLUESCRLPT-mBRK3. Sequencing of the 5' portion of the insert that was amplified by PCR shows a sequence identical to that of clone R6-11-1, indicating that no mutations are introduced during the amplification. For mammalian expression, m-BRK-3 is subcloned into the mammalian

expression vector pJT6. This vector is a derivative of pJT3, described in example 4 above, in which the Not I site at the 5' end of the multiple cloning site has been deleted, and a spacer inserted between the Pst I and BamHI restriction sites in the multiple cloning site. To accomplish the subcloning, m-BRK-3 is excised from pBLUESCRJPT- mBRK3 using Not I and Sal I, then subcloned into pJT6 at the Not I and Sal I sites to generate pJT6-mBRK3.

However, resequencing of the 3' end of pJT6-mBRK3 and the original cDNA in pSPORTl/N89-5 results in an altered reading frame at the 3' end, and shows that the stop codon is actually located 3' to the Pst I site. Thus, pJT6-mBRK3 does not contain a stop codon. Accordingly, two new constructs are prepared as follows.

First, pJT6-mBRK3 is digested with Spel (site at position 2306 in SEQ ID NO: 7) and Not I (in the multiple cloning site of pJT6), removing the 3 ' end of the insert. The longest clone isolated during the screening of the NLH-3T3 library, pSPORTl/N89- 5, is also digested with Spe I and Not I. The 1.2 kb fragment liberated from pSPORTl/N89-5 is subcloned into the Spe I/Not I digested pJT6-mBRK3, regenerating both sites. This construct is designated pJT6-mBRK-3L, and contains the entire 3' end of the pSPORTl N89-5 clone. A map of the construct is shown in Figure 10.

The 3' end of the clone contains 403 nucleotides in the untranslated region 3' to the stop codon. This region is very A-T rich, which might possibly lead to decreased expression levels. To remove this region, a second construct is prepared. The pSPORTl/N89-5 plasmid is digested with Hind III (site at nucleotide 3168 in SEQ LD NO: 7, 21 bases 3' to the stop codon). The linearized plasmid is treated with Klenow fragment of DNA polymerase (Boehringer Mannheim, Indianapolis, IN) to fill in overhangs, then cut with Spe I to liberate an 863 bp fragment at the 3' end of the insert. At the same time, pJT6-mBRK3 is digested with Not I. The linearized plasmid is treated with Klenow fragment, then cut with Spe I, releasing the 3' end of the insert. The Not I/Spe I digested pJT6-mBRK3 is then ligated to the fragment liberated from pSPORTl/N89-5 by Hind III/Spe I. This regenerates the Spe I site; the Hind III and Not I sites are destroyed. The resulting construct is designated pJT6-mBRK3S, and is shown in Figure 11.

The construct pJT6-mBRK-3S is also constructed directly from the partial cDNA clone of m-BRK-3, pSPORTl/N89-5, and the construct containing the 5' end of the cDNA, clone R6-11-1. This is accomplished by digestion of clone R6-11-1 with Sal I and Eel XI, digestion of pSPORTl/N89-5 with Eel XI and Hind III, and digestion of BLUESCRIPT JJ SK (-) with Sal I and Hind III. These fragments are then subjected to a three-way ligation to generate the full length m-BRK-3 cDNA in the BLUESCRIPT II vector. The full length cDNA is then excised from this construct using Sal I and Not I, then subcloned into the Sal I and Not I sites of the pJT6 vector. The resulting plasmid

has exactly the same cDNA for BRK-3 as does pJT6-mBRK3S described in the above example. However, it carries additional vector sequence at the 3' end of the cDNA, comprising the region between the Hind III and Not I sites in the multiple cloning site of BLUESCRIPT II SK(-).

Example 10 Sequence analysis of mouse BRK-3 The DNA sequence of the full length mouse BRK-3 insert from pJT6-mBRK3L is shown in SEQ LD NO: 7, and the deduced protein sequence is shown in SEQ LD NO: 8. The deduced amino acid sequence of mouse BRK-3 is searched against all translated protein sequences in GenBank release 84.0, dated Aug. 15, 1994, using a standard Needleman-Wunsch algorithm (S.B. Needleman and CD. Wunsch, J. Mol. Biol, 48: 443-453 (1970)). It is found to be a unique sequence. It encodes a protein of 1038 amino acids. Comparing mouse BRK-3 with the truncated human receptor over the region encoded by t-BRK-3 (amino acids 1-582 in SEQ LD N0:4; amino acids 1-582 in SEQ LD NO: 8), the two receptors are 98% identical in sequence. Like t-BRK-3, m- BRK-3 contains a predicted transmembrane region encompassing amino acids 151-172. As with t-BRK-3, the intracellular domain contains all of the consensus sequences that characterize a protein kinase domain with predicted specificity for serine/threonine residues (S.K. Hanks, A.M. Quinn, and T. Hunter, Science, 241 : 42-52 (1988)). The kinase domain is followed by an extremely long carboxy terminus (534 amino acids). Indeed, due to the presence of this carboxy terminus, the intracellular domain in BRK-3 (866 amino acids) is much larger than that of any other receptor in the TGF-β receptor family. It is nearly twice as long as the intracellular domain of DAF-4 (490 amino acids), which has the longest intracellular domain known in the TGF-β family until the present invention.

Example 11

Demonstration of rI ²⁵ I1-BMP-4 binding to m-BRK-3 In order to demonstrate that [ ¹²⁵ I]-BMP-4 binds specifically to m-BRK-3,

COS-1 cells are transfected as described in Example 5 using the constructs pJT6- mBRK-3S and pJT6-mBRK-3L. In addition, the cells are also co-transfected with cDNA for the type I receptor BRK-2, using the construct pJT3-BRK-2, to determine whether the presence of a type I BMP receptor affects binding of [ ^I2 5l]-BMP-4. Whole cell binding with [ ¹²⁵ I]-BMP-4 is carried out as described in Example 7.

The results are shown in Figure 12, which shows specific binding of [ ^I2 ^I]- BMP-4 normalized to cell number. When cells are transfected with mouse BRK-3 alone, using either of the two constructs tested, specific binding of [ ¹² ^I]-BMP-4 is increased

to 4-7 times the level seen with mock transfected cells. Transfection of BRK-2 alone shows increased binding at a similar level to that seen with mouse BRK-3 alone. When cells are co-transfected with BRK-2 as well as mouse BRK-3, the binding is further increased to 9-1 1 times that of mock-transfected cells, consistent with the results obtained with BRK-2 in combination with t-BRK-3 (Figure 6 in Example 7 above).

As an additional demonstration that m-BRK-3 binds to[l 5l]-BMP-4, a crosslinking experiment is carried out. COS-1 cells are transfected with the cDNA for m-BRK-3, using the construct pJT6-mBRK-3 S, and/or with cDNAs for BRK-1 (using pJT4-J159F) or BRK-2 (using pJT3-BRK-2) as described in Example 5. The transfected cells are incubated with [ ¹²⁵ I]-BMP-4 and crosslinked as described in Example 7, except that disuccinimidyl suberate (DSS) is used as the crosslinking agent rather than disuccinimidyl glutarate. The results of such an experiment are shown in Figure 13. Cells transfected with m-BRK-3 alone show no crosslinked band, consistent with the results obtained with t-BRK-3 (Figure 7). Cells transfected with the cDNA for BRK-1 alone show a single species migrating at an apparent molecular weight of 81 kD, consistent with the predicted molecular weight of BRK- 1 plus the crosslinked BMP-4 monomer. Cells transfected with the cDNAs for BRK-1 and m-BRK-3 show three labeled bands, one of which is consistent with the band seen with BRK-1 alone (81 kD). The other bands migrate with an apparent molecular weight of 159 kD and 128 kD. The larger of these is consistent with the predicted molecular weight of m-BRK-3 plus the crosslinked BMP-4 monomer. Note that the intensity of the crosslinked band identified with BRK-1 is considerably increased, compared to that seen with BRK-1 alone.

Similarly, transfection of cells with the cDNA for BRK-2 alone yields a crosslinked band migrating at an apparent molecular weight of 78 kD, consistent with the predicted molecular weight of BRK-2 plus the crosslinked BMP -4 monomer. In cells transfected with the cDNAs for BRK-2 and mBRK3, the 78 kD species identified with BRK-2 is observed, as well as crosslinked bands at 159 kD and 128 kD, comigrating with the higher molecular weight bands seen in cells transfected with the cDNAs for BRK-1 and m-BRK-3. As with BRK-1, the intensity of crosslinking to the band identified with BRK-2 is considerably increased compared to that seen with BRK- 2 alone. Finally, no labeled bands are observed in cells transfected with vector alone.

An immunoprecipitation experiment is carried out to demonstrate the ability of m-BRK-3 to form a complex with type I BMP receptors. COS-1 ceils are transfected with the cDNA for m-BRK-3, using the construct pJT6-mBRK-3S, and/or with cDNAs for BRK-1 (using pJT4-J159F) or BRK-2 (using pJT3-BRK-2) as described in Example 5. The transfected cells are incubated with [ ¹²⁵ I]-BMP-4, crosslinked , and subjected to immunoprecipitation with antibodies to the appropriate type I receptor or preimmune

serum as described in example 8, except that DSS is used as the crosslinking agent rather than disuccinimidyl glutarate. The results of this experiment are shown in figure

14. In cells transfected with cDNA for BRK-1 alone, a single band is precipitated by antibodies to BRK-1, migrating at an apparent molecular weight of 81 kD. In cells transfected with cDNAs for BRK-1 and m-BRK-3, antibodies to BRK-1 precipitate the

81 kD band, which is now increased in intensity. In addition, however, a band migrating at an apparent molecular weight of 159 kD is observed, consistent with the predicted molecular weight of m-BRK-3 plus crosslinked BMP -4 monomer. Similarly, in cells transfected with cDNA for BRK-2 alone, antibodies to BRK-2 precipitate a labeled species migrating at an apparent molecular weight of 78 kD. In cells transfected with cDNAs for BRK-2 and m-BRK-3 and precipitated with antibodies to BRK-2, the 78 kD band identified with BRK-2 is again observed, at increased intensity. In addition, a labeled species is seen at 159 kD, consistent with m-BRK-3 and comigrating with _* the higher molecular weight band seen in cells transfected with cDNAs for BRK-1 and m- BRK-3. In cells transfected with cDNAs for BRK-2 and m-BRK-3, an additional labeled band is observed at 94 kD. As a control, cells are transfected with the cDNAs for BRK-1 and m-BRK-3, or BRK-2 and m-BRK-3, then subjected to immunoprecipitation with preimmune sera (lanes far left and far right); no labeled bands are observed. This experiment shows that when m-BRK-3 is co-expressed with the type I

BMP receptors BRK-1 or BRK-2, antibodies which precipitate the type I receptor also precipitate m-BRK-3. Thus, m-BRK-3 can form a complex with either of these mammalian type I BMP receptors, as expected for a mammalian type II BMP receptor. This is consistent with results obtained with t-BRK-3 described in Example 8 above.

Example 12 Isolation of full length human BRK-3 cDNA Since clone HSK723, described in Example 2, does not contain an in-frame stop codon, it is desired to obtain additional sequence 3' to the end of this cDNA. Accordingly, the human foreskin fibroblast library prepared in Example 1 is rescreened with the HSK7-2 PCR fragment, using labeling and screening conditions exactly as described in Example 2. This results in isolation of a longer clone, designated pHSK1030, which contains additional human BRK-3 sequence (total of 3355 base pairs) subcloned in BLUESCRIPT SK(-). Sequencing of the insert from pHSK1030 discloses a coding region of 982 amino acids, but the insert still does not contain an in- frame stop codon.

The remainder of the coding region is cloned by PCR as follows. Two forward primers are derived from the plus strand of clone pHSK1030. The sequences of these

primers are as follows: primer RPK3-1, 5' CCTGTCACATAATAGGCGTGTGCC-3' (identical to nucleotides 1998-2021 in SEQ LD NO: l); primer RPK3-2, 5' CGCGGATCCATCATACTGACAGCATCG 3' (which incorporates a BamHI site followed by nucleotides 2078-2095 in SEQ LD NO: l). Two additional primers are derived from the minus strand of λgtIO. These primers are: GlOFl, 5' GCTGGGTAGTCCCCACCTTT 3' and G10F2, 5" GAGCAAGTTCAGCCTGGT 3".

The human fibroblast cDNA library prepared in Example 1 is used as the template for PCR. The library (0.3 μg) is incubated with the RPK3-1 and GlOFl primers (1 μM each), Tth polymerase ( 1.2 units), all four deoxynucleotides (200 μM each) , buffer for the Tth polymerase, and water in a total of 50 μl. Conditions for the PCR cycle are as follows: initial melting at 94°C for 2 min, followed by 20 cycles of melting, 94 ^β C for 1.5 min; annealing, 52 ^β C for 2 min; and extension, 72 ^β C for 3 min. After cycle 20, the sample is held at 72°C for an additional 8 min to insure complete extension. To increase specificity and reduce background, a second round of nested PCR is carried out. The incubation mixture is the same as described in this example for the first round, except that (1) an aliquot of the first PCR reaction (0.5 μl) is used as the template; and (2) RPK3-2 and G10F2 primers are used, instead of RPK3-1 and GlOFl. Conditions for the PCR run are identical to those described in this example for the first round of PCR.

The second round of PCR results in the amplification of a 1.6 kb fragment, which is isolated from an agarose gel by QIAEX. This fragment is digested with EcoRI and BamHI, and subcloned into BLUESCRIPT SK(-) at the EcoRI and Bam HI sites. The resulting construct, pHSK723-3U, is sequenced and found to encode the remaining coding region of BRK- 3 with an in-frame stop codon.

In order to assemble the full length human BRK-3, the inserts from pHSK1030 and pHSK723-3U are joined at a unique Stu I site (located at nucleotide 3219 in SEQ ID NO:l) in the vector BLUESCRIPT II SK(-). This yields the complete construct pHSK1040, which contains the complete coding sequence of human BRK-3. The pHSK1040 is shown in Figure 15. The DNA sequence of human BRK-3 is shown in SEQ LD NO: 1, and the deduced amino acid sequence for human BRK-3 is shown in SEQ LD NO: 2.

The amino acid sequence of human BRK-3 (SEQ LD NO:2) is compared to the amino acid sequence for m-BRK-3 (SEQ LD NO:8) and found to be 96.7% identical.

96/14412 PCΪ7US95/14085

37

Example 13 Use of the BRK-3 in a ligand binding assay for the identification of BMP receptor agonists and antagonists Identification of ligands that interact with BRK-3 can be achieved through the use of assays that are designed to measure the interaction of ligands with BRK-3. An example of a receptor binding assay that is adapted to handle large numbers of samples is carried out as follows.

COS-1 cells are transfected with the cDNA for m-BRK-3 using the construct pJT6-mBRK-3L as described in example 1 1 above, except that cells are grown in a 12 well culture dish. At 48-68 hr after transfection, the cells are washed once with 1.0 ml binding buffer (50 mM HEPES, pH 7.4, 128 mM NaCI, 5 mM KCL, 5 mM MgSO4, 1.2 mM CaCl2, 2 mg/mi BSA), then equilibrated in the same buffer at 4°C for 60 min. with gentle shaking. After equilibration, the buffer is aspirated, and to each well is added, 500 μl of 4°C binding buffer containing [l ² 5l]BMP-4 tracer (100-400 pM) in the presence or absence of varying concentrations of unlabeled test compounds (i.e., putative ligands), for a period of 4 hours at 4°C with gentle shaking. For determination of nonspecific binding and complete displacement from the BMP receptor complex, BMP-2 is added at a final concentration of 10 nM. To prevent degradation of ligand, a protease inhibitor cocktail is also added, to give a final concentration of 10 μg/ml leupeptin, 10 μg/ml antipain, 50 μg/ml aprotinin, 100 μg/ml benzamidine, 100 μg/ml soybean trypsin inhibitor, 10 μg ml bestatin, 10 μg/ml pepstatin, and 300 μM phenylmethylsulfonyl fluoride (PMSF). At the end of the incubation period, the buffer is aspirated, and the cells are rinsed 4 times with 1 ml washing buffer (50 mM HEPES, pH 7.4, 128 mM NaCI, 5 mM KC1, 5 mM MgSO4, 1.2 mM CaCl2, 0.5 mg/ml BSA). After the final wash is aspirated, 200 μl of soiubilization buffer (10 mM Tris Cl, pH 7.4, 1 mM EDTA, 1% (v/v) Triton X-100) is added to each well and incubated at room temperature for 15-30 min. The solubilized cells are then transferred to fresh tubes and counted in a Packard Model 5005 COBRA Gamma Counter (Packard Instruments, Meriden, CT).

Test compounds which interact with the m-BRK-3 receptor are observed to compete with binding to the receptor with the [125i]BMP-4 tracer in the cells expressing m-BRK-3, such that less [l ² 5l]BMP-4 tracer is bound in the presence of the test compound in comparison to the binding observed when the tracer is incubated in the absence of the novel compound. A decrease in binding of the [l ² 5l]BMP-4 tracer by > 30% at the highest concentration of the test compound that is studied demonstrates that the test compound binds to m-BRK-3.

Similar results are obtained when other, related BRK-3 protein receptor kinases of the present invention are used according to the method of this example.

Example 14 Use of m-BRK-3 and BRK-2 in a ligand binding assay for the identification of BMP receptor agonists and antagonists

Identification of ligands that interact with BRK-3 complexed to a type I BMP receptor can be achieved through the use of assays that are designed to measure the interaction of the ligands with this BMP receptor complex. A receptor binding assay that uses the m-BRK-3/BRK-2 complex and is adapted to handle large numbers of samples is carried out as follows.

COS-1 cells are transfected with the cDNAs for m-BRK-3, using the construct pJT6-mBRK-3L, and BRK-2, using the construct pJT3-BRK-2, as described in example 1 1 above, except that the cells are grown in a 12 well culture dish. The DNA mixture used to transfect the cells contains 2 μg ml of pJT3-BRK-2 and 4 μg/mi of pJT6- mBRK-3L. At 48-68 hours after transfection, the cells are washed once with .l ml binding buffer (50 mM HEPES, pH 7.4, 128 mM NaCI, 5 mM KCL, 5 mM MgSO4, 1.2 mM CaCl2, 2 mg/ml BSA), then equilibrated in the same buffer at 4°C for 60 min with gentle shaking. After equilibration, the buffer is aspirated, and to each well is added 500 μl of 4°C binding buffer containing [l 5l]BMP-4 tracer (100-400 pM) in the presence or absence of varying concentrations of test compounds (i.e., putative ligands), for a period of 4 hours at 4°C with gentle shaking. For determination of nonspecific binding and complete displacement from the BMP receptor complex, BMP-2 is added at a final concentration of 10 nM. To prevent degradation of ligand, a protease inhibitor cocktail is also added, to give a final concentration of 10 μg/ml leupeptin, 10 μg ml antipain, 50 μg/ml aprotinin, 100 μg/ml benzamidine, 100 μg/ml soybean trypsin inhibitor, 10 μg/ml bestatin, 10 μg/ml pepstatin, and 300 μM phenylmethylsutfonyl fluoride (PMSF). At the end of the incubation period, the buffer is aspirated, and the cells are rinsed 4 times with 1 ml washing buffer (50 mM HEPES, pH 7.4, 128 mM NaCI, 5 mM KC1, 5 mM MgSO4, 1.2 mM CaCl2, 0.5 mg/ml BSA). After the final wash is aspirated, 200 μl of soiubilization buffer ( 10 mM Tris Cl, pH 7.4, 1 mM EDTA, 1% (v/v) Triton X-100) is added to each well and incubated at room temperature for 15-30 min. The solubilized cells are then transferred to fresh tubes and counted in a Packard Model 5005 COBRA Gamma Counter (Packard Instruments, Meriden, CT).

Test compounds which interact with the m-BRK-3/BRK-2 receptor complex are observed to compete for binding to the receptor complex with the [l ² 5l]BMP-4 tracer, such that less [ _* 5l]BMP-4 tracer is bound in the presence of the test compound in comparison to the binding observed when the tracer is incubated in the absence of the novel compound. A decrease in binding of the [l 5l]BMP-4 tracer by > 30% at the highest concentration of the test compound that is studied demonstrates that the test compound binds to the m-BRK-3/BRK-2 receptor complex.

Similar results are obtained when the other BRK-3 protein receptor kinases of the present invention, or homologues thereof, are used in combination with BRK-2 or other BMP type I receptors.

Deposit of BRK-3. t-BRK-3 and m-BRK-3

£. coli transformed with pJT4-J159F (SEQ LD NO: 1 1 subcloned into expression vector pJT4) was deposited with the ATCC on October 7, 1993, and assigned ATCC Designation No. 69457.

E. coli transformed with pJT4-hBRK3T (SEQ LD NO: 3 subcloned into expression vector pJT4) was deposited with the ATCC on August 16, 1994 and assigned ATCC designation No. 69676.

E coli transformed with pJT6-mBRK-3S (SEQ LD NO: 7 subcloned into expression vector pJT6) was deposited with the ATCC on September 28, 1994 and assigned ATCC designation No. 69694. E. coli transformed with pJT6-mBRK-3L (SEQ LD NO:7 subcloned into expression vector pJT6) was deposited with the ATCC on September 28, 1994 and assigned ATCC designation No. 69695.

E. coli transformed with pHSK1040 (SEQ LD NO 1 subcloned into BLUESCRIPT JJ SK(-) was deposited with the ATCC on October 12, 1994, and assigned ATCC designation No. 69703.

As is recognized in the art, there are occasionally errors in DNA and amino acid sequencing methods. As a result, the sequences encoded in the deposited material are incorporated herein by reference and controlling in the event of an error in any of the sequences found in the written description of the present invention. It is further noted that one of ordinary skill in the art reproducing Applicants' work from the written disclosure can discover any sequencing errors using routine skill. The deposit of ATCC No. 69457, ATCC No. 69676, ATCC No. 69694, ATCC No. 69695 and ATCC No. 69703 is not to be considered as an admission that the deposited material is essential to the practice of the present invention. All publications mentioned hereinabove are hereby incorporated in their entirety by reference.

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to one skilled in the art and are to be included in the spirit and purview of this application and scope of the appended claims.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: ROSENBAUM, JAN S. NOHNO, TSUTONU

(ii) TITLE OF INVENTION: cONA ENCCOING A BMP TYPE II RECEPTOR (iii) NUMBER OF SEQUENCES: 14

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: THE PROCTER t GAMBLE COMPANY (B) STREET: 11810 EAST MIAMI RIVER ROAD

(C) CITY: ROSS

(D) STATE: OH

(E) COUNTRY: USA

(F) ZIP: 45061

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: loppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE: (C) CLASSIFICATION:

(viϋ) ATTORNEY/AGENT INFORMATION:

(A) NAME: ROOF, CARL J.

(B) REGISTRATION NUMBER: 37,708 (C) REFERENCE/DOCKET NUMBER: 5473

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 513-627-0081

(B) TELEFAX: 513-627-0260

(2) INFORMATION FOR SEQ ID NO:1:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 3601 but pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cONA

(ix) FEATURE:

(A) NAMEKEY: CDS (B) LOCATION: 409..3525

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: CGCCCCCCGA CCCCGGATCC AATCCCCCCC CTCCGCACCC TGGATATGTT TTCTCCCAGA 60

CCTGGATATT TTTTTGATAT CGTGAAACTA CGAGGGAAAT AATTTGGGGG ATTTCTTCTT 120

GGCTCCCTGC TTTCCCCACA GACATGCCTT CCGTTTGGAG GGCCGCGGCA CCCCGTCCGA 180

GGCGAAGGAA CCCCCCCAGC CGCGAGGGAG AGAAATGAAG GGAATTTCTG CAGCGGCATG 240

AAAGCTCTGC AGCTAGGTCC TCTCATCACC CATTTGTCCT TTCAAACTGT ATTGTGATAC 300 GGGCAGGATC AGTCCACGGβ AGACAAGACC AGCCTCCCGG CTGTTTCTCC GCCGGTCTAC 360

TTCCCATATT TCTTTTCTTT CCCCTCCTGA TTCTTGCCTG GCCCAGCG ATG ACT TCC 417

Met Thr Ser

1

TCG CTC CAG CGG CCC TGG CGG GTG CCC TGG CTA CCA TGG ACC ATC CTG 465 Ser Leu Gin Arg Pro Trp Arg Val Pro Trp Leu Pro Trp Thr lie Leu 5 10 15

CTG GTC AGC ACT GCG GCT GCT TCG CAG AAT CAA GAA CGG CTA TGT GCG 513 Leu Val Ser Thr Ala Ala Ala Ser Gin Asn Gin Glu Arg Leu Cys Ala 20 25 30 35

TTT AAA GAT CCG TAT CAG CAA GAC CTT GGG ATA GGT GAG AGT AGA ATC S61 Phe Lys Asp Pro Tyr Gin Gin Asp Leu Gly He Gly Glu Ser Arg He 40 45 50

TCT CAT GAA AAT GGG ACA ATA TTA TGC TCG AAA GGT AGC ACC TGC TAT 609 Ser His Glu Asn Gly Thr He Leu Cys Ser Lys Gly Ser Thr Cys Tyr 55 60 65

GGC CTT TGG GAG AAA TCA AAA GGG GAC ATA AAT CTT GTA AAA CAA GGA 657 Gly Leu Trp Glu Lys Ser Lys Gly Asp He Asn Leu Val Lys Gin Gly 70 75 80

TGT TGG TCT CAC ATT GGA GAT CCC CAA GAG TGT CAC TAT GAA GAA TGT 705 Cys Trp Ser His He Gly Asp Pro Gin Glu Cys His Tyr Glu Glu Cys 85 90 95 GTA CTA ACT ACC ACT CCT CCC TCA ATT CAG AAT GGA ACA TAC CGT TTC 753 Val Val Thr Thr Thr Pro Pro Ser He Gin Asn Gly Thr Tyr Arg Phe 100 105 110 115

TGC TCT TGT AGC ACA GAT TTA TGT AAT GTC AAC TTT ACT GAC AAT TTT 801 Cys Cya Cys Ser Thr Asp Leu Cys Asn Val Asn Phe Thr Glu Asn Phe 120 125 130

CCA CCT CCT GAC ACA ACA CCA CTC AGT CCA CCT CAT TCA TTT AAC CGA 849 Pro Pro Pro Asp Thr Thr Pro Leu Ser Pro Pro His Ser Phe Asn Arg 135 140 145

GAT GAG ACA ATA ATC ATT GCT TTG CCA TCA GTC TCT GTA TTA GCT GTT 897 Asp Clu Thr He He He Ala Leu Ala Ser Val Ser Val Leu Ala Val 150 155 160

TTG ATA GTT GCC TTA TGC TTT GGA TAC AGA ATG TTG ACA GGA GAC CGT 945 Leu He Val Ala Leu Cys Phe Gly Tyr Arg Net Leu Thr Gly Asp Arg 165 170 175 AAA CAA GGT CTT CAC ACT ATG AAC ATG ATG GAG CCA CCA GCA TCC GAA 993 Lys Gin Gly Leu His Ser Met Asn Net Met Glu Ala Ala Ala Ser Glu 180 185 190 195

CCC TCT CTT CAT CTA GAT AAT CTG AAA CTG TTC CAG CTG ATT GGC CGA 1041 Pro Ser Leu Asp Leu Aap Asn Leu Lys Leu Leu Glu Leu Ile Gly Arg 200 205 210

CCT CGA TAT GCA CCA CTA TAT AAA CGC TCC TTG GAT GAC CGT CCA GTT 1089 Gly Arg Tyr Gly Ala Val Tyr Lys Gly Ser Leu Asp Glu Arg Pro Val 215 220 225

GCT CTA AAA GTG TTT TCC TTT GCA AAC CGT CAG AAT TTT ATC AAC GAA 1137 Ala Val Lys Val Phe Ser Phe Ala Asn Arg Gin Asn Phe Ile Asn Glu 230 235 240

AAC AAC ATT TAC AGA GTG CCT TTG ATG GAA CAT CAC AAC ATT GCC CGC 1185 Lys Asn He Tyr Arg Val Pro Leu Met Glu Hia Asp Asn He Ala Arg 245 250 255 TTT ATA GTT GGA GAT GAC ACA GTC ACT GCA GAT GGA CGC ATG CAA TAT 1233 Phe He Val Cly Asp Glu Arg Val Thr Ala Asp Gly Arg Met Glu Tyr 260 265 270 275

TTG CTT GTG ATG GAG TAC TAT CCC AAT GGA TCT TTA TGC AAG TAT TTA 1281 Leu Leu Val Net Glu Tyr Tyr Pro Asn Cly Ser Leu Cys Lys Tyr Leu 280 285 290

AGT CTC CAC ACA AGT GAC TCG GTA AGC TCT TGC CGT CTT GCT CAT TCT 1329 Ser Leu His Thr Ser Asp Trp Val Ser Ser Cys Arg Leu Ala His Ser 295 300 305

GTT ACT AGA GGA CTG GCT TAT CTT CAC ACA GAA TTA CCA CGA GGA GAT 1377 Val Thr Arg Gly Leu Ala Tyr Leu His Thr Glu Leu Pro Arg Gly Asp 310 315 320

CAT TAT AAA CCT GCA ATT TCC CAT CGA GAT TTA AAC AGC AGA AAT GTC 1425

His Tyr Lys Pro Ala He Ser His Arg Asp Leu Asn Ser Arg Asn Val

325 330 335

CTA GTG AAA AAT GAT GGA ACC TGT GTT ATT AGT GAC TTT GGA CTG TCC 1473 Leu Val Lys Asn Asp Gly Thr Cys Val He Ser Asp Phe Gly Leu Ser 340 345 350 355

ATG ACG CTG ACT GGA AAT AGA CTG GTG CGC CCA GGG GAG GAA GAT AAT 1521 Met Arg Leu Thr Gly Asn Arg Leu Val Arg Pro Gly Glu Glu Asp Asn 360 365 370

GCA GCC ATA AGC GAG GTT GGC ACT ATC AGA TAT ATG GCA CCA GAA GTG 1569 Ala Ala He Ser Glu Val Gly Thr He Arg Tyr Met Ala Pro Glu Val

375 380 385

CTA GAA GCA GCT GTG AAC TTG AGC GAC TGT GAA TCA GCT TTG AAA CAA 1617 Leu Glu Gly Ala Val Asn Leu Arg Asp Cys Glu Ser Ala Leu Lys Gin 390 395 400

GTA CAC ATG TAT GCT CTT CGA CTA ATC TAT TGG GAC ATA TTT ATG AGA 1665 Val Asp Met Tyr Ala Leu Gly Leu He Tyr Trp Glu He Phe Met Arg 405 410 415 TCT ACA GAC CTC TTC CCA GGC GAA TCC GTA CCA GAG TAC CAG ATG GCT 1713 Cya Thr Asp Leu Phe Pro Gly Glu Ser Vel Pro Glu Tyr Gin Met Ala 420 425 430 435

TTT CAG ACA GAG GTT GGA AAC CAT CCC ACT TTT GAG GAT ATG CAG GTT 1761 Phe Gin Thr Clu Val Gly Asn His Pro Thr Phe Glu Asp Met Gin Val 440 445 450

CTC GTC TCT AGG CAA AAA CAC AGA CCC AAG TTC CCA GAA GCC TGG AAA 1809 Leu Val Ser Arg Glu Lys Gin Arg Pro Lys Phe Pro Glu Ala Trp Lys 455 460 465

GAA AAT AGC CTG GCA CTC ACG TCA CTC AAG GAG ACA ATC GAA GAC TGT 1857 Glu Asn Ser Leu Ala Val Arg Ser Leu Lys Glu Thr He Glu Asp Cys 470 475 480

TCG GAC CAG GAT GCA CAG GCT CGG CTT ACT GCA CAG TGT GCT GAG GAA 1905 Trp Asp Gin Aap Ala Glu Ala Arg Leu Thr Ala Gin Cys Ala Glu Glu 485 490 495 AGG ATG GCT GAA CTT ATC ATG ATT TGG GAA AGA AAC AAA TCT GTG AGC 1953 Art Net Ala Glu Lou Mot Mot He Trp Glu Arg Asn Lys Ser Val Ser 500 505 510 515

CCA ACA CTC AAT CCA ATC TCT ACT GCT ATG CAG AAT GAA CGC AAC CTG 2001 Pro Thr Val Asn Pro Not Ser Thr Ala Net Gin Asn Glu Arg Asn Leu 520 525 530

TCA CAT AAT AGC CCT GTG CCA AAA ATT GGT CCT TAT CCA GAT TAT TCT 2049 Ser His Asn Arg Arg Val Pro Lys He Gly Pro Tyr Pro Asp Tyr Ser 535 540 545

TCC TCC TCA TAC ATT CAA GAC TCT ATC CAT CAT ACT GAC AGC ATC GTG 2097 Sor Ser Ser Tyr He Glu Asp Ser He His His Thr Asp Ser He Val 550 555 560

AAG AAT ATT TCC TCT GAG CAT TCT ATG TCC AGC ACA CCT TTG ACT ATA 2145 Lys Asn He Sor Sor Glu His Sor Net Ser Ser Thr Pro Leu Thr He 565 570 575 GGC CAA AAA AAC CGA AAT TCA ATT AAC TAT GAA CGA CAC CAA GCA CAA 2193 Gly Glu Lys Asn Arg Asn Sor Ho Asn Tyr Glu Arg Gin Gin Ala Gin 580 585 590 595

CCT CGA ATC CCC AGC CCT GAA ACA AGT GTC ACC AGC CTC TCC ACC AAC 2241 Ala Arg He Pro Ser Pro Glu Thr Ser Val Thr Ser Leu Ser Thr Asn 600 605 610

ACA ACA ACC ACA AAC ACC ACA GGA CTC ACG CCA AGT ACT GGC ATG ACT 2289 Thr Thr Thr Thr Asn Thr Thr Gly Leu Thr Pro Ser Thr Gly Met Thr 615 620 625

ACT ATA TCT GAG ATG CCA TAC CCA GAT GAA ACA AAT CTG CAT ACC ACA 2337 Thr He Ser Glu Net Pro Tyr Pro Asp Glu Thr Asn Leu His Thr Thr 630 635 640

AAT GTT GCA CAG TCA ATT GGG CCA ACC CCT GTC TGC TTA CAG CTG ACA 2385 Asn Val Ala Gin Ser He Gly Pro Thr Pro Val Cys Leu Gin Leu Thr 645 650 655

GAA GAA GAC TTG GAA ACC AAC AAG CTA GAC CCA AAA GAA GTT GAT AAG 2433 Glu Glu Asp Leu Glu Thr Asn Lys Leu Asp Pro Lys Glu Val Asp Lys 660 665 670 675

AAC CTC AAG GAA AGC TCT GAT GAG AAT CTC ATG GAG CAC TCT CTT AAA 2481 Asn Leu Lys Glu Ser Ser Asp Glu Asn Leu Net Glu His Ser Leu Lys 680 685 690 CAG TTC AGT GGC CCA GAC CCA CTG AGC AGT ACT AGT TCT AGC TTG CTT 2529 Gin Phe Ser Gly Pro Asp Pro Leu Ser Ser Thr Ser Ser Ser Leu Leu 695 700 705

TAC CCA CTC ATA AAA CTT GCA GTA GAA CCA ACT GGA CAG CAG GAC TTC 2577 Tyr Pro Lou He Lys Leu Ala Val Glu Ala Thr Gly Gin Gin Asp Phe 710 715 720

ACA CAG ACT GCA AAT GGC CAA GCA TGT TTG ATT CCT GAT GTT CTG CCT 2625 Thr Gin Thr Ala Aan Gly Gin Ala Cys Leu Ho Pro Asp Val Leu Pro 725 730 735

ACT CAG ATC TAT CCT CTC CCC AAG CAG CAG AAC CTT CCC AAG AGA CCT 2673 Thr Gin He Tyr Pro Leu Pro Lys Gin Gin Asn Leu Pro Lys Arg Pro 740 745 750 755

ACT AGT TTG CCT TTG AAC ACC AAA AAT TCA ACA AAA GAG CCC CGG CTA 2721 Thr Ser Leu Pro Leu Asn Thr Lys Asn Ser Thr Lys Glu Pro Arg Leu 760 765 770 AAA TTT GGC AGC AAG CAC AAA TCA AAC TTG AAA CAA CTC GAA ACT GGA 2769 Lys Phe Gly Ser Lys His Lys Ser Asn Lou Lys Gin Val Glu Thr Gly 775 780 785

GTT GCC AAC ATG AAT ACA ATC AAT GCA GCA GAA CCT CAT GTG GTG ACA 2817 Val Ala Lys Not Aan Thr He Asn Ala Ala Glu Pro Hia Val Val Thr 790 795 800

CTC ACC ATC AAT CGT CTG GCA GCT AGA AAC CAC AGT GTT AAC TCC CAT 2865 Val Thr Not Asn Gly Val Ala Gly Arg Asn His Ser Val Asn Ser His 805 810 815

GCT GCC ACA ACC CAA TAT GCC AAT GCG ACA GTA CTA TCT GGC CAA ACA 2913 Ala Ala Thr Thr Gin Tyr Ala Asn Gly Thr Val Leu Ser Gly Gin Thr 820 825 830 835

ACC AAC ATA GTC ACA CAT AGG GCC CAA CAA ATC TTG CAC AAT CAG TTT 2961 Thr Asn He Val Thr His Arg Ala Gin Clu Not Leu Gin Asn Gin Phe 840 845 850 ATT GGT GAG GAC ACC CGG CTG AAT ATT AAT TCC AGT CCT GAT GAG CAT 3009 He Gly Clu Asp Thr Arg Leu Asn He Asn Sor Ser Pro Asp Glu His 855 860 865

GAG CCT TTA CTG AGA CGA GAC CAA CAA GCT GGC CAT GAT GAA GGT GTT 3057 Glu Pro Leu Leu Arg Arg Glu Gin Gin Ala Gly Hia Asp Glu Gly Val 870 875 880

CTG GAT CGT CTT GTG GAC AGG ACG GAA CGG CCA CTA GAA GGT GGC CGA 3105 Leu Asp Arg Leu Val Asp Arg Arg Glu Arg Pro Leu Glu Gly Gly Arg 885 890 895

ACT AAT TCC AAT AAC AAC AAC AGC AAT CCA TGT TCA GAA CAA GAT GTT 3153 Thr Asn Ser Asn Asn Asn Asn Ser Asn Pro Cys Ser Glu Gin Asp Val 900 905 910 915

CTT GCA CAG GGT GTT CCA AGC ACA GCA GCA GAT CCT GGG CCA TCA AAG 3201 Leu Ala Gin Gly Val Pro Ser Thr Ala Ala Asp Pro Gly Pro Ser Lys 920 925 930

CCC AGA AGA GCA CAG AGG CCT AAT TCT CTG GAT CTT TCA GCC ACA AAT 3249 Pro Arg Arg Ala Gin Arg Pro Asn Ser Leu Asp Leu Ser Ala Thr Asn 935 940 945

GTC CTG GAT CGC AGC AGT ATA CAC ATA GGT GAG TCA ACA CAA GAT GGC 3297 Val Leu Asp Gly Ser Sor He Gin He Gly Glu Ser Thr Gin Asp Gly 950 955 960

AAA TCA GGA TCA GGT GAA AAG ATC AAC AAA CGT CTG AAA ACT CCC TAT 3345 Lys Ser Gly Ser Gly Glu Lys He Lys Lys Arg Val Lys Thr Pro Tyr 965 970 975

TCT CTT AAG CGG TGG CGC CCC TCC ACC TGG GTC ATC TCC ACT GAA TCG 3393 Sor Leu Lys Arg Trp Arg Pro Ser Thr Trp Val He Ser Thr Glu Ser 980 985 990 995

CTG GAC TGT GAA GTC AAC AAT AAT GCC AGT AAC AGG GCA CTT CAT TCC 3441 Leu Asp Cys Glu Val Aan Asn Asn Gly Sor Asn Arg Ala Val His Ser 1000 1005 1010 AAA TCC AGC ACT CCT GTT TAC CTT GCA GAA GGA GGC ACT GCT ACA ACC 3489 Lys Ser Ser Thr Ala Val Tyr Leu Ala Glu Gly Gly Thr Ala Thr Thr 1015 1020 1025

ATG GTG TCT AAA GAT ATA GGA ATG AAC TGT CTG TGAAATGTTT TCAAGCCTAT 3542 Net Val Ser Lys Asp He Gly Not Asn Cys Leu 1030 1035

GGAGTGAAAT TATTTTTTCC ATCATTTAAA CATGCAGAAG ATGTTTAAAA AAAAAAAAA 3601

(2) INFORMATION FOR SEQ ID 110:2:

(1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1038 aaino acids (8) TYPE: aarinø acid

(D) TOPOLOGY: linear

(ii) NOLECULE TYPE: protein (xi ) SEOUENCE DESCRIPTION: SEQ 10 NO:2:

Net Thr Ser Sor Lou Gin Arg Pro Trp Arg Val Pro Trp Leu Pro Trp 1 5 10 15 Thr He Leu Lou Val Ser Thr Ala Ala Ala Ser Gin Asn Gin Glu Arg 20 25 30

Leu Cys Ala Phe Lys Asp Pro Tyr Gin Gin Asp Leu Gly He Gly Glu 35 40 45

Sor Arg He Sor His Clu Asn Cly Thr He Leu Cys Ser Lys Gly Sor 50 55 60

Thr Cya Tyr Gly Leu Trp Glu Lys Ser Lys Gly Asp He Asn Leu Val 65 70 75 80

Lys Gin Gly Cys Trp Sor His Ho Gly Asp Pro Gin Clu Cys His Tyr 85 90 95 Glu Glu Cys Vol Val Thr Thr Thr Pro Pro Ser I le Gin Asn Gly Thr 100 105 110

Tyr Arg Phe Cys Cys Cys Sor Thr Asp Leu Cys Asn Val Asn Phe Thr 115 120 125

Glu Aan Phe Pro Pro Pro Asp Thr Thr Pro Leu Ser Pro Pro His Ser

130 135 140

Phe Asn Arg Asp Glu Thr He He He Ala Leu Ala Ser Val Ser Val

145 ISO 155 160

Leu Ala Val Leu He Val Ala Leu Cys Phe Gly Tyr Arg Met Leu Thr 165 170 175

Gly Asp Arg Lys Gin Gly Leu His Ser Met Asn Met Met Glu Ale Ala 180 185 190

Ala Sor Glu Pro Ser Leu Asp Leu Asp Asn Leu Lys Leu Leu Glu Leu 195 200 205

He Gly Arg Gly Arg Tyr Gly Ala Val Tyr Lys Gly Ser Leu Asp Glu 210 215 220

Arg Pro Val Ala Val Lys Val Phe Ser Phe Ala Asn Arg Gin Asn Phe 225 230 235 240

He Asn Glu Lys Asn Ho Tyr Arg Val Pro Leu Met Glu His Asp Asn 245 250 255 He Ala Arg Phe He Val Gly Asp Glu Arg Val Thr Ala Asp Gly Arg 260 265 270

Net Glu Tyr Leu Leu Val Met Glu Tyr Tyr Pro Asn Gly Ser Leu Cys 275 280 285

Lys Tyr Leu Sor Leu His Thr Ser Asp Trp Val Ser Sor Cys Arg Leu 290 295 300

Ala His Ser Val Thr Arg Gly Leu Ala Tyr Leu His Thr Glu Leu Pro 305 310 315 320

Arg Gly Aap His Tyr Lys Pro Ala He Ser Hia Arg Asp Leu Asn Ser 325 330 335 Arg Asn Val Leu Val Lys Asn Asp Gly Thr Cys Val Ho Ser Asp Phe 340 345 350

Gly Lou Sor Mot Arg Leu Thr Gly Asn Arg Leu Vsl Arg Pro Gly Glu 355 360 365

Glu Asp Asn Ala Ala He Ser Glu Val Gly Thr Ho Arg Tyr Net Ala 370 375 380

Pro Clu Val Leu Clu Cly Ala Val Aan Leu Arg Asp Cys Glu Ser Ale 3*5 390 395 400

Lou Lys Gin Val Asp Not Tyr Ala Leu Gly Leu He Tyr Trp Glu He 405 410 415 PHβ Net Arg Cys Thr Asp Leu Phe Pro Gly Glu Ser Val Pro Glu Tyr 420 425 430

Gin Not Ala Phe Gin Thr Glu Val Gly Asn His Pro Thr Phe Glu Asp 435 440 445

Not Gin Val Leu Val Ser Arg Glu Lys Gin Arg Pro Lys Phe Pro Glu 450 455 460

Ala Trp Lys Clu Aan Sor Lou Ala Val Arg Ser Leu Lys Glu Thr He 465 470 475 480

Clu Asp Cys Trp Asp Gin Asp Ala Glu Ala Arg Leu Thr Ala Gin Cys 485 490 495 Ala Clu Glu Arg Net Ala Glu Leu Net Net He Trp Glu Arg Asn Lys 500 505 510

Sor Val Sor Pro Thr Val Asn Pro Net Ser Thr Ala Met Gin Asn Glu 515 520 525

Arg Asn Leu Ser His Asn Arg Arg Val Pro Lys He Gly Pro Tyr Pro

530 535 540

Asp Tyr Ser Ser Ser Ser Tyr He Glu Asp Ser He His His Thr Asp

545 550 555 560

Ser He Vsl Lys Asn He Ser Ser Glu His Ser Net Ser Ser Thr Pro 565 570 575

Leu Thr He Gly Glu Lys Asn Arg Asn Ser He Asn Tyr Glu Arg Gin 580 585 590

Gin Ala Gin Ala Arg He Pro Ser Pro Glu Thr Ser Val Thr Ser Leu 595 600 60S

Ser Thr Aan Thr Thr Thr Thr Asn Thr Thr Gly Leu Thr Pro Ser Thr 610 615 620

Gly Met Thr Thr He Ser Glu Met Pro Tyr Pro Asp Glu Thr Asn Leu 625 630 635 640

His Thr Thr Asn Vsl Ala Gin Ser He Gly Pro Thr Pro Val Cys Leu 645 650 655 Gin Leu Thr Glu Glu Asp Leu Glu Thr Asn Lys Leu Asp Pro Lys Glu 660 665 670

Val Asp Lys Asn Leu Lys Glu Ser Sor Asp Glu Asn Leu Met Glu His 675 680 685

Sor Lou Lys Cln Phe Sor Cly Pro Asp Pro Leu Ser Ser Thr Ser Ser 690 695 700

Sor Leu Leu Tyr Pro Leu He Lys Leu Ale Val Glu Ala Thr Gly Gin 705 710 715 720

Gin Asp Phe Thr Gin Thr Ala Asn Gly Gin Ala Cys Leu He Pro Asp

725 730 735 Val Leu Pro Thr Gin He Tyr Pro Leu Pro Lys Gin Gin Asn Leu Pro 740 745 750

Lys Arg Pro Thr Sor Leu Pro Leu Asn Thr Lys Asn Ser Thr Lys Glu

755 760 765

Pro Arg Leu Lys Phe Cly Sor Lys Nis Lys Ser Asn Leu Lys Gin Val 770 775 780

Glu Thr Gly Val Ala Lys Hat Asn Thr He Asn Ala Ala Glu Pro His 785 790 795 800

Val Val Thr Val Thr Not Asn Gly Val Ala Gly Arg Asn His Ser Val 805 810 815 Asn Sor His Ala Ala Thr Thr Gin Tyr Ala Asn Gly Thr Val Leu Ser 820 825 830

Gly Gin Thr Thr Aan He Val Thr Hia Arg Ala Gin Clu Net Leu Gin 835 840 845

Asn Cln Phe He Cly Glu Asp Thr Arg Leu Asn He Asn Ser Ser Pro 850 855 860

Asp Clu His Clu Pro Lou Lou Arg Arg Glu Gin Gin Ala Gly His Asp 865 870 875 880

Glu Gly Val Leu Aap Arg Leu Val Asp Arg Arg Glu Arg Pro Leu Glu 885 890 895 Gly Gly Arg Thr Asn Ser A n Asn Asn Asn Ser Asn Pro Cys Ser Glu 900 905 910

Gin Asp Val Leu Ala Gin Gly Val Pro Ser Thr Ala Ala Asp Pro Gly 915 920 925

Pro Ser Lys Pro Arg Arg Ala Gin Arg Pro Asn Ser Leu Asp Leu Ser 930 935 940

Ala Thr Asn Val Leu Asp Gly Ser Ser He Gin He Gly Glu Ser Thr 945 950 955 960

Gin Aap Gly Lys Ser Gly Ser Gly Glu Lys He Lys Lys Arg Val Lys 965 970 975

Thr Pro Tyr Ser Leu Lys Arg Trp Arg Pro Ser Thr Trp Val He Ser 980 985 990

Thr Glu Ser Leu Asp Cys Glu Val Asn Asn Asn-Gly Ser Asn Arg Als 995 1000 1005

Val His Ser Lys Ser Ser Thr Ala Val Tyr Leu Ala Glu Gly Gly Thr 1010 1015 1020

Ala Thr Thr Net Val Ser Lys Asp He Gly Net Asn Cys Leu 1025 1030 1035

(2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2156 base pairs

(B) TYPE: nucleic acid

(C) STRAN0EDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cONA

(ix) FEATURE: (A) NAME/KEY: CDS

(B) LOCATION: 409..2154

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

CGCCCCCCGA CCCCGCATCG AATCCCCGCC CTCCGCACCC TGGATATGTT TTCTCCCAGA 60

CCTGCATATT TTTTTGATAT CGTGAAACTA CGAGGGAAAT AATTTGGGCG ATTTCTTCTT 120 GGCTCCCTGC TTTCCCCACA GACATCCCTT CCGTTTGGAG CGCCGCGCCA CCCCGTCCGA 180

CGCCAACCAA CCCCCCCAGC CGCGAGGCAG AGAAATGAAG GGAATTTCTG CAGCGGCATG 240

AAAGCTCTGC AGCTAGGTCC TCTCATCACC CATTTCTCCT TTCAAACTGT ATTGTGATAC 300

GGGCAGGATC AGTCCACCCG AGACAAGACC AGCCTCCCGG CTGTTTCTCC GCCGGTCTAC 360

TTCCCATATT TCTTTTCTTT GCCCTCCTGA TTCTTGGCTG GCCCAGGG ATG ACT TCC 417

Met Thr Ser 1

TCG CTC CAC CGG CCC TGC CGG GTG CCC TGC CTA CCA TGG ACC ATC CTG 465 Sor Lou Gin Arg Pro Trp Arg Vel Pro Trp Leu Pro Trp Thr He Leu 5 10 15

CTG GTC AGC ACT CCC GCT GCT TCG CAC AAT CAA GAA CGC CTA TGT GCG 513 Leu Val Ser Thr Ala Ala Ala Ser Gin Asn Gin Glu Arg Leu Cys Ala 20 25 30 35 TTT AAA GAT CCG TAT CAC CAA CAC CTT GCG ATA GGT GAG AGT AGA ATC 561 Phe Lys Aap Pro Tyr Gin Gin Asp Leu Gly He Gly Glu Ser Arg He 40 45 SO

TCT CAT CAA AAT GGC ACA ATA TTA TCC TCC AAA GGT AGC ACC TGC TAT 609 Sor His Glu Asn Gly Thr He Leu Cys Ser Lys Gly Ser Thr Cys Tyr 55 60 65

CCC CTT TCG GAG AAA TCA AAA GGG GAC ATA AAT CTT GTA AAA CAA GGA 657 Gly Lou Trp Glu Lys Sor Lys Gly Asp He Asn Leu Vsl Lys Gin Gly 70 75 80

TGT TGG TCT CAC ATT GGA GAT CCC CAA GAG TGT CAC TAT GAA GAA TGT 705 Cys Trp Ser His He Gly Asp Pro Gin Glu Cys His Tyr Glu Glu Cys 85 90 95

GTA GTA ACT ACC ACT CCT CCC TCA ATT CAG AAT GGA ACA TAC CGT TTC 753 Val Val Thr Thr Thr Pro Pro Ser He Gin Asn Gly Thr Tyr Arg Phe 100 105 110 115

TGC TGT TGT AGC ACA GAT TTA TGT AAT GTC AAC TTT ACT GAG AAT TTT 801 Cys Cys Cys Ser Thr Asp Leu Cys Asn Val Asn Phe Thr Glu Asn Phe 120 125 130

CCA CCT CCT GAC ACA ACA CCA CTC AGT CCA CCT CAT TCA TTT AAC CGA 849 Pro Pro Pro Asp Thr Thr Pro Leu Ser Pro Pro His Ser Phe Asn Arg 135 140 145

GAT GAG ACA ATA ATC ATT GCT TTG GCA TCA GTC TCT GTA TTA GCT GTT 897 Asp Glu Thr He He He Ala Leu Ala Ser Vel Ser Val Leu Ala Val 150 155 160

TTG ATA GTT GCC TTA TGC TTT GGA TAC AGA ATG TTG ACA GGA GAC CGT 945 Leu He Val Ala Leu Cys Phe Gly Tyr Arg Net Leu Thr Gly Asp Arg 165 170 175

AAA CAA GGT CTT CAC AGT ATG AAC ATG ATG GAG GCA CCA GCA TCC GAA 993 Lys Gin Gly Leu His Ser Met Asn Net Met Glu Als Als Als Ser Glu 180 185 190 195 CCC TCT CTT GAT CTA GAT AAT CTG AAA CTG TTC GAG CTG ATT CGC CGA 1041 Pro Ser Leu Asp Leu Asp Asn Leu Lys Leu Leu Glu Leu He Gly Arg 200 205 210

GCT CGA TAT GGA GCA GTA TAT AAA GCC TCC TTG GAT GAG CGT CCA GTT 1089 Gly Arg Tyr Gly Ala Val Tyr Lys Gly Ser Leu Asp Glu Arg Pro Val 215 220 225

CCT CTA AAA GTG TTT TCC TTT GCA AAC CGT CAG AAT TTT ATC AAC GAA 1137 Ala Val Lys Val Phe Ser Phe Ala Asn Arg Gin Asn Phe He Asn Glu 230 235 240

AAG AAC ATT TAC AGA GTG CCT TTG ATG GAA CAT GAC AAC ATT GCC CGC 1185 Lys Asn He Tyr Arg Val Pro Leu Net Glu His Asp Asn He Ala Arg 245 250 255

TTT ATA GTT CGA GAT GAC AGA GTC ACT GCA GAT GGA CGC ATG GAA TAT 1233 Phe He Val Gly Aap Clu Arg Val Thr Ala Asp Gly Arg Met Glu Tyr 260 265 270 275 TTC CTT CTG ATC GAC TAC TAT CCC AAT GCA TCT TTA TGC AAG TAT TTA 1281

Lou Lou Val Not Glu Tyr Tyr Pro Aan Gly Sor Leu Cys Lys Tyr Leu

280 285 290

AGT CTC CAC ACA ACT GAC TCG GTA AGC TCT TGC CGT CTT CCT CAT TCT 1329 Sor Lou His Thr Sor Asp Trp Val Ser Ser Cys Arg Leu Ala His Ser 295 300 305

GTT ACT AGA CGA CTG GCT TAT CTT CAC ACA GAA TTA CCA CGA GGA GAT 1377

Vol Thr Arg Gly Leu Ala Tyr Leu His Thr Glu Leu Pro Arg Gly Asp 310 315 320

CAT TAT AAA CCT GCA ATT TCC CAT CGA CAT TTA AAC AGC AGA AAT GTC 1425

Nis Tyr Lys Pro Ala Ho Sor His Arg Asp Leu Asn Ser Arg Asn Val 325 330 335

CTA CTC AAA AAT GAT GCA ACC TCT CTT ATT AGT CAC TTT GGA CTG TCC 1473 Leu Val Lys Asn Asp Cly Thr Cys Val He Ser Asp Phe Gly Leu Ser 340 345 350 355 ATG ACG CTG ACT GGA AAT ACA CTG GTC CCC CCA GGG CAG GAA GAT AAT 1521 Net Arg Leu Thr Gly Asn Arg Leu Val Arg Pro Gly Glu Glu Asp Asn 360 365 370

GCA GCC ATA AGC GAG GTT GGC ACT ATC AGA TAT ATG GCA CCA GAA GTG 1569 Ala Ala He Ser Glu Val Gly Thr He Arg Tyr Met Ala Pro Glu Val 375 380 385

CTA GAA GGA GCT GTG AAC TTG AGG GAC TGT GAA TCA GCT TTG AAA CAA 1617 Leu Glu Gly Ala Val Asn Leu Arg Asp Cys Glu Ser Ala Leu Lys Gin 390 395 400

GTA GAC ATG TAT GCT CTT GGA CTA ATC TAT TGG GAG ATA TTT ATG AGA 1665 Val Asp Met Tyr Ala Leu Gly Leu He Tyr Trp Glu He Phe Met Arg 405 410 415

TGT ACA GAC CTC TTC CCA GGG GAA TCC GTA CCA GAG TAC CAG ATG GCT 1713 Cys Thr Asp Leu Phe Pro Gly Glu Ser Val Pro Glu Tyr Gin Met Ala 420 425 430 435

TTT CAG ACA GAG GTT GGA AAC CAT CCC ACT TTT GAG GAT ATG CAG GTT 1761 Phe Gin Thr Glu Vel Gly Asn His Pro Thr Phe Glu Asp Met Gin Val 440 445 450

CTC GTG TCT AGG GAA AAA CAG AGA CCC AAG TTC CCA GAA GCC TGG AAA 1809 Leu Val Ser Arg Glu Lys Gin Arg Pro Lys Phe Pro Glu Ala Trp Lys 455 460 465 GAA AAT AGC CTG GCA GTG AGG TCA CTC AAG GAG ACA ATC GAA GAC TGT 1857 Glu Asn Ser Leu Ala Val Arg Ser Leu Lys Glu Thr He Glu Asp Cys 470 475 480

TCG GAC CAG GAT GCA GAG GCT CGG CTT ACT GCA CAG TGT GCT GAG GAA 1905 Trp Asp Cln Asp Ala Clu Ala Arg Leu Thr Ala Gin Cya Ala Glu Glu 485 490 495

AGG ATG GCT GAA CTT ATG ATG ATT TGC GAA AGA AAC AAA TCT GTG AGC 1953 Arg Not Ala Glu Leu Net Net He Trp Glu Arg Asn Lys Ser Val Ser 500 505 510 515.

CCA ACA GTC AAT CCA ATG TCT ACT GCT ATG CAG AAT GAA CGC AAC CTG 2001 Pro Thr Val Asn Pro Net Ser Thr Ala Net Gin Asn Glu Arg Asn Leu 520 525 530

TCA CAT AAT AGG CGT GTG CCA AAA ATT GGT CCT TAT CCA GAT TAT TCT 2049 Sor His Asn Arg Arg Val Pro Lys He Cly Pro Tyr Pro Asp Tyr Ser 535 540 545 TCC TCC TCA TAC ATT GAA GAC TCT ATC CAT CAT ACT GAC ACC ATC GTG 2097 Sor Sor Sor Tyr Ho Glu Asp Ser He His His Thr Asp Ser He Vsl 550 555 560

AAG AAT ATT TCC TCT GAG CAT TCT ATG TCC AGC ACA CCT TTG ACT ATA 2145 Lys Aan Ho Ser Sor Glu His Ser Not Ser Ser Thr Pro Leu Thr He 565 570 575

G66 GAA AAA AA 2156

Cly Clu Lys 580

(2) INFORMATION FOR SEQ ID NO:4: (1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 582 aarino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein

(Xi) SEQUENCE DESCRIPTION: SEQ ID 1*0:4:

Net Thr Ser Ser Leu Gin Arg Pro Trp Arg Vel Pro Trp Leu Pro Trp 1 5 10 15

Thr Ho Leu Leu Val Ser Thr Ala Ala Ala Ser Gin Asn Gin Glu Arg 20 25 30

Leu Cys Ale Phe Lys Asp Pro Tyr Gin Gin Asp Leu Gly He Gly Glu 35 40 45

Ser Arg He Ser His Glu Asn Gly Thr He Leu Cys Ser Lys Gly Ser 50 55 60

Thr Cys Tyr Gly Leu Trp Glu Lys Ser Lys Gly Asp He Asn Leu Val 65 70 75 80

Lys Gin Gly Cys Trp Ser Nis He Gly Asp Pro Gin Glu Cys His Tyr 85 90 95

Glu Glu Cys Val Val Thr Thr Thr Pro Pro Ser He Gin Asn Gly Thr 100 105 110

Tyr Arg Phe Cys Cys Cys Ser Thr Asp Leu Cys Asn Val Asn Phe Thr 115 120 125

Glu Asn Phe Pro Pro Pro Asp Thr Thr Pro Leu Ser Pro Pro His Ser 130 135 140

Phe Asn Arg Asp Glu Thr Ho He He Ala Leu Ala Ser Val Ser Val 145 150 155 160

Leu Ala Val Leu He Val Ala Leu Cys Phe Gly Tyr Arg Met Leu Thr 165 170 175

Gly Asp Arg Lys Gin Gly Leu His Ser Net Asn Net Net Glu Ala Ala 180 185 190

Ala Ser Glu Pro Ser Leu Asp Leu Asp Asn Leu Lys Leu Leu Glu Leu 195 200 205

He Gly Arg Gly Arg Tyr Gly Ala Val Tyr Lys Gly Ser Leu Asp Glu 210 215 220

Arg Pro Vel Ala Val Lys Val Phe Sor Phe Ala Aan Arg Gin Asn Phe 225 230 235 240 He Asn Glu Lys Asn He Tyr Arg Val Pro Leu Net Glu His Asp Asn 245 250 255

He Ala Arg Phe He Vel Gly Asp Glu Arg Val Thr Ala Asp Gly Arg 260 265 270

Net Glu Tyr Leu Leu Vel Net Glu Tyr Tyr Pro Asn Gly Ser Leu Cys 275 280 285

Lys Tyr Leu Ser Leu Nis Thr Ser Asp Trp Val Ser Ser Cys Arg Leu 290 295 300

Ala Hia Sor Val Thr Arg Gly Leu Ale Tyr Leu His Thr Glu Leu Pro 305 310 315 320 Arg Cly Asp His Tyr Lys Pro Ala He Ser Hia Arg Asp Leu Asn Ser 325 330 335

Arg Asn Val Leu Val Lys Asn Asp Gly Thr Cys Vsl He Ser Asp Phe 340 345 3S0

Gly Leu Ser Net Arg Leu Thr Gly Asn Arg Leu Val Arg Pro Gly Glu 355 360 365

Glu Asp Asn Ala Ala He Ser Glu Val Gly Thr He Arg Tyr Met Ala 370 375 380

Pro Glu Val Leu Glu Gly Ala Val Asn eu Arg Aap Cys Glu Ser Ala 385 390 395 400 Leu Lys Gin Val Aap Net Tyr Ale Leu Gly Leu He Tyr Trp Glu He 405 410 415

Phe Not Arg Cys Thr Asp Leu Phe Pro Gly Glu Ser Val Pro Glu Tyr 420 425 430

Gin Met Ala Phe Gin Thr Glu Val Gly Asn His Pro Thr Phe Glu Asp 435 440 445

Met Gin Val Leu Val Ser Arg Glu Lys Gin Arg Pro Lys Phe Pro Glu 450 455 460

Ala Trp Lys Glu Asn Ser Leu Ala Val Arg Ser Leu Lys Glu Thr He 465 470 475 480

Glu Asp Cys Trp Asp Gin Asp Ala Glu Ala Arg Leu Thr Ala Gin Cys 485 490 495

Ala Glu Glu Arg Met Ala Glu Leu Met Met He Trp Glu Arg Asn Lys 500 505 510

Sor Val Ser Pro Thr Vel Asn Pro Net Ser Thr Ale Met Gin Asn Glu 515 520 525

Arg Asn Leu Ser His Asn Arg Arg Vat Pro Lys He Gly Pro Tyr Pro 530 535 540

Asp Tyr Ser Ser Ser Ser Tyr He Glu Asp Ser He His His Thr Asp

545 550 555 560 Sor He Vel Lys Asn He Ser Ser Glu His Ser Met Ser Ser Thr Pro 565 570 575

Leu Thr He Gly Glu Lys 580

(2) INFORMATION FOR SEQ ID N0:S:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 471 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cONA

(ix) FEATURE:

(A) NAME/KEY: COS

(B) LOCATION: 19..471

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

TTCTTGGCTG CCCCAGCG ATC ACT TCC TCG CTG CAG CGG CCC TGC GG GTG 51 Net Thr Sor Sor Leu Gin Arg Pro Trp Arg Vel

1 5 10

CCC TGG CTA CCA TGC ACC ATC CTG CTG GTC AGC ACT GCG GCT GCT TCG 99 Pro Trp Leu Pro Trp Thr He Leu Leu Val Ser Thr Ala Ala Ale Ser 15 20 25

CAC AAT CAA CAA CGG CTA TGT GCG TTT AAA GAT CCG TAT CAG CAA GAC 147 Gin Aan Gin Glu Arg Leu Cys Ala Phe Lys Asp Pro Tyr Gin Gin Asp 30 35 40

CTT GGG ATA GGT GAG AGT AGA ATC TCT CAT GAA AAT GGG ACA ATA TTA 195 Lou Gly He Gly Glu Ser Arg He Ser His Glu Asn Gly Thr He Leu 45 50 55 TGC TCG AAA CGT ACC ACC TGC TAT GCC CTT TGG GAG AAA TCA AAA GCG 243 Cys Ser Lys Gly Ser Thr Cys Tyr Gly Leu Trp Glu Lys Ser Lys Gly 60 65 70 75

GAC ATA AAT CTT CTA AAA CAA CCA TCT TGG TCT CAC ATT GCA GAT CCC 291 Asp He Asn Leu Val Lys Gin Gly Cys Trp Ser His He Gly Asp Pro 80 85 90

CAA GAG TGT CAC TAT GAA GAA TGT GTA GTA ACT ACC ACT CCT CCC TCA 339 Gin Glu Cys His Tyr Glu Glu Cys Val Val Thr Thr Thr Pro Pro Ser 95 100 105

ATT CAG AAT GGA ACA TAC CGT TTC TGC TGT TGT AGC ACA GAT TTA TGT 387 He Gin Asn Gly Thr Tyr Arg Phe Cys Cys Cys Ser Thr Asp Leu Cys 110 115 120

AAT GTC AAC TTT ACT GAG AAT TTT CCA CCT CCT GAC ACA ACA CCA CTC 435 Asn Val Asn Phe Thr Glu Asn Phe Pro Pro Pro Asp Thr Thr Pro Leu 125 130 135 AGT CCA CCT CAT TCA TTT AAC CGA GAT GAG ACA TG 471

Ser Pro Pro His Ser Phe Asn Arg Asp Glu Thr 140 145 150

(2) INFORMATION FOR SEQ ID N0:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 150 waino acids

(B) TYPE: asnno acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

Not Thr Sor Ser Leu Gin Arg Pro Trp Arg Val Pro Trp Leu Pro Trp 1 5 10 15

Thr He Leu Leu Vel Ser Thr Ala Ala Ala Ser Gin Asn Gin Glu Arg 20 25 30

Leu Cys Ala Phe Lys Asp Pro Tyr Gin Gin Asp Leu Gly He Gly Glu 35 40 45 Ser Arg He Ser His Glu Asn Cly Thr He Leu Cys Ser Lys Gly Ser SO 55 60

Thr Cys Tyr Gly Leu Trp Glu Lys Sor Lys Gly Asp He Asn Leu Val 65 70 75 80

Lys Gin Gly Cys Trp Sor His He Gly Asp Pro Gin Glu Cys His Tyr 85 90 95

Glu Clu Cys Vol Val Thr Thr Thr Pro Pro Ser He Gin Aan Gly Thr 100 105 110

Tyr Arg Phe Cys Cys Cys Ser Thr Asp Leu Cys Asn Vel Asn Phe Thr 115 120 125 Glu Asn Phe Pro Pro Pro Asp Thr Thr Pro Leu Ser Pro Pro His Ser 130 135 140

Phe Asn Arg Asp Glu Thr 145 150

(2) INFORMATION FOR SEQ ID N0:7:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 3508 base peirs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double (0) TOPOLOGY: linear (ii) MOLECULE TYPE: cONA

(ix) FEATURE:

(A) NAME/KEY: CSS (B) LOCATION: 17..3133

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

CTTTGCTGGC CCAGGG ATG ACT TCC TCG CTG CAT CGG CCC TTT CGG GTG 49 Not Thr Ser Ser Leu His Arg Pro Phe Arg Val 1 5 10 CCC TGG CTG CTA TGG GCC GTC CTG CTG GTC AGC ACT ACG GCT GCT TCT 97 Pro Trp Leu Leu Trp Ala Val Leu Leu Val Ser Thr Thr Ala Ala Ser 15 20 25

CAG AAT CAA GAA CGG CTG TGT GCA TTT AAA GAT CCA TAT CAA CAA GAT 145 Gin Asn Gin Glu Arg Leu Cys Ala Phe Lys Asp Pro Tyr Gin Gin Asp 30 35 40

CTT GGG ATA GGT GAG AGT CGA ATC TCT CAT GAA AAT GGG ACA ATA TTA 193 Leu Gly He Gly Glu Ser Arg He Ser His Glu Asn Gly Thr He Leu 45 50 55

TGT TCC AAA GGG AGC ACG TGT TAT GGT CTG TGG GAG AAA TCA AAA GGG 241 Cys Ser Lys Gly Ser Thr Cys Tyr Gly Leu Trp Glu Lys Ser Lys Gly 60 65 70 75

GAC ATC AAT CTT GTG AAA CAA CCA TGT TGG TCT CAC ATC GGT GAT CCC 289 Asp He Asn Leu Val Lys Gin Gly Cys Trp Ser His He Gly Asp Pro 80 85 90 CAA GAG TGC CAC TAT GAA GAG TGT GTA GTA ACT ACC ACC CCA CCC TCA 337 Gin Glu Cys His Tyr Glu Glu Cys Val Val Thr Thr Thr Pro Pro Ser 95 100 105

ATT CAG AAT GCA ACG TAC CGC TTT TCC TGC TGT AGT ACA GAT TTA TGT 385 He Gin Aan Gly Thr Tyr Arg Phe Cys Cys Cys Ser Thr Asp Leu Cys 110 115 120

AAT GTC AAC TTT ACT GAG AAC TTT CCA CCC CCT GAC ACA ACA CCA CTC 433 Asn Val Asn Phe Thr Glu Asn Phe Pro Pro Pro Asp Thr Thr Pro Leu 125 130 135

AGT CCA CCT CAT TCA TTT AAT CGA GAT GAA ACG ATA ATC ATT GCT TTG 481 Sor Pro Pro His Sor Phe Asn Arg Asp Glu Thr He He He Ala Leu 140 145 150 155

GCA TCA CTT TCT GTG TTA GCT GTT TTG ATA GTC GCC TTA TGT TTT GGA 529 Ala Ser Val Ser Vel Leu Ale Val Leu He Vel Ala Leu Cys Phe Gly 160 165 170 TAC AGA ATC TTG ACA GCA GAC CGG AAA CAC GGT CTT CAC AGC ATG AAC 577 Tyr Arg Not Lou Thr Gly Asp Arg Lys Gin Gly Leu His Ser Net Asn 175 180 185

ATG ATG CAG GCG CCA GCA GCA GAG CCC TCC CTT CAC CTG GAT AAC CTG 625 Not Not Glu Ala Ala Ala Ala Clu Pro Sor Leu Asp Leu Asp Asn Leu - 190 195 200

AAG CTC CTG GAG CTG ATT GGA CGG GGT CGA TAC GGA GCA GTA TAT AAA 673 Lys Lou Lou Clu Lou He Gly Arg Gly Arg Tyr Gly Ala Val Tyr Lys 205 210 215

GGT TCC TTG GAT GAG CGT CCA GTT CCT GTA AAA GTA TTT TCT TTT GCA 721 Gly Sor Leu Asp Clu Arg Pro Val Ala Val Lys Val Phe Ser Phe Ala 220 225 230 235

AAC CGT CAG AAT TTT ATA AAT GAA AAA AAC ATT TAC AGA GTG CCT TTG 769 Aan Arg Cln Aan Phe He Asn Glu Lys Asn He Tyr Arg Val Pro Leu 240 245 250 ATG GAA CAT GAC AAC ATT GCT CCC TTC ATA GTT GGA GAC GAG ACC CTC 817 Not Glu His Asp Asn He Ala Arg Phe He Val Gly Asp Glu Arg Leu 255 260 265

ACT GCA GAC CGC CCC ATG GAC TAT TTG CTT GTG ATG CAC TAT TAT CCC 865 Thr Ala Aap Gly Arg Net Clu Tyr Leu Leu Val Net Clu Tyr Tyr Pro 270 275 280

AAT CCA TCT CTG TGC AAA TAT CTG AGT CTC CAC ACA AGT GAT TGG GTA 913 Aan Cly Ser Leu Cys Lys Tyr Leu Ser Leu His Thr Ser Asp Trp Val 285 290 295

AGC TCT TGC CGT CTG GCT CAT TCT GTG ACT AGA GGA CTG GCT TAT CTT 961 Ser Ser Cys Arg Leu Ala His Ser Val Thr Arg Gly Leu Ala Tyr Leu 300 305 310 315

CAC ACA GAA TTA CCA CGA GCA GAT CAT TAT AAA CCC GCA ATC TCC CAC 1009 His Thr Glu Leu Pro Arg Gly Asp His Tyr Lys Pro Ala He Ser His 320 325 330

CGA GAT TTA AAC AGC AGG AAT GTC CTG CTA AAG AAT GAC GGC GCG TGT 1057 Arg Asp Leu Asn Ser Arg Asn Val Leu Val Lys Asn Asp Gly Ala Cys 335 340 345

GTT ATC AGT GAC TTT GGT TTA TCC ATG AGG CTA ACT GGA AAT CGG CTG 1105 Val He Ser Asp Phe Gly Leu Ser Met Arg Leu Thr Gly Asn Arg Leu 350 355 360

CTG CGC CCA GGG GAA GAA GAT AAT GCG GCT ATA AGT GAG GTT GCC ACA 1153 Val Arg Pro Gly Glu Glu Asp Asn Ala Ala He Ser Glu Val Gly Thr 365 370 375

ATT CGC TAT ATG GCA CCA GAA GTG CTA GAA GGA GCT GTG AAC CTG AGG 1201 He Arg Tyr Met Ala Pro Glu Val Leu Glu Gly Ala Val Asn Leu Arg 380 385 390 395

GAC TGT GAG TCA GCT CTG AAG CAA GTC GAC ATG TAT GCG CTT GGA CTC 1249 Asp Cys Clu Ser Ala Leu Lys Gin Val Asp Met Tyr Als Leu Gly Leu 400 405 410 ATC TAC TCG GAG GTG TTT ATG ACG TGT ACA GAC CTC TTC CCA GGT GAA 1297 He Tyr Trp Glu Val Phe Not Arg Cys Thr Asp Leu Phe Pro Gly Glu 415 420 425

TCT GTA CCA GAT TAC CAG ATG GCT TTT CAG ACA GAA GTT GGA AAC CAT 1345 Sor Val Pro Asp Tyr Gin Net Ala Phe Gin Thr Glu Val Gly Asn His 430 435 440

CCC ACA TTT CAG GAT ATG CAG GTT CTT GTG TCC AGA GAG AAG CAG AGA 1393 Pro Thr Phe Glu Asp Net Gin Vet Leu Vel Ser Arg Glu Lys Gin Arg 445 450 455

CCC AAG TTC CCA GAA GCC TCG AAA GAA AAT AGC CTG GCA GTG AGG TCA 1441 Pro Lys Phe Pro Glu Ala Trp Lys Glu Asn Ser Leu Ala Val Arg Ser 460 465 470 475

CTC AAG GAA ACA ATT GAA GAC TGC TGG GAC CAG GAT CCA GAG GCT CGG 1489 Leu Lys Glu Thr He Clu Asp Cys Trp Asp Cln Asp Ale Glu Ala Arg 480 485 490 CTC ACT GCA CAG TCT GCT GAC CAG AGG ATG GCT GAA CTC ATG ATG ATA 1537 Lou Thr Ala Gin Cys Ala Clu Clu Arg Net Ala Glu Leu Met Net He 495 500 505

TGβ GAC AGA AAC AAC TCT GTG AGC CCA ACG CTC AAC CCA ATG TCA ACT 1585 Trp Clu Arg Aan Lys Sor Val Ser Pro Thr Val Asn Pro Net Ser Thr 510 515 520

GCT ATC CAC AAT GAA CGC AAC CTG TCA CAT AAT AGC CGT GTG CCA AAA 1633 Ala Not Gin Aan Clu Arg Asn Leu Ser His Asn Arg Arg Vsl Pro Lys 525 530 535

ATC GGC CCT TAC CCA GAT TAT TCC TCT TCC TCA TAT ATT GAA GAC TCT 1681 He Gly Pro Tyr Pro Asp Tyr Ser Ser Ser Ser Tyr He Glu Asp Ser 540 545 550 555

ATC CAT CAT ACT GAC AGC ATT CTG AAG AAT ATT TCC TCT GAG CAT TCG 1729 He His His Thr Asp Ser He Val Lys Asn He Ser Ser Glu His Ser 560 565 570 ATG TCC AGC ACA CCA TTG ACA ATA GGA GAA AAG AAT CGA AAT TCA ATT 1777 Not Sor Sor Thr Pro Leu Thr He Gly Glu Lys Asn Arg Asn Ser He 575 580 585

AAT TAT GAA CGA CAG CAA GCA CAA GCT CGA ATC CCT AGC CCA GAA ACA 1825 Aan Tyr Glu Arg Gin Gin Ala Gin Ala Arg He Pro Ser Pro Glu Thr 590 595 600

AGC GTC ACA AGC CTG TCC ACA AAC ACA ACC ACC ACA AAC ACC ACC GGC 1873 Ser Val Thr Ser Leu Ser Thr Asn Thr Thr Thr Thr Asn Thr Thr Gly 605 610 615

CTC ACT CCA AGT ACT GGC ATG ACC ACT ATA TCT GAG ATG CCA TAC CCA 1921 Leu Thr Pro Ser Thr Gly Met Thr Thr He Ser Glu Met Pro Tyr Pro

620 625 630 635

GAT GAG ACA CAT TTG CAC GCC ACA AAT GTT GCA CAG TCA ATC GGG CCA 1969 Asp Glu Thr His Leu His Ala Thr Asn Val Ala Gin Ser He Gly Pro 640 645 650

ACC CCT GTC TGC TTA CAG CTG ACA GAA GAA GAC TTG GAG ACT AAT AAG 2017 Thr Pro Vol Cys Leu Gin Leu Thr Glu Clu Asp Leu Glu Thr Asn Lys 655 660 665

CTA GAT CCA AAA GAA GTT GAT AAG AAC CTC AAG GAA AGC TCT GAT GAG 2065 Leu Asp Pro Lys Glu Val Asp Lys Asn Leu Lys Glu Ser Ser Asp Glu 670 675 680 AAT CTC ATG GAG CAT TCT CTG AAG CAG TTC AGT GGG CCA GAC CCA TTG 2113 Asn Leu Met Glu His Ser Leu Lys Gin Phe Ser Cly Pro Asp Pro Leu 685 690 695

ACC ACT ACC ACT TCT AGC TTG CTT TAT CCA CTC ATA AAG CTC CCA GTG 2161 Ser Ser Thr Sor Sor Ser Leu Leu Tyr Pro Leu Ile Lys Leu Ala Val 700 705 710 715

GAA GTG ACT GCA CAA CAG GAC TTC ACA CAG GCT GCA AAT GGG CAA GCA 2209 Glu Val Thr Gly Gin Gin Asp Phe Thr Gin Ala Ala Asn Gly Gin Ala 720 725 730

TGT TTA ATT CCT GAT CTT CCA CCT GCT CAG ATC TAT CCT CTC CCT AAG 2257 Cys Leu He Pro Asp Val Pro Pro Ala Gin He Tyr Pro Leu Pro Lys 735 740 745

CAA CAG AAC CTT CCT AAG AGA CCT ACT AGT TTG CCT TTG AAC ACC AAA 2305 Gin Gin Asn Leu Pro Lys Arg Pro Thr Ser Leu Pro Leu Asn Thr Lys 750 755 760 AAT TCA ACA AAA CAA CCC CGC CTA AAA TTT CCC AAC AAG CAC AAA TCA 2353 Asn Sor Thr Lys Glu Pro Arg Lou Lys Phe Gly Asn Lys His Lys Ser 765 770 775

AAC TTG AAA CAA CTA CAA ACT GGA CTT GCC AAG ATG AAT ACA ATC AAT 2401 Aan Lou Lys Cln Val Glu Thr Cly Val Ala Lys Not Asn Thr Ho Asn 780 785 790 795

CCA CCA GAC CCT CAT CTG CTC ACA CTA ACT ATC AAT GGT GTC CCA GGT 2449 Ala Ala Clu Pro Nis Val Val Thr Val Thr Net Asn Gly Val Ala Gly 800 805 810

AGA AGC CAC AAT GTT AAT TCT CAT GCT GCC ACA ACC CAC TAT CCC AAT 2497 Arg Sor Nis Asn Val Asn Sor His Ala Ala Thr Thr Gin Tyr Ala Asn 815 820 825

GGC GCA GTC CCA GCT GGC CAG GCA CCC AAC ATA GTG GCA CAT AGG TCC 2545 Gly Ala Val Pro Ala Gly Gin Ala Ala Asn He Val Ala His Arg Ser 830 835 840 CAA CAA ATG CTG CAC AAT CAA TTT ATT CCT GAG GAT ACC AGG CTG AAT 2593 Gin Clu Not Leu Gin Asn Gin Phe He Gly Glu Asp Thr Arg Leu Asn 845 850 855

ATC AAT TCC ACT CCT GAT GAG CAT CAA CCT TTA CTG AGA CCA GAG CAA 2641 He Aan Sor Sor Pro Aap Glu His Glu Pro Leu Leu Arg Arg Glu Gin 860 865 870 875

CAC GCT GGC CAT GAT CAA GCG GTT CTG GAT CCT TTG GTA GAT AGG AGG 2689 Gin Ala Gly His Asp Glu Gly Val Leu Asp Arg Leu Val Asp Arg Arg 880 885 890

GAA CGG CCA TTA GAA GGT GGC CGA ACA AAT TCC AAT AAC AAC AAC AGC 2737 Glu Arg Pro Leu Glu Gly Gly Arg Thr Asn Ser Asn Asn Asn Asn Ser 895 900 905

AAT CCA TGT TCA GAA CAA GAT ATC CTT ACA CAA GGT GTT ACA AGC ACA 2785 Asn Pro Cys Ser Glu Gin Asp He Leu Thr Gin Gly Val Thr Ser Thr 910 915 920

GCT GCA GAT CCT GCG CCA TCA AAG CCC AGA AGA GCA CAG AGG CCC AAT 2833 Ala Ala Asp Pro Gly Pro Ser Lys Pro Arg Arg Ala Gin Arg Pro Asn 925 930 935

TCT CTG GAT CTT TCA GCC ACA AAT ATC CTG GAT CGC AGC AGT ATA CAG 2881 Sor Leu Asp Leu Ser Ala Thr Asn He Leu Asp Gly Ser Ser He Gin

940 945 950 955

ATA GGT GAG TCA ACA CAA GAT GCC AAA TCA GGA TCA GGT GAA AAG ATC 2929 He Gly Glu Ser Thr Gin Asp Gly Lys Ser Gly Ser Gly Glu Lys He 960 965 970

AAG AGA CGT GTG AAA ACT CCA TAC TCT CTT AAG CGG TGG CGC CCG TCC 2977 Lys Arg Arg Val Lys Thr Pro Tyr Ser Leu Lys Arg Trp Arg Pro Ser 975 980 985

ACC TGG GTC ATC TCC ACC GAG CCG CTC GAC TGT GAG GTC AAC AAC AAT 3025 Thr Trp Val He Ser Thr Glu Pro Leu Asp Cys Glu Val Asn Asn Asn 990 995 1000 GCC ACT GAC ACC GCA CTC CAT TCT AAA TCT AGC ACT GCT GTG TAC CTT 3073 Gly Ser Asp Arg Aio Val Hia Sor Lys Ser Ser Thr Ala Val Tyr Leu 1005 1010 1015

GCA GAG CGA GGC ACT GCC ACG ACC ACA GTG TCT AAA GAT ATA GCA ATG 3121 Ale Glu Gly Gly Thr Ala Thr Thr Thr Val Ser Lys Asp He Gly Met 1020 1025 1030 1035

AAT TGT CTG TGAGATGTTT TCAAGCTTAT GGAGTGAAAT TATTTTTTTG 3170

Asn Cys Leu

CATCATTTAA ACATCCACAA GACATTTAAA AAAAAAACTG CTTTAACCTC CTGTCAGCAC 3230

CCCTTCCCAC CCCTGCAGCA ACGACTTGCT TTAAATAGAT TTCAGCTATG CAGAAAATTT 3290

TAGCTTATGC TTCCATAATT TTTAATTTTC TTTTTTAACT TTTCCACTTT TGTTTAGTCT 3350

TGCTAAAGTT ATATTTGTCT CTTATCACCA CATTATATGT GTGCTTATCC AAAGTCCTCT 3410 CCAAATATTT TTTTAAGAAA AAAGCCCAAA CAATGCATTG CTCATAATCA GTTTGGACCA 3470

TTTTCTAAAG GTCATTAAAA CAGAAGCAAA TTCACACC 3508

(2) INFORMATION FOR SEQ ID N0:β:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1038 aanno acids

(B) TYPE: oaino acid (D) TOPOLOGY: linear

(ii) NOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

Net Thr Ser Ser Leu His Arg Pro Phe Arg Val Pro Trp Leu Leu Trp 1 5 10 15

Ala Val Leu Lβu Vol Sor Thr Thr Ala Ala Ser Gin Asn Gin Glu Arg 20 25 30

Lou Cys Ala Phe Lys Asp Pro Tyr Gin Gin Asp Leu Gly He Gly Glu 35 40 45

Ser Arg He Ser His Glu Asn Gly Thr He Leu Cys Ser Lys Gly Ser

50 55 60

Thr Cys Tyr Gly Leu Trp Glu Lys Ser Lys Gly Asp He Asn Leu Val

65 70 75 80

Lya Gin Gly Cys Trp Ser His He Gly Asp Pro Gin Glu Cys His Tyr 85 90 95

Glu Glu Cys Val Val Thr Thr Thr Pro Pro Ser He Gin Asn Gly Thr 100 105 110

Tyr Arg Phe Cys Cys Cys Ser Thr Asp Leu Cys Asn Val Asn Phe Thr 115 120 125

Glu Asn Phe Pro Pro Pro Asp Thr Thr Pro Leu Ser Pro Pro His Ser 130 135 140

Phe Asn Arg Asp Glu Thr He He He Ala Leu Ala Ser Val Ser Val 145 150 155 160

Leu Ala Val Leu Ho Vol Ala Leu Cys Phe Gly Tyr Arg Met Leu Thr 165 170 175 Gly Asp Arg Lys Gin Gly Leu His Ser Net Asn Net Net Glu Ala Ala 180 185 190

Ala Ala Glu Pro Ser Leu Asp Leu Asp Asn Leu Lys Leu Leu Glu Leu 195 200 205

He Gly Arg Gly Arg Tyr Gly Ala Val Tyr Lys Gly Ser Leu Asp Glu 210 215 220

Arg Pro Val Ala Val Lys Vat Phe Ser Phe Ala Asn Arg Gin Asn Phe 225 230 235 240

He Asn Glu Lys Asn He Tyr Arg Val Pro Leu Net Glu His Asp Asn 245 250 255 He Ala Arg Phe He Val Gly Asp Glu Arg Leu Thr Ala Asp Gly Arg 260 265 270

Net Glu Tyr Leu Leu Val Net Glu Tyr Tyr Pro Asn Gly Ser Leu Cys 275 280 285

Lys Tyr Leu Sor Lou His Thr Ser Asp Trp Val Ser Ser Cys Arg Leu 290 295 300

Ala Hia Ser Val Thr Arg Gly Leu Ala Tyr Leu His Thr Glu Leu Pro 305 310 315 320

Arg Gly Aap Hia Tyr Lys Pro Ala Ha Sor Hia Arg Aap Leu Asn Ser 325 330 335 Arg Aan Val Leu Val Lys Asn Asp Gly Ala Cys Val He Ser Asp Phe 340 345 350

Gly Lou Sor Not Arg Lou Thr Gly Asn Arg Leu Vsl Arg Pro Gly Glu 355 360 365

Glu Asp Asn Ala Ala He Ser Glu Val Gly Thr He Arg Tyr Met Ala 370 375 380

Pro Glu Val Leu Clu Gly Ala Val Asn Leu Arg Asp Cys Glu Ser Ala 385 390 395 400

Leu Lya Gin Val Aap Not Tyr Ala Leu Gly Leu He Tyr Trp Glu Val 405 410 415 Ptto Not Arg Cys Thr Asp Lou Phe Pro Gly Glu Ser Val Pro Asp Tyr 420 425 430

Gin Net Ala Phe Cln Thr Glu Val Gly Asn His Pro Thr Phe Glu Asp 435 440 445

Net Gin Val Leu Val Ser Arg Glu Lys Gin Arg Pro Lys Phe Pro Glu 450 455 460

Als Trp Lys Glu Asn Ser Leu Ala Val Arg Ser Leu Lys Glu Thr He 465 470 475 480

Glu Asp Cys Trp Asp Gin Asp Ala Glu Ala Arg Leu Thr Ala Gin Cys 485 490 495

Ala Glu Glu Arg Met Ala Glu Leu Net Met He Trp Glu Arg Asn Lys 500 505 510

Ser Val Ser Pro Thr Val Asn Pro Met Ser Thr Ala Net Gin Asn Glu 515 520 525

Arg Asn Leu Ser His Asn Arg Arg Val Pro Lys He Gly Pro Tyr Pro 530 535 540

Asp Tyr Ser Ser Ser Ser Tyr He Glu Asp Ser He His His Thr Asp 545 550 555 560

Ser He Val Lys Asn He Ser Ser Glu His Ser Met Ser Ser Thr Pro 565 570 575 Leu Thr He Gly Glu Lys Asn Arg Asn Ser He Asn Tyr Glu Arg Gin

580 585 590

Gin Ala Gin Ala Arg He Pro Ser Pro Glu Thr Ser Val Thr Ser Leu 595 600 605

Ser Thr Aan Thr Thr Thr Thr Aan Thr Thr Gly Leu Thr Pro Ser Thr 610 615 620

Gly Met Thr Thr He Ser Glu Net Pro Tyr Pro Asp Glu Thr His Leu 625 630 635 640

His Aio Thr Asn Val Ala Gin Ser He Gly Pro Thr Pro Val Cys Leu 645 650 655 Gin Leu Thr Glu Glu Asp Leu Glu Thr Asn Lys Leu Asp Pro Lys Glu 660 665 670

Vol Asp Lys Asn Leu Lys Glu Ser Ser Asp Glu Asn Leu Net Glu His 675 680 685

Sor Lou Lys Gin Phe Sor Gly Pro Asp Pro Lou Ser Ser Thr Ser Ser 690 695 700

Sor Lou Leu Tyr Pro Lou He Lys Leu Ala Val Glu Val Thr Gly Gin 705 710 715 720

Cln Aap Phe Thr Gin Ala Ala Asn Gly Gin Ala Cys Leu He Pro Asp 725 730 735 Val Pro Pro Ala Cln He Tyr Pro Leu Pro Lys Gin Gin Asn Leu Pro 740 745 750

Lys Arg Pro Thr Ser Lβu Pro Leu Asn Thr Lys Asn Ser Thr Lys Glu 755 760 765

Pro Arg Leu Lys Phe Gly Asn Lys His Lys Ser Asn Leu Lys Gin Val 770 775 780

Glu Thr Gly Val Ala Lya Net Aan Thr He Asn Ala Ala Glu Pro His 785 790 795 800

Val Val Thr Val Thr Net Asn Gly Vsl Ala Gly Arg Ser His Asn Val 805 810 815 Aan Ser Hia Ala Ala Thr Thr Gin Tyr Ala Asn Gly Ala Val Pro Ala 820 825 830

Gly Gin Ala Ala Asn He Vol Ala Hia Arg Sor Gin Glu Met Leu Gin 835 840 845

Asn Gin Phe He Gly Glu Asp Thr Arg Leu Asn He Asn Ser Ser Pro 850 855 860

Asp Glu His Glu Pro Leu Leu Arg Arg Glu Gin Gin Ala Gly His Asp 865 870 875 880

Glu Gly Val Leu Asp Arg Leu Val Asp Arg Arg Glu Arg Pro Leu Glu 885 890 895

Gly Gly Arg Thr Asn Ser Asn Asn Asn Asn Ser Asn Pro Cys Ser Glu 900 905 910

Gin Asp He Leu Thr Gin Gly Val Thr Ser Thr Ala Ala Asp Pro Gly 915 920 925

Pro Ser Lys Pro Arg Arg Ala Gin Arg Pro Asn Ser Leu Asp Leu Ser 930 935 940

Ala Thr Asn He Leu Asp Gly Ser Ser He Gin He Gly Glu Ser Thr 945 950 955 960

Cln Asp Gly Lys Ser Gly Ser Gly Glu Lys He Lys Arg Arg Val Lys 965 970 975 Thr Pro Tyr Sor Leu Lys Arg Trp Arg Pro Ser Thr Trp Val Ile Ser 980 985 990

Thr Glu Pro Leu Asp Cys Glu Val Asn Asn Asn Gly Ser Asp Arg Ala 995 1000 1005

Val His Sor Lys Ser Ser Thr Ala Val Tyr Leu Ala Glu Gly Gly Thr 1010 1015 1020

Ala Thr Thr Thr Val Ser Lys Asp He Gly Net Asn Cys Leu 1025 1030 1035

(2) INFORMATION FOR SEQ ID NO:9: (1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 469 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(i ) FEATURE: (A) NAME/KEY: CDS

(8) LOCATION: 17..69

(X<) SEQUENCE DESCRIPTION: SEQ ID NO:9:

CTTTGCTCCC CCAGCG ATC ACT TCC TCG CTG CAT CCG CCC TTT CGG GTG 49 Mot Thr Ser Sor Leu Hia Arg Pro Phe Arg Val 1 5 10 CCC TGfi CTG CTA TGG GCC GTC CTG CTG GTC AGC ACT ACG GCT GCT TCT 97

Pro Trp Leu Leu Trp Ala Val Leu Leu Val Ser Thr Thr Ala Ala Ser

15 20 2S

CAG AAT CAA GAA CGG CTG TGT GCA TTT AAA GAT CCA TAT CAA CAA GAT 145 Cln Aan Cln Glu Arg Leu Cys Ala Phe Lys Asp Pro Tyr Gin Gin Asp

30 35 40

CTT GGG ATA GGT GAC AGT CGA ATC TCT CAT GAA AAT GGG ACA ATA TTA 193

Lou Cly He Cly Glu Sor Arg He Ser His Glu Asn Gly Thr He Leu 45 50 55

TGT TCC AAA GGG ACC ACC TGT TAT GCT CTC TGG GAG AAA TCA AAA CCG 241 Cys Sor Lys Gly Sor Thr Cys Tyr Gly Leu Trp Glu Lys Ser Lys Gly

60 65 70 75

GAC ATC AAT CTT GTG AAA CAA GGA TGT TGG TCT CAC ATC GGT GAT CCC 289 Asp He Asn Leu Val Lys Gin Gly Cys Trp Ser His He Gly Asp Pro 80 85 90

CAA GAG TGC CAC TAT GAA GAG TGT GTA GTA ACT ACC ACC CCA CCC TCA 337 Gin Glu Cys His Tyr Glu Glu Cys Val Val Thr Thr Thr Pro Pro Ser 95 100 105

ATT CAG AAT GGA ACG TAC CGC TTT TGC TGC TGT AGT ACA GAT TTA TGT 385 He Gin Asn Gly Thr Tyr Arg Phe Cys Cys Cys Ser Thr Asp Leu Cys 110 115 120

AAT CTC AAC TTT ACT GAG AAC TTT CCA CCC CCT GAC ACA ACA CCA CTC 433 Asn Vel Asn Phe Thr Glu Asn Phe Pro Pro Pro Asp Thr Thr Pro Leu 125 130 135

AGT CCA CCT CAT TCA TTT AAT CGA GAT GAA ACG TG 469 Ser Pro Pro His Ser Phe Asn Arg Asp Glu Thr 140 145 150

(2) INFORMATION FOR SEQ ID NO:10:

(i) SEQUENCE CHARACTERISTICS: (A) LEHGTH: 150 aarino acids

(B) TYPE: aaino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

Mot Thr Ser Ser Lou His Arg Pro Phe Arg Val Pro Trp Leu Leu Trp 1 5 10 15

Ala Val Leu Leu Val Ser Thr Thr Ala Ala Ser Gin Asn Gin Glu Arg 20 25 30

Leu Cys Ala Phe Lys Asp Pro Tyr Gin Gin Asp Leu Gly He Gly Glu 35 40 45

Sor Arg He Ser His Glu Asn Gly Thr He Leu Cys Ser Lys Gly Ser 50 55 60 Thr Cys Tyr Gly Lou Trp Glu Lys Sor Lys Gly Asp He Asn Leu Val 65 70 75 80

Lys Gin Gly Cys Trp Sor His Ho Gly Asp Pro Gin Glu Cys His Tyr 85 90 95

Glu Clu Cys Val Val Thr Thr Thr Pro Pro Ser 1le Gin Asn Gly Thr 100 105 110

Tyr Arg Phe Cys Cys Cys Ser Thr Asp Leu Cys Asn Vsl Asn Phe Thr 115 120 125

Glu Aan Phe Pro Pro Pro Asp Thr Thr Pro Leu Ser Pro Pro His Ser 130 135 140 Phe Aen Arg Aap Clu Thr 145 150

(2) INFORMATION FOR SEQ ID NO:11:

(1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2402 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double (0) TOPOLOGY: linear

(ix) FEATURE:

(A) NAME/KEY: CDS (B) LOCATION: join(11..1609)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: GAATCAGACA ATG ACT CAG CTA TAC ACT TAC ATC AGA TTA CTG GGA GCC 49 Net Thr Gin Leu Tyr Thr Tyr He Arg Leu Leu Gly Ala 1 5 10

TGT CTG TTC ATC ATT TCT CAT GTT CAA GGG CAG AAT CTA GAT AGT ATG 97 Cys Leu Phe He He Ser His Val Gin Gly Gin Asn Leu Asp Ser Met 15 20 25

CTC CAT GGC ACT GGT ATG AAA TCA GAC TTG GAC CAG AAG AAG CCA GAA 145 Leu His Gly Thr Gly Net Lys Ser Asp Leu Asp Gin Lys Lys Pro Glu 30 35 40 45

AAT GGA GTG ACT TTA GCA CCA GAG GAT ACC TTG CCT TTC TTA AAG TGC 193 Asn Gly Val Thr Leu Ala Pro Glu Asp Thr Leu Pro Phe Leu Lys Cys 50 55 60

TAT TGC TCA GGA CAC TGC CCA GAT GAT GCT ATT AAT AAC ACA TGC ATA 241 Tyr Cys Sor Gly His Cys Pro Asp Asp Ala He Asn Asn Thr Cys He 65 70 75 ACT AAT GGC CAT TGC TTT GCC ATT ATA CAA GAA GAT GAT CAG GGA GAA 289 Thr Asn Gly His Cys Phe Ala He He Glu Glu Asp Asp Gin Gly Glu 80 85 90

ACC ACA TTA ACT TCT CCG TGT ATG AAG TAT GAA GGC TCT GAT TTT CAA 337 Thr Thr Leu Thr Ser Gly Cys Not Lys Tyr Glu Gly Ser Asp Phe Gin 95 100 105

TGC AAG GAT TCA CCG AAA GCC CAG CTA CGC AGG ACA ATA GAA TGT TGT 385 Cys Lys Asp Ser Pro Lys Ala Gin Leu Arg Arg Thr He Glu Cys Cys 110 115 120 125

CGG ACC AAT TTG TGC AAC CAG TAT TTG CAG CCT ACA CTG CCC CCT GTT 433 Arg Thr Asn Leu Cys Asn Gin Tyr Leu Gin Pro Thr Leu Pro Pro Vsl 130 135 140

GTT ATA GGT CCG TTC TTT GAT GGC AGC ATC CGA TGG CTG GTT GTG CTC 481 Val He Gly Pro Phe Phe Asp Gly Sor Ho Arg Trp Leu Vel Val Leu 145 150 155 ATT TCC ATC GCT GTC TGT ATA CTT GCT ATG ATC ATC TTC TCC AGC TGC 529 Ho Sor Not Ala Val Cys Ho Val Ala Not He He Phe Ser Ser Cys 160 165 170

TTT TCC TAT AAG CAT TAT TGT AAG AGT ATC TCA AGC AGG GGT CGT TAC 577 Pho Cys Tyr Lys His Tyr Cys Lys Sor He Ser Sor Arg Gly Arg Tyr 175 180 185

AAC CGT GAT TTG GAA CAC GAT CAA GCA TTT ATT CCA GTA GGA GAA TCA 625 Aan Arg Aap Leu Glu Cln Aap Clu Ala Pho Ho Pro Val Gly Glu Ser 190 195 200 205

TTG AAA GAC CTC ATT GAC CAC TCC CAA AGC TCT GCG ACT CGA TCT GCA 673 Lou Lys Asp Law He Aap Gin Sor Gin Ser Ser Gly Sor Gly Ser Gly 210 215 220

TTG CCT TTA TTG GTT CAG CCA ACT ATT CCC AAA CAG ATT CAG ATG GTT 721 Lou Pro Leu Leu Val Cln Arg Thr He Ala Lys Gin He Gin Net Val 225 230 235 CGC CAC GTT GGT AAA GCC CGC TAT GCA GAA GTA TGG ATG GGT AAA TGG 769 Arg Gin Val Gly Lys Gly Arg Tyr Gly Glu Vsl Trp Met Gly Lys Trp 240 245 250

CGT CCT CAA AAA CTC CCT CTC AAA CTG TTT TTT ACC ACT GAA GAA GCT 817 Arg Gly Glu Lys Val Ala Val Lya Val Phe Phe Thr Thr Glu Glu Ala

2S5 260 265

ACC TGC TTT ACA GAA ACA GAA ATC TAC CAG ACG GTG TTA ATG CGT CAT 865

Sor Trp Pho Arg Glu Thr Glu He Tyr Cln Thr Val Leu Net Arg His 270 275 280 285

GAA AAT ATA CTT GGT TTT ATA GCT GCA GAC ATT AAA GGC ACT GGT TCC 913

Glu Aan He Leu Gly Phe He Ala Als Asp He Lys Gly Thr Gly Ser

290 295 300

TGG ACT CAG CTG TAT TTG ATT ACT GAT TAC CAT GAA AAT GGA TCT CTC 961

Trp Thr Gin Leu Tyr Leu He Thr Asp Tyr His Glu Asn Gly Ser Leu 305 310 315 TAT GAC TTC CTG AAA TGT GCC ACA CTA GAC ACC AGA GCC CTA CTC AAG 1009

Tyr Aap Phe Leu Lys Cys Ale Thr Leu Asp Thr Arg Ala Leu Leu Lys 320 325 330

TTA GCT TAT TCT GCT CCT TGT GGT CTG TGC CAC CTC CAC ACA GAA ATT 1057 Leu Ala Tyr Ser Ala Ala Cys Gly Leu Cys His Leu His Thr Glu He

335 340 345

TAT GGT ACC CAA CGG AAG CCT CCA ATT GCT CAT CGA GAC CTG AAG AGC 1105

Tyr Gly Thr Gin Gly Lys Pro Ale He Ala His Arg Asp Leu Lys Ser 350 355 360 365

AAA AAC ATC CTT ATT AAG AAA AAT GGA AGT TGC TGT ATT GCT GAC CTG 1153

Lys Asn He Leu He Lys Lys Asn Gly Ser Cys Cys He Als Asp Leu

370 375 380

GCC CTA GCT GTT AAA TTC AAC AGT GAT ACA AAT GAA GTT GAC ATA CCC 1201 Gly Leu Ala Val Lys Phe Asn Ser Asp Thr Asn Glu Val Asp He Pro 385 390 395 TTC AAT ACC AGG GTG GGC ACC AAC CGG TAC ATG GCT CCA GAA CTG CTG 1249 Leu Aan Thr Arg Val Gly Thr Lys Arg Tyr Net Als Pro Glu Val Leu 400 405 410

GAT CAA AGC CTG AAT AAA AAC CAT TTC CAG CCC TAC ATC ATG CCT GAC 1297 Asp Glu Ser Leu Asn Lys Asn His Phe Gin Pro Tyr He Net Ala Asp 415 420 425

ATC TAT AGC TTT GCT TTG ATC ATT TGG GAA ATG GCT CGT CGT TGT ATT 1345 He Tyr Ser Phe Gly Leu He He Trp Glu Not Ala Arg Arg Cys He 430 435 440 445

ACA CGA GGA ATC GTG GAC GAA TAT CAA TTA CCA TAT TAC AAC ATG CTG 1393 Thr Gly Gly He Val Glu Glu Tyr Gin Leu Pro Tyr Tyr Asn Net Vsl 450 455 460

CCC ACT GAC CCA TCC TAT GAC GAC ATC CGT GAG GTT GTG TGT GTG AAA 1441 Pro Sor Asp Pro Sor Tyr Glu Asp Not Arg Glu Val Val Cys Val Lys 465 470 475 CCC TTC CGC CCA ATC GTC TCT AAC CCC TGG AAC AGC GAT GAA TGT CTT 1489 Arg Leu Arg Pro Ho Vol Sor Aan Arg Trp Asn Ser Asp Glu Cys Leu 480 485 490

CGA GCA GTT TTC AAC CTA ATG TCA GAA TGT TGG CCC CAT AAT CCA GCC 1537 Arg Ala Val Lβu Lys Leu Net Ser Glu Cys Trp Als His Asn Pro Ala 495 500 SOS

TCC AGA CTC ACA CCT TTG AGA ATC AAG AAC ACA CTT GCA AAA ATG GTT 1585 Sor Arg Leu Thr Ale Lou Arg Ile Lys Lys Thr Leu Ala Lys Net Vel 510 515 520 525

GAA TCC CAC GAT GTA AAG ATT TGACAATTAA ACAATTTTGA GCGACAATTT 1636 Glu Sor Gin Asp Val Lys Ho

AGACTGCAAG AACTTCTTCA CCCAAGGAAT GGGTCCCATT AGCATGGAAT AGCATGTTGA 1696

CTTGGTTTCC AGACTCCTTC CTCTACATCT TCACACGCTG CTAACAGTAA ACCTTACCGC 1756 ACTCTACAGA ATACAAGATT GGAACTTGGA ACTTGGAACT TCAAACATGT CATTCTTTAT 1816

ATATGCACAG CTGTCTTTTA AATGTCCGCT TTTTGTCTTT TGCTTTCTTT CTTTTGTTTT 1876

CGTTTTGATC CTTTTTTGCT TTTTATCAAC TGCATCAAGA CTCCAATCCT GATAAGAAGT 1936

CTCTG5TCAA CCTCTGGGTA CTCACTATCC TGTCCATAAA GTGGTGCTTT CTGTGAAAGC 1996

CTTAAGAAAA TTAATGAGCT CAGCAGAGAT GGAAAAAGGC ATATTTGGCT TCTACCAGAG 2056 AAAACATCTG TCTGTGTTCT GTCTTTGTAA ACAGCCTATA GATTATGATC TCTTTGGGAT 2116

ACTGCCTGGC TTATGATGGT GCACCATACC TTTGATATAC ATACCAGAAT TCTCTCCTGC 2176

CCTAGGGCTA AGAAGACAAG AATGTAGAGG TTGCACAGGA GGTATTTTGT GACCAGTGGT 2236

TTAAATTGCA ATATCTAGTT CGCAATCGCC AATTTCATAA AAGCCATCCA CCTTGTAGCT 2296

GTAGTAACTT CTCCACTGAC TTTATTTTTA GCATAATAGT TGTGAAGGCC AAACTCCATG 2356 TAAAGTGTCC ATAGACTTGG ACTGTTTTCC CCCAGCTCTG ATTACC 2402

(2) INFORMATION FOR SEQ ID NO:12: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 532 aaiino acids

(B) TYPE: aaiino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:

Not Thr Gin Leu Tyr Thr Tyr He Arg Leu Leu Gly Ala Cys Leu Phe 1 5 10 15

He Ho Ser His Vsl Gin Gly Gin Asn Leu Asp Ser Net Leu His Gly 20 25 30 Thr Gly Net Lys Ser Asp Leu Asp Gin Lys Lys Pro Glu Asn Gly Val 35 40 45

Thr Leu Ala Pro Glu Asp Thr Leu Pro Phe Leu Lys Cys Tyr Cys Ser 50 55 60

Gly His Cys Pro Asp Asp Ala He Asn Asn Thr Cys He Thr Asn Gly 65 70 75 80

His Cys Phe Ala He He Glu Glu Asp Asp Gin Gly Glu Thr Thr Leu 85 90 95

Thr Ser Gly Cys Net Lys Tyr Glu Gly Ser Asp Phe Gin Cys Lys Asp 100 105 110 Sor Pro Lys Ala Gin Lβu Arg Arg Thr He Glu Cys Cys Arg Thr Asn 115 120 125

Lau Cys Aan Gin Tyr Leu Gin Pro Thr Leu Pro Pro Val Val He Gly 130 135 140

Pro Pho Pho Asp Gly sor Ho Arg Trp Leu Val Val Lau He Ser Net 145 150 155 160

Ala Val Cys He Val Ala Net He He Phe Ser Ser Cys Phe Cys Tyr 165 170 175

Lys His Tyr Cys Lys Ser He Ser Ser Arg Gly Arg Tyr Asn Arg Asp 180 185 190 Lou Glu Gin Asp Glu Ala Phe He Pro Val Gly Glu Ser Leu Lys Asp 195 200 205

Lou Ho Asp Gin Sor Cln Sor Sor Gly Sor Gly Sor Gly Leu Pro Leu 210 215 220

Leu Vol Cln Arg Thr Ho Ala Lys Gin He Gin Net Val Arg Gin Val

225 230 235 240

Gly Lys Gly Arg Tyr Cly Clu Val Trp Not Gly Lya Trp Arg Gly Glu 245 250 255

Lys Val Ala Val Lys Vsl Phe Phe Thr Thr Glu Glu Ala Ser Trp Phe 260 265 270

Arg Glu Thr Glu He Tyr Gin Thr Val Leu Net Arg His Glu Asn He 275 280 285

Leu Gly Phe He Als Ala Asp He Lys Gly Thr Gly Ser Trp Thr Gin 290 295 300

Leu Tyr Leu He Thr Asp Tyr His Glu Asn Gly Ser Leu Tyr Asp Phe 305 310 315 320

Leu Lys Cys Ale Thr Leu Asp Thr Arg Ala Leu Leu Lys Leu Ala Tyr 325 330 335

Ser Ala Ala Cya Gly Leu Cys His Leu His Thr Glu He Tyr Gly Thr 340 345 350 Gin Gly Lys Pro Ala He Ala His Arg Asp Leu Lys Ser Lys Asn He 355 360 365

Leu He Lys Lys Asn Gly Ser Cys Cys He Ala Asp Leu Gly Leu Ala 370 375 380

Val Lys Phe Asn Ser Asp Thr Asn Glu Val Asp He Pro Leu Asn Thr 385 390 395 400

Arg Val Gly Thr Lys Arg Tyr Net Ala Pro Glu Val Leu Asp Glu Ser 405 410 415

Lau Aan Lys Asn His Phe Gin Pro Tyr He Net Ala Asp He Tyr Ser 420 425 430 Phe Gly Leu He He Trp Glu Not Ala Arg Arg Cys He Thr Gly Gly 435 440 445 lie Vel Glu Glu Tyr Gin Leu Pro Tyr Tyr Asn Net Vel Pro Ser Asp 450 455 460

Pro Sor Tyr Glu Asp Hot Arg Glu Vsl Val Cys Val Lys Arg Leu Arg 465 470 475 480

Pro He Val Sor Aan Arg Trp Aan Sor Asp Glu Cys Leu Arg Ale Vel 485 490 495

Leu Lys Leu Not Sor Clu Cys Trp Ala Hia Aan Pro Ala Ser Arg Leu 500 SOS 510 Thr Ala Lau Arg He Lys Lya Thr Lau Ala Lya Net Val Glu Ser Gin 515 520 525

Aap Val Lys Ile 530

(2) INFORMATION FOR SEQ ID NO:13:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2252 base pairs

(B) TYPE: nucleic acid

(C) STRAN0EDNESS: double

(D) TOPOLOGY: linoor (ii) MLECULE TYPE: CDNA

(ix) FEATURE:

(A) NAME/KEY: CDS (B) LOCATION: 355..1863

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: GTTTTCCACC ACACTCATCC TATAAATGCT CCACAACATG GAGAATGGTT TGGGTTGGAA 60

GTAGACTTAA AGACCATCTA TGTGTGGGGA TACCTCCCAC TAGATCAGGC TGCTCAGGGC 120

CCCATTCACC ACCTCCAGGG ACGGGGTAGC CACTGCTTCT CTGAGCAACC TGAGCAACTT 180

CCTCACAGTG AAGAGTTCCT CCTGTATCCG ACGGTGGAGT TCATTTCTTT TGTCCTTGGA 240

AGTTGAATAG CAGAAAGCGA CATTTCAGCT TTTCTTGATA AAGGTTACAT CCATTTTACT 300 TAGACTACAA GACGAAGATT TCTGAAAATT GAGATCTTTA GTTTTCTGGA CAAG ATG 357

Met

1

CCC TTG CTT AGC TCC AGC AAG TTG AGC ATG GAG AGC AGA AAA GAA GAT 405 Pro Leu Leu Ser Ser Ser Lys Leu Ser Met Glu Ser Arg Lys Glu Asp 5 10 15

AGT GAG GGC ACA GCA CCT GCC CCT CCA CAG AAG AAG CTG TCA TGT CAG 453 Ser Glu Gly Thr Ala Pro Ala Pro Pro Gin Lys Lys Leu Ser Cys Gin 20 25 30

TCC CAC CAC CAT TGT CCT GAG GAC TCA GTC AAC ACC ACC TGC AGC ACT 501 Cys His His His Cys Pro Glu Asp Ser Val Asn Ser Thr Cys Ser Thr 35 40 45

GAT GGC TAC TGC TTC ACC ATA ATA CAA GAA GAT GAT TCT GGT GGA CAT 549

Asp Gly Tyr Cys Phe Thr He He Glu Glu Asp Asp Ser Gly Gly His

50 55 60 65 TTG CTC ACC AAA GGA TCT CTA GGA TTA GAG GGC TCG GAC TTC CAG TCT 597 Lou Val Thr Lys Gly Cys Leu Gly Leu Glu Gly Ser Asp Phe Gin Cys 70 75 80

CGG GAC ACT CCT ATT CCA CAC CAA AGA AGA TCT ATT GAA TGC TGC ACA 645 Arg Asp Thr Pro He Pro His Gin Arg Arg Ser He Glu Cys Cys Thr 85 90 95

CCC CAA GAT TAC TGT AAC AAA CAT CTT CAC CCA ACG CTG CCA CCA CTG 693 Gly Cln Asp Tyr Cys Asn Lys His Leu His Pro Thr Leu Pro Pro Leu 100 105 110

AAA AAT CGA GAC TTT GCT GAA GGA AAC ATT CAC CAT AAG GCC CTG CTG 741 Lys Asn Arg Asp Phe Ala Glu Gly Asn He His His Lys Ala Leu Leu 115 120 125

ATC TCG GTG ACT GTC TGT ACT ATA CTA CTG GTG CTT ATC ATC ATA TTC 789 He Ser Vel Thr Val Cys Ser He Lou Leu Val Leu He He He Phe 130 135 140 145 TGC TAC TTC AGC TAC AAG CGC CAA GAA CCC ACC CCC CGC TAC AGC ATC 837 Cys Tyr Phe Arg Tyr Lys Arg Gin Glu Ala Arg Pro Arg Tyr Ser He 150 155 160

CCC CTC GAG CAG GAC GAG ACC TAC ATT CCC CCT GGA GAA TCC CTG AAG 885 Cly Lou Clu Cln Aap Clu Thr Tyr Ho Pro Pro Cly Glu Ser Leu Lys 165 170 175

GAT CTG ATC GAG CAG TCC CAG AGC TCA CGC AGC GCC TCC GGG CTC CCT 933 Asp Lou He Glu Cln Ser Gin Ser Ser Gly Ser Gly Ser Gly Leu Pro 180 185 190

CTC CTG GTT CAA AGC ACC ATA CCA AAA CAG ATT CAG ATG GTA AAA CAG 981 Leu Leu Vsl Gin Arg Thr He Ala Lys Gin He Cln Met Val Lys Gin 195 200 205

ATT CGA AAA GGT CGC TAT GGC GAA GTC TGG ATG GGA AAG TGG CGT GGC 1029 He Cly Lys Gly Arg Tyr Gly Clu Val Trp Not Gly Lys Trp Arg Gly 210 215 220 225 GAA AAG CTA GCT CTC AAA GTG TTT TTT ACC ACG GAG GAG GCC AGC TGG 1077 Clu Lys Val Ala Val Lys Val Pho Pho Thr Thr Glu Clu Ala Ser Trp 230 235 240

TTC AGA GAA ACA GAA ATC TAC CAA ACT GTC CTG ATG AGG CAT GAA AAT 1125 Pho Arg Glu Thr Glu He Tyr Gin Thr Val Leu Net Arg His Glu Asn 245 250 255

ATT CTC GGA TTC ATT GCG GCA GAC ATT AAA GGC ACA GGC TCT TGG ACC 1173 He Leu Gly Phe He Ala Ala Asp He Lys Gly Thr Gly Ser Trp Thr 260 265 270

CAA CTG TAT CTC ATC ACT GAC TAT CAT GAG AAT GGC TCC CTT TAC GAT 1221 Gin Leu Tyr Leu He Thr Asp Tyr His Glu Asn Gly Ser Leu Tyr Asp 275 280 285

TAC CTA AAA TCC ACC ACC CTG GAC ACA AAA GGC ATG CTA AAA TTG GCT 1269 Tyr Leu Lys Ser Thr Thr Leu Asp Thr Lys Gly Net Leu Lys Leu Ala 290 295 300 305

TAC TCC TCT GTT AGT GGC TTG TGC CAC CTA CAT ACA GCG ATC TTC AGT 1317 Tyr Sor Ser Vel Ser Gly Leu Cys His Leu His Thr Gly He Phe Ser 310 315 320

ACC CAA CGC AAA CCG GCT ATT GCC CAC CGT GAT CTA AAA AGT AAG AAC 1365 Thr Gin Gly Lys Pro Ala He Ala His Arg Asp Leu Lys Ser Lys Asn 325 330 335 ATC CTG GTG AAA AAG AAC GGA ACC TGC TGT ATA GCA GAT TTG GGC TTG 1413 He Leu Val Lys Lys Asn Gly Thr Cys Cys He Ala Asp Leu Gly Leu 340 345 350

CCT GTT AAA TTT ATT ACT GAT ACA AAT GAG GTA GAC ATC CCT CCA AAC 1461 Ala Val Lys Phe He Sor Asp Thr Asn Glu Vel Asp He Pro Pro Asn 355 360 365

ACC CGC GTA GGA ACA AAA CCC TAT ATC CCT CCT GAG GTG CTG GAT GAA 1509 Thr Arg Vol Gly Thr Lys Arg Tyr Net Pro Pro Glu Val Leu Asp Glu 370 375 380 385

AGC TTC AAC AGA AAT CAC TTT CAG TCG TAC ATC ATG GCT GAT ATG TAC 1557 Sor Lou Asn Arg Asn His Phe Gin Ser Tyr He Net Als Asp Net Tyr 390 395 400

AGC TTT GGA CTC ATC CTT TGG CAG ATA GCC AGG AGA TGT GTG TCA GGA 1605 Ser Phe Gly Leu He Leu Trp Glu He Als Arg Arg Cys Val Ser Gly 405 410 415 GGA ATA GTG GAA GAA TAC CAG CTC CCA TAT CAC GAC CTT GTC CCC AGT 1653 Gly Ho Val Clu Glu Tyr Cln Lau Pro Tyr His Asp Leu Val Pro Ser 420 425 430

CAC CCC TCC TAC GAC GAC ATG ACC GAC ATT GTG TGC ATC AAA AGG CTA 1701 Aap Pro Sor Tyr Glu Aap Not Arg Glu lie Vol Cya He Lys Arg Leu 435 440 445

CGT CCT TCA TTC CCC AAC AGA TGG AGC AGC GAT GAG TGC CTG CGC CAG 1749 Arg Pro Sor Pho Pro Asn Arg Trp Ser Ser Asp Glu Cys Leu Arg Gin 450 455 460 465

ATG CCC AAC CTC ATC ATC GAG TGC TGG GCC CAT AAC CCT CCA TCC CGG 1797 Not Gly Lys Leu Net Net Glu Cys Trp Als His Asn Pro Ala Ser Arg 470 475 480

CTC ACA GCC CTA CGA GTC AAA AAA ACA CTT GCC AAA ATG TCA GAG TCG 1845 Lou Thr Ala Leu Arg Val Lys Lys Thr Leu Als Lys Net Ser Glu Ser 485 490 495 CAG CAC ATT AAG CTC TCATGCAGCA AAAACAGCTC CTTCTCGTGA AGACCCATGG 1900 Cln Aap He Lys Lou 500

AAACAGACTT TCTCTTCCAG GCAGAAGTCA TGGAGAGGTG CTGATAAGTA CCCTGAGTGC 1960

AGTCATATTT AAGAGCAACT CTTTGTTTGA CAGCTTTGAG GAGACTGTTC TTGGCAAAAT 2020

CAGCTCAATT TTGGCATGCA AGGTTCGGAG ACGCTTATCT GCCCTTGTTT ACACAGGGAT 2080 ATACAGTTTT AGTAACTGGT TTAAGCTTAT GCATGTTGCT TTCCGTCAAA GCCACTTATT 2140

ATTTTATTAT TATTGTTATT ATTATTATTT TGATTGTTTT AAAAGATACT GCTTTAAATT 2200 TTATGAAAAT AAAACCCTTT GGTTAGAAGA AAAAAAGATG TATATTGTTA CA 2252

(2) INFORMA ION FOR SEQ ID NO:14:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 502 amino acids

(B) TYPE: aerino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:

Net Pro Leu Leu Ser Ser Ser Lys Leu Ser Net Glu Ser Arg Lys Glu 1 5 10 15

Asp Ser Glu Gly Thr Ala Pro Ala Pro Pro Gin Lys Lys Leu Ser Cys 20 25 30

Gin Cys His His His Cys Pro Glu Asp Ser Val Asn Ser Thr Cys Ser 35 40 45

Thr Asp Gly Tyr Cys Phe Thr He He Glu Glu Asp Asp Ser Gly Gly 50 55 60 His Leu Val Thr Lys Gly Cys Leu Gly Leu Glu Gly Ser Asp Phe Gin 65 70 75 80

Cys Arg Asp Thr Pro He Pro His Gin Arg Arg Ser He Glu Cys Cys 85 90 95

Thr Gly Gin Asp Tyr Cys Asn Lys His Leu His Pro Thr Leu Pro Pro 100 105 110

Lou Lys Asn Arg Asp Phe Ala Glu Gly Asn He His His Lys Ala Leu 115 120 125

Leu He Ser Val Thr Val Cys Ser He Leu Leu Vel Leu lie He He 130 135 140 Pho Cys Tyr Pho Arg Tyr Lys Arg Gin Glu Ala Arg Pro Arg Tyr Ser 145 150 155 160

Ho Gly Lou Glu Gin Asp Glu Thr Tyr He Pro Pro Gly Glu Ser Leu 165 170 175

Lya Asp Leu Ho Glu Cln Ser Cln Ser Ser Gly Ser Gly Ser Gly Leu 180 185 190

Pro Lou Leu Val Gin Arg Thr He Ala Lys Gin He Gin Met Vsl Lys 195 200 205

Gin Ho Cly Lys Cly Arg Tyr Gly Glu Val Trp Net Gly Lys Trp Arg 210 215 220 Gly Glu Lys Val Ala Val Lys Val Phe Phe Thr Thr Glu Glu Ala Ser 225 230 235 240

Trp Pho Arg Glu Thr Glu He Tyr Gin Thr Val Leu Net Arg His Glu 245 250 255

Asn He Leu Gly Phe He Ala Ala Asp He Lys Gly Thr Gly Ser Trp 260 265 270

Thr Gin Leu Tyr Leu He Thr Asp Tyr His Glu Asn Gly Ser Leu Tyr 275 280 285

Asp Tyr Lou Lys Sor Thr Thr Lou Aap Thr Lys Gly Net Leu Lys Leu 290 295 300

Ala Tyr Ser Ser Val Ser Gly Leu Cys His Leu His Thr Gly He Phe

305 310 315 320

Sor Thr Gin Gly Lys Pro Ala He Ala His Arg Asp Leu Lys Ser Lys

325 330 335

Asn He Leu Vet Lys Lys Asn Gly Thr Cys Cys He Ala Asp Leu Gly 340 345 350 Leu Ala Val Lys Phe He Ser Asp Thr Asn Glu Val Asp He Pro Pro 355 360 365

Aan Thr Arg Val Gly Thr Lys Arg Tyr Net Pro Pro Glu Val Leu Asp 370 375 380

Glu Sor Leu Asn Arg Asn His Phe Gin Ser Tyr He Net Ala Asp Net 385 390 395 400

Tyr Sor Phe Gly Leu He Leu Trp Glu He Ala Arg Arg Cys Val Ser 405 410 415

Cly Gly He Val Glu Glu Tyr Gin Leu Pro Tyr His Asp Leu Vat Pro 420 425 430 Ser Aap Pro Ser Tyr Glu Asp Net Arg Glu He Val Cys He Lys Arg 435 440 US

Leu Arg Pro Ser Phe Pro Asn Arg Trp Ser Ser Asp Glu Cys Leu Arg

450 455 460

Gin Net Gly Lys Lou Not Net Glu Cys Trp Ala His Asn Pro Als Ser

465 470 475 480

Arg Lou Thr Ala Lau Arg Val Lys Lys Thr Leu Ala Lys Net Ser Glu 485 490 495

Sor Gin Asp He Lys Leu 500