IE83288B1

IE83288B1 - Domains of extracellular region of human platelet-derived growth factor receptor polypeptides

Info

Publication number: IE83288B1
Application number: IE1992/0317A
Authority: IE
Inventors: A. Escobedo Jaime; T. Williams Lewis; Wolf David; E. Tomlinson James; J. Fretto Larry; A. Giese Neill
Original assignee: Cor Therapeutics Inc
Filing date: 1992-01-30

Description

DOMAINS OF EXTRACELLULAR REGION OF HUMAN PLATELET-DERIVED GROWTH FACTOR RECEPTOR POLYPEPTIDES COR THERAPEUTICS, INC and THE REGENTS OF THE UNIVERSITY OF CALIFORNIA PATENT Attorney Docket No. 12418-14 DOMAINS OF EXTRACELLULAR REGION OF HUMAN PLATELET DERIVED GROWTH FACTOR RECEPTOR POLYPEPTIDES FIELD OF THE INVENTION The present invention relates to receptors for growth factors, particularly to human platelet-derived growth factor receptors (hPDGF-R). More particularly, it provides various composite constructs of human platelet-derived growth factor receptors, these constructs retaining ligand binding regions found in the natural extracellular region of the receptors. It also provides recombinant nucleic acids encoding these polypeptides, typically also comprising a promoter for expression, and fusion peptides on the amino or carboxy terminus of the expressed extracellular composite structure.

Antibodies are provided which recognize epitopes containing amino acids contained in different domains of the extracellular region. Cells comprising these polypeptides and nucleic acids, and diagnostic uses of these reagents are also provided.

BACKGROUND OF THE INVENTION Polypeptide growth factors are mitogens that act on cells by specifically binding to receptors located on the cell plasma membrane. The platelet-derived growth factor (PDGF) stimulates a diverse group of biochemical responses, e.g., changes in ion fluxes, activation of various kinases, alteration of cell shape, transcription of various genes, and modulation of enzymatic activities associated with phospholipid metabolism. e.g., Bell et al. (1989) "Effects of Platelet Factors on Migration of Cultured Bovine Aortic Endothelial and Smooth Muscle Cells," Circulation Research 65:l057—l065.

See, Platelet—derived growth factors are found in higher animals, particularly in warm blooded animals, e.g., mammals.

In vitro, PDGF is a major polypeptide mitogen in serum for cells of mesenchymal origin such as fibroblasts, smooth muscle cells, and glial cells. In vivo, PDGF does not normally circulate freely in blood, but is stored in the alpha granules of circulating blood platelets. During blood clotting and platelet adhesion the granules are released, often at sites of injured blood vessels, thereby implicating PDGF in the repair of blood vessels. PDGF may stimulate migration of arterial smooth muscle cells from the medial to the intimal layer of the artery where the muscle cells may proliferate. This is likely to be an early response to injury.

PDGF has also been implicated in wound healing, in atherosclerosis, in myeloproliferative disease, and in stimulating genes associated with cancerous transformation of cells, particularly c-myc and c-ﬁgs.

The platelet-derived growth factor is composed of two homologous polypeptide chains; it is a dimer of 16 kilodalton proteins which are disulfide connected. These polypeptides are of two types, the type B chain and the type A chain. Three forms of the growth factor dimer are found corresponding to a homodimer of two type A chains, a homodimer of two type B chains, and a heterodimer of the type A chain with the type B chain. Each of these three different combinations is referred to as a PDGF isoform. See, for a review on PDGF, Ross et al. (1986) "The Biology of Platelet—Derived Growth Factor," gel; 46:155-169. The growth factor sequences from mouse and human are highly homologous.

The PDGF acts by binding to the platelet-derived growth factor receptor (PDGF-R). The receptor is typically found on cells of mesenchymal origin. The functional receptor acts while in a form comprising of two transmembrane glycoproteins, each of which is about 180 kilodaltons. Two different polypeptides have been isolated, a type B receptor polypeptide and a type A receptor polypeptide.

A sequence of a type B receptor polypeptide of the mouse platelet-derived growth factor receptor polypeptide is published in Yarden et al. (1986) Nature 323:226-232. A sequence of an type A human platelet-derived growth factor receptor (hPDGF-R) polypeptide is disclosed in Matsui et al. (1989) Science 243: 800-803.

These PDGF receptors usually have three major identifiable regions. The first is a transmembrane region (TM) which spans the plasma membrane once, separating the regions of the receptor exterior to the cell from the regions interior to the cell. The second region is an extracellular region (XR) which contains the domains that bind the polypeptide growth factor (i.e., the ligand binding domains). The third is an intracellular region (IR) which possesses a tyrosine kinase activity. This tyrosine kinase domain is notable in having an insert of about 100 amino acids, as compared with most other receptor tyrosine kinase domains which are contiguous or have shorter insert segments.

The complete sequences of the human type B and human type A receptor polypeptides are reported elsewhere, e.g., U.S.S.N. 07/309,322, which is hereby incorporated herein by reference. However, for many purposes, a smaller or less than full length functional protein would be desired. For example, smaller molecules may be more easily targeted to areas of compromised circulation, or present fewer epitopes or extraneous domains unrelated to various activities of interest.

Functional analogues with a slightly modified spectrum of activity, or different specificity would be very useful.

Thus, the use of new composite constructs exhibiting biological activity in common with platelet-derived growth factor receptor polypeptides will have substantial use as research reagents, diagnostic reagents, and therapeutic reagents. In particular, the identification of important polypeptide features in the extracellular region of the platelet-derived growth factor receptor polypeptides will allow substitutions and deletions of particular features of the domains. Moreover, use of an in vitro assay system provides the ability to test cytotoxic or membrane disruptive compounds.

SUMARY OF THE INVENTION In accordance with the present invention, defined constructs of modified human platelet-derived growth factor receptor polypeptides are provided. Extracellular region domain structures are identified and modifications and combinatorial rearrangements of the receptor segments are furnished. Both cell bound and soluble forms of modified segments are made available, as are methods for assays using them, thereby allowing for screening of ligand analogues.

The present invention provides a platelet-derived growth factor receptor (hPDGF-R) fragment of between about 8 and 400 amino acids comprising one or more platelet-derived growth factor (PDGF) ligand binding regions (LBR's) from extracellular domains D1, D2, or D3, wherein the fragment binds a platelet-derived growth factor ligand. Generally, the fragment will exhibit a binding affinity of about 5 nM or better and will have a sequence of at least about 6 or 8 contiguous amino acids, preferably at least about 15 or more contiguous amino acids from a domain D3 intra-cysteine region.

The fragment will often lack a transmembrane region. In other embodiments, the fragment is soluble, is substantially pure, or has at least one ligand binding region derived from a domain D3. The fragment may be derived from a type B, or from a type A PDGF-R LBR fragment, e.g., from Table 1 or Table 2. In particular embodiments, the fragment is selected from the group of formulae consisting of: a) Xa—Dm—Xc; b) Xa-Dm-X1-Dn—Xc; c) Xa-Dm-Xl—Dn-X2-Dp-Xc; and d) Xa-Dm-X1-Dn-X2-Dp-X3-Dq-XC7 e) Xa-Dm-Xl—Dn-X2-Dp-X3-Dq-X4-Dr-Xc7 where the fragment is not D1-D2-D3-D4—DS: each of Xa, X1, X2, X3, and Xc is, if present, a polypeptide segment lacking a D domain; and each of Dm, Dn, Dp, and Dq is, independently of one another, selected from the group consisting of D1, D2, D3, D4, and D5. Preferred fragments are selected from the group consisting of: a) D1-D2-D3 or D3-D4-D5; and b) D1-D2-D3-D4 or D2-D3-D4-D5.

The present invention also embraces a soluble human platelet-derived growth factor receptor (hPDGF-R) fragment of between about 10 and 350 amino acids comprising at least one platelet-derived growth factor (PDGF) ligand binding region (LBR) from a domain D3, wherein the fragment specifically binds to a platelet-derived growth factor ligand. Usually the fragment comprises a sequence of at least about 15 contiguous amino acids from the intra-cysteine portion of domain D3 and has a binding affinity of better than about 5 nM. other useful fragment embodiments will be soluble, substantially pure, or a type B or type A PDGF—R LBR, e.g., from Table 1 or Table 2.

The invention also includes nucleic acid sequences, including those encoding the above described polypeptide fragments. Often the nucleic acid sequences incorporate a promoter, generally operably linked to the sequence encoding the fragments.

Cells comprising the nucleic acids or peptides of the invention are also embraced. In particular cell embodiments, the cell will be a mammalian cell, and often will contain both a nucleic acid and a protein expression product of the nucleic acid.

The compositions described above provide antibodies which recognize an epitope of a described PDGF—R fragment, but not a natural PDGF—R epitope. The antibody will often be a monoclonal antibody.

The present invention also provides a method for measuring the PDGF receptor binding activity of a biological sample comprising the steps of: a) contacting an aliquot of a sample to a PDGF ligand in the presence of a described PDGF-R fragment in a first analysis; b) contacting an aliquot of the sample to a PDGF ligand in the absence of the PDGF-R fragment in a second analysis; and c) comparing the amount of binding in the two analyses.

In some instances, the PDGF—R fragment is attached to a cell, or a solid substrate, e.g., a microtiter dish.

The invention also embraces a method for measuring the PDGF ligand content of a biological sample comprising the steps of: a) contacting an aliquot of the sample to a ligand binding region (LBR) in the presence of a described PDGF-R fragment in a first analysis: b) contacting an aliquot of the sample to a LBR in the absence of the PDGF-R fragment in a second analysis; and c) comparing the amount of binding in the two analyses.

In some embodiments, the contacting steps are performed simultaneously.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 illustrates a strategy for oligonucleotide directed in vitro deletion mutagenesis of soluble hPDGF—R extracellular domains. Many of these constructs will be soluble peptides, or can be modified to be such.

The abbreviations used are: PR = PDGF-R7 intact P = PDGF-R; extracellular region TM = transmembrane K = kinase S = signal sequence Fig. 2 illustrates the structure of a plasmid derived form pcDL-Sa296 used for expressing various deletion polypeptides.

Fig. 3 illustrates the structure of a plasmid pBJA derived from pcDLa296. See Takabe et al. (1988) Mol. Cell. mi 8:466-472.

. The pcDL-SRa296 is cut with XhoI.

. A polylinker (XhoI-Xbal-SfiI-NotI-EcoRI— EcoRV-HindIII—ClaI-SalI) is inserted into the XhoI cut vector.

. SalI is compatible with the XhoI site; and generates both a SalI and an XhoI site.

. The SV40 16s splice junction is no longer present.

Fig. 4 illustrates the inhibition of receptor phosphorylation by a human type B PDGF receptor polypeptide.

Labeling with a reagent which binds to phosphorylated tyrosine shows that phosphorylation activity is decreased in the presence of the receptor polypeptide fragment.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT I. General Description A. PDGF-R 1. structural features a. extracellular domain (XR) i. signal sequence ii. D domains (Ig-like) transmembrane segment (TM) c. intracellular domain (IR) i. tyrosine kinase ii. insert 2. function a. bind ligands (PDGF analogues) b. tyrosine kinase activity c. bind to PDGF-R peptide (dimer formation) d. phosphorylated segments B. Physiological Functions 1. cellular 2. tissue differentiation 3. organismal II. Polypeptides A. D domains . ﬂ-sheet strands . cysteine residues B. Soluble Forms, extracellular region C. Truncated/Deletion Forms D. Fusion Proteins E. Genetic Variants (site-directed mutagenized) F Compositions Comprising Proteins Nucleic Acids III.

A. Isolated Nucleic Acids B. Recombinant Nucleic Acids C. Compositions Comprising Nucleic Acids IV. Methods for Making PDGF-R Constructs A. Protein Purification _ 1. affinity with derivatized PDGF 2. various ligands, same receptor B. Expression of Nucleic Acids C. Synthetic methods V. Antibodies VI. Methods for Use A. Diagnostic B. Therapeutic I. General Description A. Platelet-derived growth factor receptor (PDGF-R) The human platelet-derived growth factor receptor (hPDGF—R) typically comprises two polypeptides. These polypeptides, which may be identical or only slightly different, associate during the functional activities of ligand binding and transducing of the ligand binding signal into the cell.

The platelet-derived growth factor receptor was identified as having a major component of an approximately 180 kilodalton protein which is glycosylated. This glycoprotein was identified as a platelet-derived growth factor receptor polypeptide. Primary structures of two homologous forms of polypeptides have been reported. A type B receptor nucleic acid and its corresponding polypeptide sequence from mouse are reported in Yarden et al. (1986) Nature 323: 226-232; and a homologous genetic sequence has been isolated from humans.

U.S.S.N. O7/309,322. reported in Matsui et al.

See A human type A receptor sequence is (1989) 800-803.

Although the two different forms of the receptor polypeptides are homologous, they are encoded by two separate genes.

Science 243: The functional receptor apparently involves a dimer of these polypeptides, either homodimers of the type B receptor polypeptide or of the type A receptor polypeptide, or a heterodimer of the type B receptor polypeptide with an type A receptor polypeptide. The specificity of binding of each of these forms of the receptor is different for each of the different forms of platelet-derived growth factor (PDGF), the AA, BB, or AB forms (from either mouse or human, or presumably other mammals).

The PDGF-R is a member of a family of related Each of these receptor polypeptides has a hydrophobic membrane spanning region (TM for transmembrane), a large extracellular region (XR) with regularly spaced cystine residues, and a cytoplasmic intracellular region (IR) having intracellular tyrosine kinase activity. The XR of the PDGF-R has a predicted structure containing 5 E-strand-rich immunoglobulin (Ig)-like domains. receptors. See, e.g., Yarden et al. supra.

Nature 323:226-232.

Each of these Ig-like domains consists of about 100 amino acids, ranging more specifically from about 88 to about 114 amino acids, and, except for the fourth domain, contains regularly spaced cysteine residues. Many of the structural features of the various growth factor receptors are homologous, including the mouse and human versions of the PDGF—R. Thus, many of the structural features defined herein are shared with other related proteins. However, in most cases, the functional relationship to particular structural features is unknown.

The intracellular region (IR) is that segment of the PDGF-R which is carboxy proximal of the transmembrane (TM) segment. The intracellular region is characterized, in part, by the presence of a split tyrosine kinase structural domain.

In the human type B receptor polypeptide, the tyrosine kinase domain is about 244 amino acids with an insert of about 104 See Table 1. polypeptide, the domain is about 244 amino acids long with a kinase insert of about 103 amino acids. See Table 2.

Functionally, this domain is defined, in part, by its tyrosine kinase activity, typically modulated by ligand binding to binding sites found in the extracellular region, and appears to function in a dimer state. amino acids. In the human type A receptor The substrate for phosphorylation includes various tyrosine residues on the accompanying receptor polypeptide chain, and other proteins which associate with the receptor. The tyrosine kinase domain is also defined, in part, by its homology to similar domains in other tyrosine kinase activity containing proteins. (1986) Each IR segment of the dimerized receptor complex appears to phosphorylate specific tyrosine residues on the other polypeptide chain.

See, e.g., Yarden et al.

Each transmembrane segment of the human receptor polypeptides is about 24 or 25 amino acids long and is characterized by hydrophobic amino acid residues. These segments have sequences characteristic of membrane spanning segments. In the human type B receptor polypeptide the transmembrane region appears about 25 amino acids long extending from about val(500) to trp(524), while in the human type A receptor polypeptide, the transmembrane segment appears to be about 24 amino acids extending from about leu(502) to trp(526). Claesson-Welsh et al.

Acad. Sci. USA, 86:49l7-4921.

See, e.g., (1989) Proc. Nat'l A polypeptide or nucleic acid is a "human" sequence if it is derived from, or originated in part from, a natural human source. For example, proteins derived from human cells, or originally encoded by a human genetic sequence, will be human proteins. A sequence is also human if it is selected on the basis of its high similarity to a sequence found in a natural human sample, or is derived therefrom.

A fusion polypeptide or nucleic acid is a molecule which results from the fusion of segments from sequences which are not naturally in continuity with one another. Thus, a chimeric protein or nucleic acid is a fusion molecule. A heterologous protein is a protein originating from a different source .

B. Physiological Functions The PDGF-R appears to have at least four major different biological functions. The first is the binding of ligands, usually the PDGF mitogenic proteins or their analogues. These ligands and analogues may also serve as either agonists or antagonists. The ligand binding sites, made up of ligand binding regions (LBR's), are localized in the extracellular region (XR). The functional receptor transduces a signal in response to ligand binding, and the resulting response is a ligand modulated activity. As the likely ligand is a PDGF, or an analogue, the signal will ordinarily be PDGF modulated.

A second biological activity relates to the tyrosine kinase enzymatic activity. This activity is typically activated intracellularly in response to ligand binding.

However, since these receptors apparently function in a dimeric state, the interchain binding interactions may be considered a third biological activity which may be mediated by blocking agents. Blocking or interference with the dimerization interactions may be mediated by receptor protein fragments, particularly in the functional ligand binding or tyrosine kinase activities. Thus, the introduction of analogues of the receptor domains to natural or other receptor polypeptides may serve as an additional means to affect PDGF mediation of ligand mediated activities.

The fourth function of the PDGF receptor is as a binding substrate for other proteins, e.g., the PI3 kinase. In particular, the PDGF receptor is phosphorylated at various positions in response to ligand binding or other events. This binding interaction activates an enzymatic activity on the part of the binding protein which activates further cellular or metabolic responses.

The term "ligand" refers to the molecules, usually members of the platelet-derived growth factor family, that are bound by the ligand binding regions (LBR's). regions are typically found in the XR.

The binding Also, a ligand is a molecule that serves either as the natural ligand to which the receptor binds, or a functional analogue of a ligand. The Typically ligands will be molecules which share structural features of analogue may serve as an agonist or antagonist. natural PDGF, e.g., polypeptides having similar amino acid sequences or other molecules sharing molecular features with a ligand. The determination of whether a molecule serves as a ligand depends upon the measurement of a parameter or response which changes upon binding of that ligand, such as dimerization or tyrosine kinase activity. e.g., (eds) (1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics, 8th Ed., Pergamon Press, which is incorporated herein by reference.

See, Gilman et al.

The receptor has ligand binding regions (LBR), or regions which are important in determining both affinity and specificity of binding of ligand, e.g., PDGF and its analogues.

The ligand binding regions determine the binding interactions between the receptors and ligand. Typically, these regions are those contact points between the ligand molecule and the receptor. These molecular interactions can be determined by crystallographic techniques, or by testing which regions of the receptor are important in ligand interaction. Various segments of the extracellular region of the PDGF receptor make up the ligand binding regions, while other segments form structural segments which spatially orient the LBR's in proper arrangement to properly bind the ligands.

Generally, the fragment will have a sequence of at least about 6 contiguous amino acids, usually at least about 8 contiguous amino acids, more usually at least about 10 A contiguous amino acids, preferably at least about 13 contiguous amino acids, and more preferably at least about 15 or more contiguous amino acids. Usually, the LBR's will be located within the intra-cysteine (or equivalent) residues of each Ig- like domain, e.g., domains D1, D2, D3, D4, and D5. They will be preferably derived from D3 sequences, but D1 and D2 derived sequences will also be common. Occasionally, sequences from D4, D5, or other proteins will provide LBR function.

The extra-cysteine (or equivalent) regions provide structural functions, as will inter-domain spacer segments.

The intra-cysteine portions, or segments, are indicated in Tables 4 and 5, and comprise the segments designated C, C‘, C", D, and E, along with portions of the B and F segments, as indicated. The extra-cysteine residues comprise the segments designated A and G, and portions of B and F.

The ligand binding regions as defined, in part, by the importance of their presence, or their effect on the affinity of PDGF ligand binding. The natural, native full length PDGF—R binds with a Kd of about 0.2 mM. e.g., et al. (1991) J. Biol. Chem. 266:413-418, which is hereby incorporated herein by reference.

See, Duan An LBR is a segment of polypeptide whose presence significantly affects ligand binding, generally by at least about a factor of two, usually by at least about a factor of four, more usually by at least a factor of about eight, and preferably by at least about a factor of twelve or more. A fragment of this invention which binds to the PDGF ligand will generally bind with a Kd of less than about 10 uM, more generally less than about 1 pM, usually less than about 0.1 uM, more usually less than about 10 nM, preferably less than about 1 nM, and more preferably less than about 0.5 nM.

An epitope is an antigenic determinant which potentially or actually has elicited an antibody response. It may also refer to a structural feature which is defined by an antibody binding region, or its equivalent. An epitope need not necessarily be immunogenic, but will serve as a binding site for an antibody molecule or its equivalent.

II. Polypeptides Table 1 discloses the sequence of one allele of a type B human platelet-derived growth factor receptor polypeptide. Both a nucleic acid sequence and its corresponding protein sequence are provided. The nucleic acid sequence corresponds to Seq. ID No. 1. The amino acid sequence corresponds to Seq. ID No. 2. A homologous mouse sequence was reported in Yarden et al. (1988) Nature 323:226-232. The sequence of a mouse PDGF receptor polypeptide also exhibits structural features in common with the regions, the domains, and the 3-strand segments of the human receptor polypeptides.

The mouse polypeptides, and those from other related receptors, will serve as a source of similar domains, homologous B-strand segments, and inter-segment sequences, and sequences of homology for general replacement or substitutions.

IALE_1 Sequence of one type B human PDGF receptor polypeptide allele and protein TGTTCTCCTGAGCCTTCAGGAGCCTGCACCAGTCCTGCCTGTCCTTCTACTC AGCTGTTACCCACTCTGGGACCAGCAGTCTTTCTGATAACTGGGAGAGGGCAGTAAGGAGGACTTCC TGGAGGGGGTGACTGTCCAGAGCCTGGAACTGTGCCCACACCAGAAGCCATCAGCAGCAAGGACACC ATG CGG CTT CCG GGT Met Arg Leu Pro Gly GCG Ala ATG Met CCA GCT Pro Ala CTG Leu GCC Ala CTC Leu AAA Lys GGC Gly GAG Glu CTG Leu CTG Leu TTG Leu CTG TCT Leu Ser CTC Leu CTG TTA Leu Leu CTT Leu CTG Leu GAA Glu CCA CAG ATC Pro Gln Ile TCT CAG Ser Gln GGC Gly CTG GTC Leu Val GTC ACA Val Thr CCC Pro CCG Pro GGG Gly CCA GAG Pro Glu CTT Leu GTC Val CTC Leu AAT Asn GTC Val TCC Ser AGC Ser ACC Thr TTC Phe GTT Val CTG ACC Leu Thr TGC TCG Cys Ser CAG Gln GGT TCA GCT Gly Ser Ala CCG PIG GTG GTG Val Val TGG TYP GAA coo ATG Glu Arg Met TCC Ser CAG GAG Gln Glu CCC Pro CCA Pro GAA ATG Glu Met GCC Ala AAG Lys GCC Ala CAG GAT Gln Asp GGC Gly ACC Thr TTC Phe TCC Ser AGC Ser GTG Val CTC Leu ACA Thr CTG ACC Leu Thr AAC Asn CTC Leu ACT Thr GGG Gly CTA Leu GAC Asp ACG Thr GGA GAA TAC TTT Gly Glu Tyr Phe GAG CGG AAA CGG Glu Arg Lys Arg TGC ACC Cys Thr CAC His AAT GAC TCC Asn Asp CGT Ser Arg GGA Gly CTG Leu GAG ACC GAT Glu Thr Asp CTC TAC Leu Tyr ATC Ile TTT Phe GTG Val CCA GAT Pro Asp ccc Pro ACC GTG Thr Val GGC Gly TTC Phe CTC Leu CCT Pro AAT Asn GAT Asp GCC Ala GAG Glu GAA Glu CTA Leu TTC Phe ATC TTT Ile Phe CTC Leu ACG GAA ATA ACT Thr Glu Ile Thr GAG Glu ATC Ile ACC ATT Thr Ile CCA TGC Pro Cys GAC Asp CGA GTA ACA GAC Arg Val Thr Asp CCA Pro CAG Gln CTG Leu GTG Val GTG Val ACA Thr CTG Leu CAC His GAG Glu AAG Lys AAA Lys GGG Gly GTT Val GCA Ala CTG Leu CCT Pro GTC Val CCC TAT Pro Tyr TGC Cys GAT Asp CAC His CAA Gln CGT Arg GGC TTT TCT GGT ATC Gly Phe Ser Gly Ile TTT Phe GAG Glu GAC Asp AGA AGC Arg Ser TAC Tyr TAT GTC TAC Tyr Val Tyr ATC Ile AAA Lys ACC Thr ACC Thr ATT Ile GGG Gly GAC Asp AGG Arg GAG GTG Glu Val GAT Asp TCT Ser GAT Asp GCC Ala TAC Tyr GCA GTG Ala Val AGA Arg CTC Leu CAG GTG Gln Val TCA TCC ATC Ser Ser Ile AAC Asn GTC Val TCT Ser GTG Val AAC Asn CAG Gln ACT GTG Thr Val GTC Val CGC Arg CAG Gln GGT Gly GAG AAC Glu Asn ATC Ile ACC Thr CTC ATG Leu Met TGC Cys ATT Ile GTG ATC Val Ile GGG Gly AAT GAT Asn Asp GTG Val GTC Val AAC Asn TTC Phe GAG Glu TGG ACA Trp Thr TAC Tyr CCC Pro CGC Arg GAA AGT Glu Ser Lys Gly 186 -15 36 CGG Arg CGC Arg ACC Thr AAC Asn ACA Thr GAG Glu GGC Gly ACC Thr GGC Gly TTC Phe CAC His CAG Gln GAG Glu GAG Glu ACA Thr ccc Arg TTG Leu Table 1, page 2 CTG Leu TCC Ser TGC Cys ATC Ile CTA Leu GCC Ala GAC Asp cos Arg CAC His CAG Gln CCT PIO CCG Pro CTG Leu ACT Thr CTG Leu AAC Asn CCC Pro GTG Val ATC Ile AAT Asn ACC Thr CAA Gln TAC Tyr TCC Ser TAT Tyr TAC Tyr CTA Leu GAC Asp AAC Asn CCG Pro AAC Asn CGT Arg GCT Ala TTT Phe GAG Glu CTG Leu GTG Val GTG Val CCA Pro AGC Ser GTG Val ACC Thr CAG Gln AGT Ser ATC Ile CCC Pro GTG Val CTG Leu GTG Val AAG Lys CCG Pro CAC His ACG Thr GTT Val GCT Ala CCG Pro GCT Ala TCA Ser ATG Met ATC Ile GGG Gly ATC Ile ACG Thr ACG Thr CAG Gln GGC Gly GTG Val GTG Val ATC Ile GAG Glu GAG Glu GAG Glu CCC Pro GGC Gly GAG Glu CGG Arg AAT Asn GAA Glu TGG Trp CTG Leu TAC Tyr CAC His CAG Gln GTG Val ACT Thr CCC Pro AGT Ser AGC Ser CTG Leu ACT Thr GAA Glu CTG Leu GCC Ala GTC Val CAG Gln TCT Ser CTG Leu TGG Trp GTG Val GAC Asp GTG Val GAC Asp AGT Ser GTG Val GGC Gly CAT His GTC Val ATC Ile ACA Thr TTC Phe CCT Pro ACA Thr GCC Ala GGG Gly GAG Glu GAT Asp ACG Thr ATC Ile TTC Phe GCC Ala AAT Asn TAC Tyr cos Arg CTG Leu GCC Ala CTG Leu CAT His GTC Val GTC Val TGC Cys AAC Asn GAG Glu CGG Arg CAG Gln TCA Ser CTC Leu GAG Glu GAC Asp GTG Val AGC Ser TGG Trp CTG Leu GTT Val GAG Glu CGA Arg CGC Arg AGA Arg AGT Ser GAG Glu CCA Pro GAG Glu GCC Ala TTG Leu TTA Leu CAT His coo Arg CGG Arg TTC Phe TCC Ser CGC Arg GAT Asp GTG Val TGT Cys GAC Asp TCC Ser CAG Gln CTG Leu GTC Val ATC Ile GAT Asp GAA Glu CAG Gln CTC Leu ACA Thr ACG Thr GTG Val GCT Ala CTG Leu CGT Arg CTC Leu GAA Glu GAG Glu TCG Ser ATC Ile CTG Leu ATG Met GAC Asp GAT Asp CTG Leu CTG Leu GAC Asp CGC Arg AAG Lys GAG Glu GAG Glu GGC Gly Lys GAG Glu Phe GTG Val GTG Val GCC Ala CCT PIG TCG Ser GAA Glu GGA Gly CAG Gln AAC Asn AAC ASH GTG Val GTC Val CTA Leu CGG Arg AGG Arg GAG Glu GAG Glu CGC Arg GTG Val CTG Leu TAC Tyr GGG Gly AAG Lys GAG Glu GTA Val CGC Arg GTG Val GCA Ala CAG Gln AGT Ser GGC Gly TGT Cys AGC Ser GTG Val TGC Cys CCA Pro GTG Val CAC His ACC Thr GCC Ala GTG Val GTG Val ACC Thr TCG Ser GAG Glu CTC Leu GAG Glu ATG Met CCA PIG CAG Gln GTG Val ACG Thr CAC His GTG Val ATC Ile TAC Tyr ATC Ile GGC Gly TTC Phe CTG Leu GAG Glu GCT Ala TCC Ser AGC Ser CCG Pro CGT Arg CTG Leu AGC Ser CTG Leu TCC Ser CTC Leu ACC Thr TAC Tyr TAC Tyr CCG Pro CAG Gln GCC Ala GTG Val ACT Thr CAC His CTC Leu TTG Leu GTG Val ATC Ile CCT Pro TAC Tyr CTG Leu Table 1, page 3 ATC Ile GAG Glu ATC Ile CGG Arg GTG Val GTG Val Leu GTC Val GAG Glu ATC Ile ATC Ile TAC Tyr GAC Asp GTG Val GCC Ala ATG Met AAC ASH TAC Tyr ACC.TTC TAC Tyr ACC Thr GAC Asp GAG Glu GAG Glu ATG Met GCC Ala AGC Ser GGG Gly TAT Tyr TCC Ser AGG Arg GAC Asp TCC Ser TCC Ser CGA Arg GTG Val CAG Gln GAG Glu GTC Val TCG Ser CTG Leu TGC Cys CTG Leu AAT Asn GAG Glu GTG Val TCC Ser ACC Thr CTC Leu AAG Lys CTT Leu was Trp GAC Asp GCC Ala AAG Lys GAG Glu TTG Leu CGC Arg CAG Gln GCT Ala AGC Ser CCC Pro AAC Asn TGC Cys GTG Val AAC Asn ATC Ile AAG Lys CCC Pro GTG Val ACA Thr ATG Met CTG Leu GGG Gly TAC Tyr CAC His CTG Leu GAC Asp ATG Met TAC Tyr CGA Arg GGC Gly TGC Cys ATC Ile GTG Val ATG Met CTG Leu GCT Ala Leu AAG Lys GCC Ala GGA Gly CAC His CCC Pro GGT Gly CTG Leu ATG Met GCA Ala TTC Phe GTC Val CTC Leu ATT Ile CAG Gln GGA Gly CAT His Lys ATC Ile TGC Cys GAC Asp TCC Ser GTT Val GGC Gly GAC Asp GCC Ala ACT Thr AGC Ser CAC His ATC Ile GAG Glu CTG Leu CGC Arg GGT Gly TCC Ser ATG Met ACC Thr CTG Leu GAC Asp GGG Gly TAC Tyr ATG Met CCT Pro TTG Leu TAC Tyr ATG Met TCT Ser CCC PIO ACC Thr CTG Leu ACA Thr AGT Ser GTG Val AAG Lys CTC Leu ATG Met AAA Lys TAC Tyr ATC Ile CAG Gln GTG Val TAT Tyr CTC Leu AGC Ser GCC Ala CAC His GGA Gly GAC Asp CGC Arg CCC Pro GAC Asp GGA Gly GAT Asp AAC Asn GTG Val Arg Asp Leu TGG TIP AGC SE1’ GAC Asp ccc Gly CAT His CGC Arg GGA Gly TAC Tyr CGC Arg CTG Leu ATG Met GAC Asp AAC Asn GAG Glu GCC Ala GCG Ala CAG Gln TCT Ser TCC Ser TCT Ser TCT Ser AGC Ser CCC P170 CTG Leu CCG Pro CCC Pro AGC Ser GTC Val TAC Tyr TCT Ser AAT Asn GCT Ala AAG Lys GAC Asp ACG Thr GGG Gly CAG Gln AGT Ser CCC Pro ATC Ile CAC His CCC Pro AGC Ser AAG Lys AAA Lys GTT Val CCA Pro GGC Gly AGG Arg AAG Lys GGC Gly TGG Trp GCC Ala GCC Ala GAG Glu CAC His TAT Tyr CGC Arg AGC Ser CAT His GAC Asp TAT Tyr CCC Pro GTG Val ATG Met AAC Asn CCA Pro CAT His GAG Glu ACG Thr AAG Lys CTG Leu ATC Ile AAC Asn GCG Ala GTG Val GAG Glu GCA Ala TCT Ser CTA Leu GAG Glu GTG val CGT Arg GAG Glu CTG Leu GGG Gly ATG Met CAA Gln AAC Asn ATC Ile GAG Glu TCC Ser TCG Ser GAC Asp GCC Ala AGC Ser TTT Phe CTC Leu ' Table 1, page 4 ATC TGT GAA GGC AAG CTG GTC AAG ATC TGT GAC TTT GGC CTG GCT CGA GAC 2736 Ile Cys Glu Gly Lys Leu Val Lys Ile Cys Asp Phe Gly Leu Ala Arg Asp 818 ATC ATG CGG GAC TCG AAT TAC ATC TCC AAA GGC AGC ACC TTT TTG CCT TTA 2787 Ile Met Arg Asp Ser Asn Tyr Ile Ser Lys Gly Ser Thr Phe Leu Pro Leu 835 AAG TGG ATG GCT CCG GAG AGC ATC TTC AAC AGC CTC TAC ACC ACC CTG AGC 2838 Lys Trp Met Ala Pro Glu Ser Ile Phe Asn Ser Leu Tyr Thr Thr Leu Ser 852 GAC GTG TGG TCC TTC GGG ATC CTG CTC TGG GAG ATC TTC ACC TTG GGT GGC 2889 Asp Val Trp Ser Phe Gly Ile Leu Leu Trp Glu Ile Phe Thr Leu Gly Gly 869 ACC CCT TAC CCA GAG CTG CCC ATG AAC GAG CAG TTC TAC AAT GCC ATC AAA 2940 Thr Pro Tyr Pro Glu Leu Pro Met Asn Glu Gln Phe Tyr Asn Ala Ile Lys 886 CGG GGT TAC CGC ATG GCC CAG CCT GCC CAT GCC TCC GAC GAG ATC TAT GAG 2991 Arg Gly Tyr Arg Met Ala Gln Pro Ala His Ala Ser Asp Glu Ile Tyr Glu 903 ATC ATG CAG AAG TGC TGG GAA GAG AAG TTT GAG ATT CGG CCC CCC TTC TCC 3042 Ile Met Gln Lys Cys Trp Glu Glu Lys Phe Glu Ile Arg Pro Pro Phe Ser 920 CAG CTG GTG CTG CTT CTC GAG AGA CTG TTG GGC GAA GGT TAC AAA AAG AAG 3093 Gln Leu Val Leu Leu Leu Glu Arg Leu Leu Gly Glu Gly Tyr Lys Lys Lys 937 TAC CAG CAG GTG GAT GAG GAG TTT CTG AGG AGT GAC CAC CCA GCC ATC CTT 3144 Tyr Gln Gln Val Asp Glu Glu Phe Leu Arg Ser Asp His Pro Ala Ile Leu 954 CGG TCC_CAG GCC CGC TTG CCT GGG TTC CAT GGC CTC CGA TCT CCC CTG GAC 3195 Arg Ser Gln Ala Arg Leu Pro Gly Phe His Gly Leu Arg Ser Pro Leu Asp 971 ACC AGC TCC GTC CTC TAT ACT GCC GTG CAG CCC AAT GAG GGT GAC AAC GAC 3246 Thr Ser Ser Val Leu Tyr Thr Ala Val Gln Pro Asn Glu Gly Asp Asn Asp 989 TAT ATC ATC CCC CTG CCT GAC CCC AAA CCT GAG GTT GCT GAC GAG GGC CCA 3297 Tyr Ile Ile Pro Leu Pro Asp Pro Lys Pro Glu Val Ala Asp Glu Gly Pro 1005 CTG GAG GGT TCC CCC AGC CTA GCC AGC TCC ACC CTG AAT GAA GTC AAC Acc 3348 Leu Glu Gly Ser Pro Ser Leu Ala Ser Ser Thr Leu Asn Glu Val Asn Thr 1022 TCC TCA ACC ATC TCC TGT GAC AGC CCC CTG GAG CCC CAG GAC GAA CCA GAG 3399 Ser Ser Thr Ile Ser Cys Asp Ser Pro Leu Glu Pro Gln Asp Glu Pro Glu 1039 CCA GAG CCC CAG CTT GAG CTC CAG GTG GAG CCG GAG CCG GAG CTG GAA CAG 3450 Pro Glu Pro Gln Leu Glu Leu Gln Val Glu Pro Glu Pro Glu Leu Glu Gln 1056 TTG CCG GAT TCG GGG TGC CCT GCG CCT CGG GCG GAA GCA GAG GAT AGC TTC 3501 Leu Pro Asp Ser Gly Cys Pro Ala Pro Arg Ala Glu Ala Glu Asp Ser Phe 1073 CTG TAGGGGGCTGGCCCCTACCCTGCCCTGCCTGAAGCTCCCCCGCTGCCAGCACCCAGCATCTCC 3567 Leu 10Table 1, page 5 TGGCCTGGCCTGGCCGGGCTTCCTGTCAGCCAGGCTGCCCTTATCAGCTGTCCCCTTCTGGAAGCTT TCTGCTCCTGACGTGTTGTGCCCCAAACCCTGGGGCTGGCTTAGGAGGCAAGAAAACTGCAGGGGCC GTGACCAGCCCTCTGCCTCCAGGGAGGCCAACTGACTCTGAGCCAGGGTTCCCCCAGGGAACTCAGT TTTCCCATATGTAAGATGGGAAAGTTAGGCTTGATGACCCAGAATCTAGGATTCTCTCCCTGGCTGA CAGGTGGGGAGACCGAATCCCTCCCTGGGAAGATTCTTGGAGTTACTGAGGTGGTAAATTAACTTTT TTCTGTTCAGCCAGCTACCCCTCAAGGAATCATAGCTCTCTCCTCGCACTTTTATCCACCCAGGAGC TAGGGAAGAGACCCTAGCCTCCCTGGCTGCTGGCTGAGCTAGGGCCTAGCCTTGAGCAGTGTTGCCT CATCCAGAAGAAAGCCAGTCTCCTCCCTATGATGCCAGTCCCTGCGTTCCCTGGCCCGAGCTGGTCT GGGGCCATTAGGCAGCCTAATTAATGCTGGAGGCTGAGCCAAGTACAGGACACCCCCAGCCTGCAGC CCTTGCCCAGGGCACTTGGAGCACACGCAGCCATAGCAAGTGCCTGTGTCCCTGTCCTTCAGGCCCA TCAGTCCTGGGGCTTTTTCTTTATCACCCTCAGTCTTAATCCATCCACCAGAGTCTAGAAGGCCAGA CGGGCCCCGCATCTGTGATGAGAATGTAAATGTGCCAGTGTGGAGTGGCCACGTGTGTGTGCCAGAT ATGGCCCTGGCTCTGCATTGGACCTGCTATGAGGCTTTGGAGGAATCCCTCACCCTCTCTGGGCCTC AGTTTCCCCTTCAAAAAATGAATAAGTCGGACTTATTAACTCTGAGTGCCTTGCCAGCACTAACATT CTAGAGTATCCAGGTGGTTGCACATTTGTCCAGATGAAGCAAGGCCATATACCCTAAACTTCCATCC TGGGGGTCAGCTGGGCTCCTGGGAGATTCCAGATCACACATCACACTCTGGGGACTCAGGAACCATG CCCCTTCCCCAGGCCCCCAGCAAGTCTCAAGAACACAGCTGCACAGGCCTTGACTTAGAGTGACAGC CGGTGTCCTGGAAAGCCCCCAGCAGCTGCCCCAGGGACATGGGAAGACCACGGGACCTCTTTCACTA CCCACGATGACCTCCGGGGGTATCCTGGGCAAAAGGGACAAAGAGGGCAAATGAGATCACCTCCTGC AGCCCACCACTCCAGCACCTGTGCCGAGGTCTGCGTCGAAGACAGAATGGACAGTGAGGACAGTTAT GTCTTGTAAAAGACAAGAAGCTTCAGATGGGTACCCCAAGAAGGATGTGAGAGGTGGGCGCTTTGGA GGTTTGCCCCTCACCCACCAGCTGCCCCATCCCTGAGGCAGCGCTCCATGGGGGTATGGTTTTGTCA CTGCCCAGACCTAGCAGTGACATCTCATTGTCCCCAGCCCAGTGGGCATTGGAGGTGCCAGGGGAGT CAGGGTTGTAGCCAAGACGCCCCCGCACGGGGAGGGTTGGGAAGGGGGTGCAGGAAGCTCAACCCCT CTGGGCACCAACCCTGCATTGCAGGTTGGCACCTTACTTCCCTGGGATCCCAGAGTTGGTCCAAGGA GGGAGAGTGGGTTCTCAATACGGTACCAAAGATATAATCACCTAGGTTTACAAATATTTTTAGGACT CACGTTAACTCACATTTATACAGCAGAAATGCTATTTTGTATGCTGTTAAGTTTTTCTATCTGTGTA CTTTTTTTTAAGGGAAAGATTTTAATATTAAACCTGGTGCTTCTCACTCAC 3768 3835 3902 3969 4036 4103 4170 4237 4304 4371 4438 4505 4572 4639 4706 4773 4840 4907 4974 5041 5108 5175 5242 5309 5376 54 Table 2 discloses the sequence of an allele of an type A human platelet-derived growth factor receptor polypeptide. Both a nucleic acid sequence and its corresponding protein sequence are provided. The nucleic acid sequence corresponds to Seq. ID No. 5. The amino acid sequence corresponds to Seq. ID No. 4. Another human type A allele sequence is reported in Matsui et al. (1989) Science 243:800- 803.

ATG Met CTG Leu AAT Asn GAG Glu GTG Val GAA Glu AAC Asn TAT Tyr TTA Leu GAT Asp TCC S81." ATC Ile GTT Val Lys AAC Asn IAELEJ Sequence of a human type A PDGF receptor polypeptide allele and protein TTGGAGCTACAGGGAGAGAAACAGAGGAGGAGACTGCAAGAGATCATTGGAGGCCGTGGGC ACGCTCTTTACTCCATGTGTGGGACATTCATTGCGGAATAACATCGGAGGAGAAGTTTCCCAGAGCT cos Gly AGC Ser GAA Glu AGT Ser GAA Glu GTG Val CAC His GTG Val GTC Val CCC Pro TAC Tyr TGT Cys TAT Tyr ACC Thr AAT Asn ACT Thr CTA Leu AAG Lys GAA Glu ATC Ile AGC Ser ACT Thr CCA Pro .ATC Ile GAG Glu GAC Asp GAG Glu GCT Ala GTG Val GAG Glu TCC Ser ATC Ile GTT Val GTG Val AGA Arg AGT Ser CAG Gln GAC Asp GTG Val ACT Thr AGC Ser GCC Ala TTA Leu TAT Tyr GTG Val CAT His CTC Leu GTG Val AGC Ser AAT Asn GCC Ala ACA Thr CCA PIC GAG Glu CCT P170 AGA Arg ACC Thr AAG Lys GTT Val CCG Pro TGC Cys CAG Gln TGG Trp GAA Glu TCG Ser GAA Glu GAT Asp GAT Asp GTA Val CAG Gln GTC Val GCA Ala TCA Ser GAC Asp GCG Ala CAG Gln GTG Leu CAG Gln GAA Glu GCG Ala GAG Glu GTA Val GAT Asp ACC Thr GGC Gly Lys ACA Thr GGG Gly CTT Leu TTC Phe CTT Leu AAT Asn TAC Tyr AAC Asn GCC Ala AAT Asn GCC Ala GAT Asp TTA Leu Phe GGA Gly TCA Ser GAA Glu CAA Gln CTG Leu TCA Ser TCA Ser CCC Pro AAC Asn CAC His GAG Glu TCT Ser CAC His AAT Asn AAG Lys GAG Glu ACG Thr TGG Trp GTC Val TTA Leu TCC SE1’ ATG Met AGC Ser ACA Thr CTT Leu GTA Val GCC Ala AAC ASH GGG Gly AAG Lys CTG Leu ATT Ile ACT Thr TTA Leu CCC Pro TTT Phe TCT Ser GGC Gly GGG Gly GAA Glu CCT Pro ATT Ile AGT Ser ACC Thr TTC Phe GAT Asp GTG Val TAC Tyr GGC Gly TCT Ser TCT Ser GAA Glu CTT Leu TTG Leu GGC Gly CTA Leu ATA Ile GAG Glu TTC Phe CAG Gln CTA Leu GTC Val CCT Pro TGT Cys ATC Ile CTG Leu GAA Glu TTT Phe TAC Tyr AGG Arg GGA Gly CCT Pro GGG Gly ACT Thr ACC Thr GAA Glu ACC Thr GGA Gly CTT Leu CTT Leu AGA Arg GAG Glu GTG Val ACT Thr CAC His ATG Met TGT Cys GTG Val GTA Val ATC Ile ATG Met TGT Cys GAA Glu CTC Leu CCA Pro TGC cys AGC Ser ACG Thr TGC cys ATT Ile ACG Thr CGC Arg GTA Val GGG Gly CCA Pro GAA Glu GCT Ala GTG Val ACA Thr AAT Asn TTT Phe TCC Ser GTC Val TAT Tyr TAC Tyr GAT Asp ACA Thr CCT Pro CCC Pro Phe GCT Ala GTT Val GGG Gly GAA Glu GGG Gly GAT Asp TTG Leu TAC Tyr ATC Ile TAT Tyr ACT Thr GCC Ala TAT Tyr AAT Asn CTT Leu TTT Phe GGC Gly -7 TAC Tyr TGT Cys ATT Ile TTG Leu GCC Ala GAA Glu TAT Tyr TAT Tyr CTG Leu GGC Gly CCT Pro ACT Thr CAC His GAG Glu AAC Asn GCT Ala Table 2, page 2 GGC Gly ACT Thr GCT Ala TCT Ser GAA Glu TAC Tyr AAT Asn CGA Arg ACT Thr TTA Leu TCA Ser GAT Asp TCC Ser TCC Ser GAG Glu CGA Arg GCT Ala ATC Ile TTG Leu GCC Ala GTC Val GCT Ala CCA Pro CTC Leu AGC Ser ATT Ile ACT Thr ACT Thr ATT Ile TGG TIP CGA Arg ACC Thr GAG Glu GCA Ala ACA Thr ACG Thr CGC Arg CAT His GTC Val CCT Pro ACT Thr GTA val CAA Gln GGG Gly GAG Glu ACT Thr GAC Asp ATC Ile CTG Leu GTC Val ATG Met GTC Val CAG Gln GAG Glu AAC Asn CCC Pro GAG Glu TTA Leu GCT Ala GTT Val GGA Gly TGG TIP ATT Ile AGG Arg GCC Ala AAG Lys CTG Leu CTG Leu CCC Pro GCT Ala CTG Leu AGG Arg ATC Ile AAG Lys CAA Gln CCT Pro CAG Gln ATG Met TTG Leu AGT Ser GTG Val CTG Leu GTG Val GAA Glu GAG Glu ACC Thr GGT Gly CAT His ATA Ile ACC Thr CTG Leu AAT Asn TCA Ser ACG Thr ATA Ile GCC Ala ACC Thr CGA Arg GTG Val CTG Leu GAA Glu GCC Ala AGG Arg TTC Phe GAA Glu TCC Ser ACT Thr ATC Ile GAA Glu TCC Ser GTG Val TGC Cys AAC Asn GTG Val TGC Cys GCT Ala TTG Leu ATC Ile ACG Thr GAG Glu ATT Ile GTC Val "rec Tr? GAT Asp CGT Arg GAT Asp ATT Ile AGG Arg AAT Asn GAG Glu CTG Leu CCC Pro GTG Val GTG val GTC Val GAA Glu CTG Leu GTG Val GCT Ala GCT Ala CTG Leu TGC Cys GAT Asp GTC Val GGC Gly GCT Ala ACC Thr ATT Ile GTC Val ATC Ile CAT His Lys GAA Glu AAG Lys GTG Val GAC Asp ACA Thr ATT Ile TCA Ser CGT Arg AAG Lys CTG Leu GTG Val CCA Pro GAC Asp GAA Glu AAC Asn AAG Lys GAA Glu AAG Lys TTG Leu GCT Ala AAG Lys AAC Asn GTG Val AAT Asn CGT Arg ATC Ile TCC Ser AGT Ser ATG Met CCC Pro GTT Val AAT Asn ATT Ile GAA Glu AGC Ser GTC Val GAA Glu ATC Ile ACT Thr CTC Leu TCT Ser ATC Ile ATC Ile GGA Gly AAG Lys ACC Thr GTA Val CTG Leu CAG Gln GAC Asp TAT Tyr GAT Asp GGC Gly TGT Cys ATC Ile TTC Phe CTT Leu GAA Glu TCA Ser GAT Asp TTC Phe GAG Glu ACT Thr GAA Glu AGT Ser ACT Thr GAT Asp ACG Thr AAT Asn ACG Thr GCC Ala GGA Gly CTC Leu CTT Leu TTG Leu TAC Ty): GTC Val AGC Ser GTG val CTG Leu ATA Ile GGC Gly TTT Phe CAC His CCG Pro AAT Asn GAG Glu Lys GCT Ala ACG Thr ATT Ile GTG Val GAA Glu ACT Thr CAG Gln ccc Arg ATT Ile AGG Arg CAT His GAA Glu CAT His CTT Leu GAA Glu ATC Ile GTG Val GAG Glu GTG Val GTC Val CTG Leu ATT Ile CAG Gln GGT Gly TAT Tyr Lys ATA Ile TGC Cys GAT Asp CCA Pro GAA Glu ATG Met GCC Ala TCA Ser TAT Tyr CGT Arg Table 2, page 3 GTT Val GAA Glu CTG Leu CGG Arg GGA Gly CCC PIC ATG Met ACC Thr TTG Leu GAG Glu AGC Ser GAC Asp GAG Glu TCA Ser GAT Asp CAA Gln GAT Asp GTC Val TCA Ser CCT Pro GTC Val TTA Leu ACG Thr ACT Thr AAG Lys GTC Val AAG Lys ACA Thr ATG Met GTT Val TAT Tyr GAT Asp GTT Val CTG Leu ATT Ile ATC Ile TAT Tyr TTG Leu AGC Ser GCC Ala CAC His TCA Ser AAC Asn CCA Pro CGG Arg AAG Lys TCT Ser AAG Lys AAC Asn GCC Ala GCT Ala TGG TIP AGC Ser GAC Asp GGG Gly CGG Arg AGA Arg CTG Leu GGC Gly TAT Tyr AAG Lys AGC Ser CAG Gln AAG Lys TCA Ser CGA Arg GCT Ala CCA Pro TCA Ser TCT Ser TCC Ser TCC Ser GGG Gly CCC Pro TTG Leu AAA Lys TAT Tyr GCT Ala TAT Tyr AAA Lys GAA Glu GGA Gly CGC Arg CAG Gln GAT Asp AGA Arg GGA Gly CAA Gln AGT Ser CCA Pro ATT Ile CAT His GAG Glu GTT Val GAT Asp TCC Ser TCT Ser sec Gly ATG Met AAC ASH GGA Gly TGG TIP GCG Ala CCT Pro GAA Glu CAT His TAC Tyr AAG Lys CTG Leu ATT Ile ACT Thr GAC Asp ATG Met CTT Leu GAG Glu GTT Val CCG Pro CAT His GAG Glu Phe GTC Val Lys TTG Leu ATC Ile AAT Asn GAT Asp TTA Leu ACA Thr ATC Ile TTA Leu ACT Thr TTT Phe CTC Leu AGG Arg GAA Glu GGG Gly ATG Met CAA Gln AAC Asn ATC Ile AGG Arg ATC Ile TCT Ser CAG Gln CAG Gln GAC Asp TTA Leu TTG Leu CTG Leu TAT Tyr TAT Tyr CCA Pro AAG Lys Lys GCT Ala ATT Ile ACA Thr GAT Asp TAT Tyr AGA Arg TCA Ser TTG Leu GCT Ala GCA Ala GAA Glu ATT Ile AGA Arg GTG Val GTT Val CTC Leu GTA Val GAG Glu AGC Ser GGA Gly GAA Glu GTC Val TCA Ser GAA Glu GAT Asp TCA Ser CAA Gln ATT Ile TAT Tyr GAT Asp GTT Val GCA Ala ATG Met AAC Asn TAT Tyr TTC Phe TTG Leu AAC A511 CCC Pro CTC Leu GTC Val TTG Leu Lys GGA Gly CGC Arg GTG Val GGA Gly GAA Glu GTG Val TCT Ser TTG Leu TGC Cys CTG Leu AAC Asn AAT Asn ATG Met TAT Tyr Lys TTG Leu AAT Asn AAA Lys TGG Trp GAC Asp CTA Leu GGA Gly AAG Lys GAA Glu CTG Leu TTC Phe AGC Ser CCT Pro GGT Gly CTA Leu GAT Asp AAC Asn AGC Ser TGT Cys ATT Ile AGG Arg CCG Pro GTG Val ACA Thr ATG Met CTG Leu GGA Gly TAT Tyr CAC His GCT Ala GAC Asp GAA Glu CGT Arg CTC Leu TTC Phe GTC Val GTG val GTC Val ATG Met GCC Ala CTA Leu AAG Lys GCC Ala GGA Gly CAC His GAT Asp TAC Tyr AGG Arg CCA Pro CTT Leu ACC Thr CAC His AAG Lys ATC Ile TCG Ser TTT Phe CTC Leu GAT Asp GAC Asp GAG Glu CTG Leu CTG Leu GCA Ala GAG Glu CCT Pro AAC Asn AGC Ser ATG Met CTG Leu Table 2, page 4 TGT Cys Lys GAC Asp TGG TIP TCT Ser CAC His CCG Pro CTG Leu AAG Lys TAC Tyr GGT Gly CTG Leu AGA Arg AGT Ser ATG Met GAC Asp GGC Gly AAC Asn GAG Glu ACT Thr GCT Ala GAG Glu CCT Pro AGT Ser ATT Ile GGT Gly CCT Pro CAC His TCC Ser GAC Asp TTT Phe AGT Ser CTC Leu ATC Ile TTC Phe ACC Thr AAG Lys GGA Gly GAC Asp GGT Gly CTG Leu GAC Asp AGC Ser ACC Thr GAC Asp GGC Gly ACC Thr TAC Tyr TTT Phe TAC Tyr AGT Ser AGA Arg CAA Gln CAT His GTC Val GAT Asp ATT Ile TCG Ser TTC Phe ATC Ile CTG Leu ACC Thr TCC Ser AAT Asn GAA Glu CCC PI'O TAT Tyr CCT Pro ACC Thr GAG Glu GAC Asp CAG Gln ATC Ile GGC Gly GCC Ala CTG Leu ACA Thr AAG Lys GTC Val TCC Ser AAA Lys GCT Ala TAC Tyr CAG Gln CCT Pro ACC Thr AAG Lys ATA Ile AGA Arg CCC Pro CTG Leu GGT Gly ATC Ile TAC Tyr TTT Phe AAG Lys GTG Val AAA Lys AGA Arg GTC Val TCT Ser AGA Arg GAC Asp GAC Asp GTG Val AGT Ser GGC Gly AAG Lys GAG Glu TAC Tyr AGT Ser GCA Ala AAC Asn CTG Leu CCT Pro GAA Glu GAG Glu TCT Ser ATC Ile AAG Lys GAT Asp ACC Thr AGT Ser ATC Ile CAC His TAT Tyr CGC Arg GAG Glu AGC Ser GAG Glu GAG Glu GAC Asp TCA Ser ATG Met TGG Trp GTC Val CCT Pro GGG Gly ATG Met CTG Leu GAA Glu ATG Met GAA Glu GCT Ala GAG Glu AGT Ser GAG Glu GAC Asp CAT His ATG Met TGG TIP TAC Tyr TAC Tyr GTG Val AGT Ser AAA Lys CGT Arg GAC Asp GAC Asp GAG Glu GCC Ala ACC Thr CTG Leu GAT Asp GCT Ala TCT Ser CCC Pro CGG Arg AAA Lys GAG Glu ATT Ile GTG Val AAG Lys AGT Ser GAC Asp ATT Ile ATT Ile GTG Val TCG Ser CCT Pro TAT Tyr GGC Gly ATG Met TGC Cys ATT Ile CAC His GAC Asp CTG Leu GGC Gly CTG Leu GAG Glu GAA Glu GAA Glu AAC Asn GAG Glu GGC Gly ATG Met GCC Ala TGG TIP GTG Val CTG Leu TCA Ser AAG Lys TAC Tyr GGC Gly ACG Thr GAC Asp GAC Asp TAT Tyr AGC Ser ATT Ile ATG Met AAG Lys AAC Asn GAG Glu GAC Asp GAC Asp GAC Asp ATC Ile AAG Lys GGT Gly ATC Ile AGC Ser GTG Val ATC Ile CTG Leu GTG Val CCT Pro AGT Ser AAT Asn TTC Phe AAT Asn TGG TIP ATT Ile AGG Arg TCC Ser GAC Asp TTC Phe TAACTGGCGGATTCGAGGGGTTCCTTCCACTTCTGGGGCCACCTCTGGATCCCGTTCAGAAAA CCACTTTATTGCAATGCGGAGGTTGAGAGGAGGACTTGGTTGATGTTTAAAGAGAAGTTCCCAGCCA AGGGCCTCGGGGAGCCTTTCTAAATATGAATGAATGGGATATTTTGAAATGAACTTTGTCAGTGTTG CCTCTTGCAATGCCTCAGTAGCATCTCAGTGGTGTGTGAAGTTTGGAGATAGATGGATAAGGGAATA ATAGGCCACAGAAGGTGAACTTTCTGCTTCAAGGACATTGGTGAGAGTCCAACAGACACAATTTATA 3659 37 Table 2, page 5 CTGCGACAGAACTTCAGCATTGTAATTATGTAAATAACTCTAACCACGGCTGTGTTTAGATTGTATT AACTATCTTCTTTGGACTTCTGAAGAGACCACTCAATCCATCCATGTACTTCCCTCTTGAAACCTGA TGTCAGCTGCTGTTGAACTTTTTAAAGAAGTGCATGAAAAACCATTTTTGACCTTAAAAGGTACTGG TACTATAGCATTTTGCTATCTTTTTTAGTGTTAAAGAGATAAAGAATAATAATTAACCAACCTTGTT TAATAGATTTGGGTCATTTAGAAGCCTGACAACTCATTTTCATATTGTAATCTATGTTTATAATACT ACTACTGTTATCAGTAATGCTAAATGTGTAATAATGTAACATGATTTCCCTCCACACAAAGCACAAT TTAAAAACAATCCTTACTAAGTAGGTGATGAGTTTGACAGTTTTTGACATTTATATTAAATAACATG TTTCTCTATAAAGTATGGTAATAGCTTTAGTGAATTAAATTTAGTTGAGCATAGAGAACAAAGTAAA AGTAGTGTTGTCCAGGAAGTCAGAATTTTTAACTGTACTGAATAGGTTCCCCAATCCATCGTATTAA AAAACAATTAACTGCCCTCTGAAATAATGGGATTAGAAACAAACAAAACTCTTAAGTCCTAAAAGTT CTCAATGTAGAGGCATAAACCTGTGCTGAACATAACTTCTCATGTATATTACCCAATGGAAAATATA ATGATCAGCGCANAAAGACTGGATTTGCAGAAGTTNTTTTTTTTTTTTCTTCTTGCCTGATGAAAGC TTTGGCGACCCCAATATATGTATTTTTTGAATCTATGAACCTGAAAAGGGTCACAAAGGATGCCCAG ACATCAGCCTCCTTCTTTCACCCCTTACCCCAAAGAGAAAGAGTTTGAAACTCGAGACCATAAAGAT ATTCTTTAGTGGAGGCTGGAAGTGCATTAGCCTGATCCTCAGTTCTCAAATGTGTGTGGCAGCCAGG TAGACTAGTACCTGGGTTTCCATCCTTGAGATTCTGAAGTATGAAGTCTGAGGGAAACCAGAGTCTG TATTTTTCTAAACTCCCTGGCTGTTCTGATCGGCCAGGTTTCGGAAACACTGACTTAGGTTTCAGGA AGTTGCCATGGGAAACAAATAATTTGAACTTTGGAACAGGGTTCTTAAGTTGGTGCGTCCTTCGGAT GATAAATTTAGGAACCGAAGTCCAATCACTGTAAATTACGGTAGATCGATCGTTAACGCTGGAATTA AATTGAAAGGTCAGAATCGACTCCGACTCTTTCGATTTCAAACCAAAACTGTCCAAAAGGTTTTCAT TTCTACGATGAAGGGTGACATACCCCCTCTAACTTGAAAGGGGCAGAGGGCAGAAGAGCGGAGGGTG AGGTATGGGGCGGTTCCTTTCCGTACATGTTTTTAATACGTTAAGTCACAAGGTTCAGAGACACATT GGTCGAGTCACAAAACCACCTTTTTTGTAAAATTCAAAATGACTATTAAACTCCAATCTACCCTCCT ACTTAACAGTGTAGATAGGTGTGACAGTTTGTCCAACCACACCCAAGTAACCGTAAGAAACGTTATG ACGAATTAACGACTATGGTATACTTACTTTGTACCCGACACTAATGACGTTAGTGACACGATAGCCG TCTACTACGAAACCTTCTACGTCTTCGTTATTATTTCATGAACTGATGGATGACCACATTAGAGTTA CGTTCGGGGTTGAAAGAATAGGTTGAAAAAGTATCATTCACGCTTCTGACTCGGTCTAACCGGTAA TTTTTCTTTTGGACTGATCCAAGACATCTCGGTTAATCTGAACTTTATGCAAACACAAAGATCTTAG TGTCGAGTTCGTAAGACAAATAGCGAGTGAGAGGGAACATGTCGGAATAAAACAACCACGAAACGTA AAACTATAACGACACTCGGAACGTACTGTAGTACTCCGGCCTACTTTGAAGAGTCAGGTCGTCAAAG GTCAGGATTGTTTACGAGGGTGGACTTAAACATATACTGACGTAAACACCCACACACACACAAAAGT CGTTTAAGGTCTAAACAAAGGAAAACCGGAGGACGTTTCAGAGGTCTTCTTTTAAACGGTTAGAAAG GATGAAAGATAAAAATACTACTGTTAGTTTCGGCCGGACTCTTTGTGATAAACACTGAAAAATTTGC TAATCACTACAGGAATTTTACACCAGACGGTTAGACATGTTTTACCAGGATAAAAACACTTCTCCCT GTATTCTATTTTACTACAATATGTAGTTATACATATATACATAAAGATATATCTGAACCTCTTATGA CGGTTTTGTAAATACTGTTCGACATAGTGACGGAAGCAAATATAAAAAAATTGACACTATTAGGGGT GTCCGTGTAATTGACAACGTGAAAACTTACAGGTTTTAAATATAAAATCTTTATTATTTTTCTTTCT ATGAATGTACAAGGGTTTTGTTACCACACCACTTACACACTCTTTTTGATTGAACTATCCCAGATGG TTATGTTTTACATAATGCTTACGGGGACAAGTACAAAAACAAAATTTTGCACATTTACTTCTAGAAA TATAAAGTTATTTACTATATATTAAATTTCCTTAAG 3927 3994 4061 4128 4195 4262 4329 4396 4463 4530 4597 4664 4731 4798 4865 4932 4999 5066 5133 5200 5267 5334 5401 5468 5535 5602 5669 5736 5803 5870 5937 6004 6071 6138 6205 6272 6339 6375 A polypeptide or nucleic acid is substantially pure, or substantially purified, when it comprises at least about 30% of the respective polymer in a composition, typically at least about 50%, more typically at least about 70%, usually at least about 80%, more usually at least about 90%, preferably at least about 95%, and more preferably about 98% or more.

The soluble fragments of the extracellular region will generally be less than about 400 amino acids, usually less than about 350 amino acids, more usually less than about 300 amino acids, typically less than about 200 amino acids, and preferably less than about 150 amino acids.

A. D Domains Based on a number of observations, the extracellular region (XR) of these PDGF receptor polypeptides comprises 5 immunoglobulin-like domains. First, the amino acid sequence contains 5 segments characteristic of Ig-like domain structures, each of the segments having an appropriate size for an immunoglobulin domain. Each segment, except for the fourth, has characteristically spaced cysteine residues that are a diagnostic feature of an immunoglobulin-like domain. The receptor polypeptide sequence displays other features of immunoglobulin-like domain structure, e.g., the presence of characteristically positioned tryptophan and tyrosine residues.

Direct sequence comparisons of segments of the receptor polypeptides with corresponding segments of true immunoglobulin domains shows a statistically significant similarity between PDGF receptor polypeptide domains and immunoglobulin domains.

See, e.g., Williams (1989) Science 243: 1564-1570. The argument that the receptor polypeptide domains assume the folding pattern of immunoglobulin domains can be strengthened by examining the predicted secondary structure of the receptor polypeptides. when a homology mapping analysis is performed, the PDGF receptor polypeptide shows five Ig-like domains in the extracellular region, each domain showing statistically significant homology to defined Ig-like domains.

Williams and Barclay (1988) Ann. Rev. Immunol. Biochem. 6: See, e.g., - . Regions of homology will show significant sequence homology to particular Ig—like domains, and exhibit particular secondary and tertiary structural motifs characteristic of Ig- like domains. The domain structures will preferably be those segments with boundaries which approximately match the boundaries of the domain structures. The boundaries will preferably match within about 9 amino acids, typically within about 7 amino acids, more typically within about 5 amino acids, usually within about 3 amino acids, and more usually within 1 amino acid. See, e.g., Cantor and Schimmel (1980) Biophysical Chemistry, Vols I-III, Freeman and Co., San Francisco: Creighton (1984) Proteins: Structure and Molecular Properties, Freeman and Co., New York; and Watson et al. (1987) The Molecular Biology of the Gene, Vols 1 and 2, Benjamin, Menlo Park, California; each of which is hereby incorporated herein by reference.

The sequences of the human type B and the human type A receptor polypeptides can be analyzed to predict their beta strand topology. Combining a Fourier analysis of hydrophobic sequence pattern and a Garnier-Robson algorithm, see, e.g., Garnier et al. (1978) J. Mol. Biol. 120: 97, with a turn predictor program, as reported in Cohen et al. (1986) Biochemistry 25: 266, produces a characteristic structural pattern. This pattern exhibits consensus B-strand segments in each domain when analysed as described.

The first two Ig—like domains of the PDGF receptor polypeptides, D1 and D2, have about seven ﬂ-strand segments, designated the A, B, C, D, E, F, and G segments, as listed from amino proximal to carboxy proximal direction. The third, fourth and fifth Ig-like domains, D3, D4 and D5, are long enough to include an extra 5-strand segment, designated C‘.

The fifth domain, D5, most closely resembles a variable heavy chain domain in length. The type B receptor polypeptide D5 further comprises an additional B-strand segment designated C".

These features and designations are based partly on the homology of segments between domains and segments in the type B and type A hPDGF-R polypeptides, and with the mouse type B PDGF receptor polypeptide, and also based upon homology to other Ig- like segments found on other proteins, particularly other growth factor receptor proteins. The csf-1 receptor and c-kit proto-oncogene have similar Ig-like domain organizations. e.g., (1989) See, Williams Science 243:1564-1570.

The domain structure is based, in part, upon features common to Ig-like domains found in other proteins, including related receptors. See, e.g., Ullrich and Schlessinger (1990) gel; 61:203—212; and Yarden and Ullrich (1988) Ann. Rev. 57:443—78. The domain boundaries for the two alleles disclosed herein are identified below, but different alleles may have slightly different positions for the boundaries.

Table 14.

Biochem.

The Ig-like domains (D domains) are characterized by the regularity of spacing of cysteine residues in the extracellular region. These five D domains, each about 100 amino acids in length, have ﬂ-sheet rich structures, resembling immunoglobulin variable or constant regions. (1989) Science 243:l964-1570.

See, Williams The natural XR domains are numbered from the amino proximal domain D1, in order, through D5, at the carboxy proximal end of the XR.

The exon structure of the mouse type B PDGF receptor polypeptide gene also matches this domain structure with reasonable fidelity. The correlation between the intron-exon structure and functional units further supports the hypothesis that the boundaries define functional units of the polypeptide.

See, e.g., Williams and Barclay (1988) Biochem. 6:381-405.

Ann. Rev. Immunol.

The boundaries for each of these segments are indicated below for the two alleles disclosed herein, and similar boundaries will be found in other alleles at locations of sequence and functional homology.

The amino-proximal Ig-like domain of the human p1ate1et—derived growth factor receptor polypeptides is designated D1. The D1 domain extends from about leu(1) to pro(91) in the type B receptor polypeptide, and from about gln(1) to pro(101) in the type A receptor polypeptide.

Table 14. segments.

See The D1 domain apparently has about seven ﬂ-sheet .oov. .~cv. .no—. .emv. .hnv. .s~v. .mdv. .aon. .«om. .vov. .oc—. .mov. .vmv. .~vv. .o~v. .oHv. .sov. .~mm. .mav. nu; :In :un :40 sac =o« lwu nun sam man I .mcv.

I ANNV.

I .omv.

I .n-v.

I .onv.

I .v~v.

I .muv.

I _oov.

I .mmm.

I .mon. :~u cad ~o> nag uh» and can you you saw sou An> an> =.u and cnu can Am> Au> Adam. own I .msn. can I .oon. nun I .mvm. aau I .nem. ugu .son. sac .vmm. non .mvm. nun .onn. us» I .o~m. :ua I .n~m. ago I .~om. :~o I .~on. cum I nuwnavczon ouaﬁ .mmm. sun .m~m. one .oan. qua .va~. and .~m~. uzu .—om. can I .mnn. non .aon. nu: I .oon. was .nmn. an: I .nvn. nun .~vm. nan I .omn. uoa Anna. >40 I .n~n. nan Adan. man I .m~n. us» .oom. =~u I .oon. ans .vo~. :_a I .oo~. =04 .vmm. Ohm I A muwumvcso mam. nu» m ounﬁwxouant u:vEmUm u:cuuIIQ uvwunvnhaom HOuQUUO .no~_ .~n~. .om~. .mv~. .~n~.

ANNNV .ma~.

AHON. .om~.

AQAN. .ne~. .av~. .hmN.

AQNN. .e-. .eo~.

ANOA. .~m~. cud I .oo«. «u> I .mo~. ran I .~a~.

Cu WADCH >~u Hhv zau n>~ all odd sun ado zau ken HUI an! and GUI nag HUI Uﬁd .om~. .054. _om~. .oq~. ..w~. .on~. .m-. .oo~. .on~. .~o~. .av~. .ov«.

.Hm«. .o-. .mo~.

Aanu.

«IOunQ< ucoﬁuom Usnnulnﬁ 0v«un0l>uOm ucunuuvx on>kI¢ :uE== I un>FIm :uE== and huh aau ~m> can can cum and man Dun one can .eo. .«n. 500. .mm. .nm. .o«. .o~. .ao~ .oo. .~n. .nm. .ov. ~u> I .oo. aaa nu; I .ns. wan nun I .co. any nun I Amvv an: act I .nn. au> ad: I .nn. nan I>A I .0. non . cum I .a. can an as: I .oo. :~u an: I .vo. >~o :nI I Adm. hon man I .ov. sun uus I .m~. ~a> you I .na. can =u~ I .~. «u> cum I an. 50A an _;: - : < D U U U G H N O ,: - r C H U U U G H E U The next Ig-like domain, in the carboxy proximal direction of natural human platelet—derived growth factor receptor polypeptides, is designated D2. The D2 domain extends from about thr(92) to ser(181) in the type B receptor polypeptide, and from about asp(102) to ser(189) receptor polypeptide. in the type A The D2 domain apparently also has about seven B-sheet strands designated A, B, C, D, E, F, and G.

The third Ig-like domain found on natural human PDGF receptor polypeptides is designated D3. The D3 domain extends from about ile(182) to gly(282) in the type B receptor polypeptide, and from about glu(190) to gly(290) in the type A receptor polypeptide. The D3 domain apparently has about eight B-sheet strands designated A, B, C, C‘, D, E, F, and G.

The fourth Ig-like domain found in the natural human PDGF receptor polypeptides is designated D4. The D4 domain extends from about tyr(283) to pro(384) in the type B receptor polypeptide, and from about phe(291) to pro(391) in the type A receptor polypeptide. ﬁ—sheet strands.

The D4 domain apparently has about eight Note that the D4 domains lack the characteristic cysteine residues, which correspond to val(306) and met(364) in the type B sequence shown, and to val(3l3) and ile(371) in the type A sequence shown.

The fifth Ig—like domain is designated D5. The D5 domain extends from about val(385) to lys(499) in the type B receptor polypeptide, and from about ser(392) to glu(501) in the type A receptor polypeptide. The D5 of the type B receptor polypeptide has about nine putative ﬁ—sheet strand segments designated A, B, C, C‘, C", D, E, F, and G, while the type A receptor polypeptide has only about eight B—strand segments, lacking a C" segment.

The approximate boundaries of the domains and B- strand segments are listed in Table 14. The apparent alignments of the segments are illustrated in Tables 4 and 5. other alleles of the receptor polypeptides may also be analyzed by either homology or the structural analysis as described above. nnnann xmm4m:....m>>H>m m>zHaqoum umm >>PH2H mm> oqm>>v> LEE >hH%AEZ¢ nan: OHGOD5< ZIHH qo>m GO=DZ>m mp>z ma>u=oo HHHI DHHADKW DZZH Ucwﬁcmwam ucwﬁvww Ucmuumln SUM: .0U:0SUmm UHUM noun m>mq axzu P%HU uwmm EVHU m DDAAAAJ mmn>:o Amuhm>> £Mt>X> :>qh4mm woman: mn~=q~m ...muum DHEAU.

HDAUPJ 2Pdh4>m mama >%¢H max» axe» muse D nnnnnnn mmm:>u> mm>zmam mtauquo m>a4<...

DO<¥¢Z :0 .U 952 nnnnnn ZFHAOw WNW MMZUAAHAL JNKLUZ ¥A.Dﬂ u H> . . . . . . . . . . . . . . . . . . ..mm>a¢ ummx: . . . . . . . . . . . . . . . . . . . . . . . . . . . ..>DDK Om . . . . . . . . . . . . . . . . . . . . . . . . . ..mwOM Q Mdﬂlh U 2208522 ucwzauz m..OLZU Qxh3d>F m..mm»< AVFIHBZ >>...DZU acme ¥E=A8>> S¢H3>> . . . . . . ..m< Anna ¢>po.

Odhﬂ ZARA BHFH PJDH ..mumnm:m mmzqucm zmoo.x> meuuaqm hmm>zq> C ADDBDDDDB mmqmu>z>. m :%ﬂEOQ OdHU>HUAH ﬂ>5 V GAHEOD >.PO>W >2“ n HAHEOQ HMJMHCDZE JhU>F N ﬂnuﬁom AHmUhhPD> J A Enason OEMEM wvmumwmwaom Hoummowh wmxulm m mamas &..<>AKJH n>ohq4mua ozm :>m~e>xx: max :q¢>>zmm~ mam >¥H¥~=¢U AMZHPOH nan: mz >m:> mz>mma< o=<< aomxxuz >h :Z>¥ ucmﬁcvwam 2222222 U ¢>¥4 hF>¢Dﬂ> H FVIU MGHHX4 zuqxuzm U HNDU mQK>F< Wm>h2F> o urmo >FhFU. zmoo U H%JU P=((m¢ mm>m4>h ucwﬁmwm Ucmuumln £uu3 ammo ¢>¢~ >4x~ «mo» >kJU D xU .U 22222222222 222 2222222 mm: . . . . . ..H HHHHZM ... >Z2 monx m>n hFuN.. FJ2...H HJHAZ .....mm>:~m HA.... . . . . . . .. IFMUK. UZ>HU m¢m>> . . . . . . . . . . . . . . . . . . . . . . . . . .. ...Uw . . . . . . . . . . . . . .. Hmwm mzzumz:~u>n mm....

W NHHGH .00:0:Uww 0205 uaxzuun m....qmau ZKAIMHK m...mm~ aahxoqo >>....mzz mz=4h>m ‘...hmmo :m»o:m> . . . . . ..mm m>ho H>>H mum: zqmm UDFmU::Q >U=d2>< amum.x» maaou>~ mmz4o>> OGMEM wuwumwmhaom uoumwowu 0Q>u|< cm n>qnuumm, m Emmﬁoo HAOWEHEKH mum m I«oEOQ >.PKJ m.ﬂwmEOn >A>DFZOdh >h(> : HEOD zmzmzm.4~ mmqmqo a ESQ The prototypical D1 domains are those sequences of the human type B receptor polypeptide and the human type A receptor polypeptide, as described. However, compatible amino acid substitutions, insertions, and deletions which preserve the desired ligand binding functions can be made. The function will usually be preserved by retaining the LBR segments in the correct orientation by use of appropriate structured segments.

Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. Substitution or exchange of B-sheet segments or sequences intermediate the segments from different domains may be performed, including between type B and A receptor polypeptides, or between different domains of another related receptor polypeptide. Segments outside the prototypical cysteines within B-segments B and F (but val(306) and met(364) in the type B D4, and val(3l3) and ile(371) in the type A D4) will he usually less critical than the sequences between those residues, e.g., the C, C’, C", D; and E B-strand segments. Also, segments homologous to these disclosed segments may be substituted, including those with compatible amino acid substitutions, insertions, and deletions. Sources of similar domains and segments include related receptor polypeptides from human or other mammalian species. Non- mammalian receptor polypeptides may also exhibit significant homology and serve as sources for similar segments. other Ig- like domains and segments may also be substituted.

The present invention embraces polypeptides which exhibit homology to the disclosed and described segments and domains. It embraces segments comprising contiguous amino acids of the sequences disclosed, typically at least about 8 contiguous amino acids, more typically at least about 11 contiguous amino acids, usually at least about 14 contiguous amino acids, more usually at least about 17 contiguous amino acids, and preferably at least about 21 or more contiguous amino acids. Constructs retaining the LBR segments are most valuable. The invention also includes modifications of those sequences, including insertions, deletions, and substitutions with other amino acids. Glycosylation modifications, either changed, increased amounts, or decreased amounts, as well as Thus, the modified proteins comprising these amino acid sequences, e.g., other sequence modifications are envisioned. analogues, will usually be substantially equivalent to these proteins in either function or structure.

The ﬂ-sheet strands may be slightly enlarged or shortened by respective insertions or deletions in the polypeptide sequence. Thus, certain embodiments will have a slightly enlarged or shortened particular domain by adding or deleting particular sequences of B-sheet strands or their inter-strand sequences. Segments may be inserted or deleted which conform to the structural requirements of retaining the proper intra- and inter-domain interactions. In particular, changes which interrupt the secondary and tertiary structure of the protein will be disfavored. e.g., (1990) and Creighton (1984). In addition, amino acids or See, Cantor and Schimmel segments may be inserted or deleted in the regions outside of the 5-sheet strands and between domains. Typically the substitutions will be of amino acids having similar properties, and additions or deletions would preferably be selected among those which retain receptor biological functions, e.g., ligand binding.

The sequence of a B-sheet segment will typically not differ from a sequence from a human type B polypeptide or a human type A polypeptide by greater than about 50%, more typically less than about 39%, usually less than about 29%, and more usually less than about 20%. Comparable similarities over each of the non-ﬂ-sheet strands of each domain will be preferred.

The boundaries between domains are defined, in part, by the definitions for domains in the Ig-like domains.

Examples of similar domains are found in immunoglobulin and growth factor receptor polypeptides. between D1 and D2; D2 and D3; The domain boundaries D3 and D4; and D4 and D5 correspond approximately to exon locations, further supporting the proposal that the domain structures correspond to evolutionary and functional units. See, e.g., Watson et al. (1987) The Molecular Biology of the Gene, vols. 1 and 2, Benjamin, Menlo Park, California.

The D2 domains have similar characteristics to the D1 domains, as shown by the alignments illustrated in Tables 4 and . Both domains have B-sheet segments designated A, B, C, D, E, F, and G. The domain 3 segments, or D3, also exhibit homology, but have an additional B-strand segment designated C’. The D4 segments, or D4, have non-cysteine residues at the positions which typically correspond to cysteines in the other domains. In the type B allele shown, the residues are val(306) and met(364), while in the type A allele shown, the residues are val(313) and ile(371). segments designated C‘.

The D4 domains also have ﬁ—strand The domain 5, or D5, have the consensus cysteine residues and the additional C‘ ﬂ-strand segments, and the type B receptor polypeptide has an additional C" B-strand segment.

The present invention provides for various constructs comprising ligand binding constructs, typically comprising substantially intact domains. These constructs will have various uses,e.g., for binding ligands, or substituting for intact receptor polypeptides. For example, each of the separate domains may comprise a separate polypeptide alone, or may be fused to another peptide, such as the TM and IR regions of a receptor polypeptide, e.g., hPDGF-R. See, e.g., Table 6.

These individual single domain polypeptides will exhibit specific activity associated with these specific domains, preferably as an agonist or antagonist for ligand binding, preferably with characteristics shared with the intact receptor polypeptide or XR. The domains may also preferably serve as competitive inhibitors of PDGF-R polypeptides, competing with natural PDGF—receptors to bind ligands. The present invention also provides repetitive sequences of a single domain. For example, a D1 domain by itself is provided, a D1-D1 dimer in a single polypeptide is provided, a D1-D1—D1 triplet repeat is also provided. Likewise up to a large number of D1 domains which will exhibit many functions, e.g., immunological properties, characteristic of various natural PDGF-R sequences.

Similar constructs of each of D2, D3, D4, and D5 are provided, along with combinations. See Tables 6, 7, 8, 9 and 10. These will often be soluble fragments of the XR, or may be fused to other polypeptides, including a PDGF-R TM segment, preferably with an IR segment also.

TABLE 6 XR domain structure of single domain forms D1 D2 D3 D4 D5 TABLE 7 XR domain structure of two domain forms D1-D1 D2-D1 D3-D1 D4-D1 D5-D1 D1-D2 D2-D2 D3-D2 D4-D2 D5-D2 D1-D3 D2-D3 D3-D3 D4-D3 D5-D3 D1-D4 D2-D4 D3-D4 D4-D4 D5-D4 D1-D5 D2-D5 D3-D5 D4-D5 D5-D5 TABLE 8 XR domain structure of three domain forms Dl-W D2-W D3-W D4-W D5-W where W is each of the 25 possible combinations listed in TABLE 2, giving a total of 125 elements in this table TABLE 9 XR domain structure of four domain forms D1-X D2-X D3—X D4-X D5-X where X is each of the 125 possible combinations listed in TABLE 5, giving a total of 625 elements in this table TABLE 10 XR domain structure of five domain forms D1-Y D2-Y D3-Y D4-Y D5-Y where Y is each of the 625 possible combinations listed in TABLE 6, but not including the combination D1-D2-D3-D4-D5, giving a total of 3124 elements in this table In addition, the present invention provides similar structures with spacer regions between the domain structures.

In particular, the regions corresponding to the intra-cysteine residues of the domains shown in Tables 4 and 5 are useful.

For example, a spacer polypeptide may be inserted between adjacent domains or do spaces between the important ligand binding segments, typically found within the intra-cysteine segments described, e.g., the B, C, C‘, C", D, E, and F 3- strand segments. Thus, for example, a polypeptide of the structure D1-X1-D2 is provided where X1 is a spacer segment which is not a D domain. The order of the domains may be reversed, and the invention also provides polypeptides such as D2-D1, or D2—X1-D1. In particular, the non—D domain character of X1 is provided to avoid the peptide D1-X1-D3 from describing, or encompassing, D1-D2-D3.

Another particularly preferred embodiment of the invention is a polypeptide having the described extracellular region domain structure combined with other segments of a human platelet-derived growth factor receptor, particularly the transmembrane segment (TM) and the intracellular region (IR).

Thus, the present invention provides for a receptor polypeptide which either has a modified order of the extracellular region domains in the amino to carboxy direction, e.g., a D5—D4—D3-D2- D1-TM-IR polypeptide, or, in some cases reversal of Various domains. It also provides for a receptor polypeptide with a deleted intact domain and for a receptor polypeptide having an additional domain added to it.

IR, or D1-D2-D3-D4-TM-IR.

Examples include D1-D2-D3-TM- In particular, fusions with the XR segments described in Tables 6, 7, 8, 9, and 10 are preferred embodiments.

The modified combinations of the D domains are expected to both simulate and differ from the natural receptor.

The modified polypeptide would be expected, in some embodiments, to exhibit a modified binding affinity, e.g., higher or lower affinity, or to exhibit a different spectrum of binding to different ligands or ligand analogues. They may also have an altered ligand binding transducing efficiency, or a modified inter—chain association affinity.

The present invention provides the means for determining the minimal structural features necessary to perform various functions of the extracellular region of platelet-derived growth factor receptors, preferably human receptors. Although similar determinations may be performed in mouse or other mammalian species, the human receptor will typically be preferred for diagnostic or therapeutic purposes.

To determine the minimal region necessary for a functional activity, e.g., ligand binding, an assay for that activity is developed. The main receptor functions, as indicated above, include ligand binding, tyrosine kinase activity, and receptor dimerization. Simple and quick assays for each of these molecular functions may be developed. Ligand binding assays are described, e.g., in Gronwald et al. (1988) Proc. Nat’l Acad. Sci. USA 85:3435-3439; Heldin et al. (1988) EMBO J. 711387-1393; 240:1532-1534. and Escobedo et al. (1988) Science Receptor dimerization assays are described, e.g., in Yarden and Schlessinger (1987) Biochemistry 26:1434- 1442 and 1443-1451.

As an alternative means for determining sites which interact with specific other proteins, physical structure determination, e.g., x-ray crystallography or 2 dimensional NMR techniques, will provide guidance as to which amino acid residues form the molecular contact regions. For a detailed description of protein structural determination, see, e.g., Blundell and Johnson (1976) Protein Crystallography, Academic Press, New York, which is hereby incorporated herein by reference.

Ligand binding assays may include binding of labeled ligand or competition assays for binding. Signal transduction may be indirectly assayed by measuring an activity modulated by ligand binding, e.g., tyrosine kinase activity, or some measure of a conformational or other change in receptor structure. example, an antibody or other binding protein which specifically binds or dissociates from the receptor polypeptide upon ligand binding may be used.

Receptor dimerization may be measured by a proximity assay, including a fluorescence quenching or other spectroscopic measurement. Various proximity assays are known, see, e.g., Ullrich and Schlessinger (1990) gel; 61:203-212; Yarden and Schlessinger (1987) Biochemistry 26:1434-1942 and 1443-1451; each of which is hereby incorporated herein by reference.

Once an assay has been developed, various combinations of domain or other segments, e.g., LBR's, can be tested for affecting that activity. A competitive inhibition assay will detect those constructs which can bind the ligand.

The first domain structures to try will ordinarily be the individual domains, either alone or linked to chimeric proteins or the TM-IR segment of the receptor. Various alleles, modifications to the individual domains, or related chimeric domains would be tested. Both deletion and chimeric proteins will be constructed.

Various combinations of each domain will be constructed and tested to select those which affect the measured activity. Repeats of those domains should be tested, e.g,., D1-D1. If no single domain does affect the function, then various 2 domain constructs, in order, would be tried, e.g., D1-D2-TM-IR, D2-D3-TM-IR, D3-D4-TM-IR, and D4-D5-TM-IR.

Selected combinations listed in Tables 6, 7, 8, 9, and 10 will be constructed and tested.

In order to produce soluble forms, it will often be desireable to attach appropriate amino terminal segments, some of which would be expected to be present in the D1 domain or in the precursor form. Correct secretion and processing may be dependent upon various amino proximal features, such as signal sequences, and other features essential for correct targeting and processing. See, e.g., Watson et al. (1987) The Molecular Biology of the Gene, vols. 1 and 2, Benjamin, Menlo Park, California. ’ When correct domains have been selected which are especially effective in modulating or competing defined functions, a more detailed analysis, to the level of the ﬁ- strand segments might be addressed. deletion, Various chimeric, insertion, or substitution constructs of each ﬁ- strand or inter-strand segment may be generated and tested, as described above. Each construct could be produced using methods of standard genetic engineering, especially using synthetic primers. Procedures for using such reagents are described, e.g., in Sambrook, et al. (1989) Molecular Cloning: A Laboratory Manual, vols. 1-3, Cold Spring Harbor Press, and Ausubel et al. (eds.) (1989) Current Protocols in Molecular Biology, Wiley, each of which is hereby incorporated herein by reference.

B. Soluble Forms In some embodiments, only the extracellular region is provided. Thus, the extracellular region alone, without the transmembrane segment, will often be a soluble polypeptide. It has been demonstrated that the entire extracellular region, separated from, and which lacks a transmembrane region and an intracellular region, still serves as a ligand binding polypeptide. In particular, the soluble polypeptide D1-D2-D3- D4-D5 has been demonstrated to bind various PDGF forms.

Although the binding specificity for the PDGF form is dependent, to some extent, on the specific domains included, modifications to the specificity of the ligand binding may be , effected by either substituting various different domains or rearranging the domains. Substitution with other homologous segments may also be performed, e.g., substituting an Ig-like domain from an antibody molecule, such as an antibody which binds a platelet-derived growth factor. Alternatively, a domain from a different related growth factor or ligand receptor may be substituted, e.g., from an FGF receptor or another PDGF receptor. The order of the domains may also be modified, e.g., D5-D4—D3-D2-D1.

In particular, the activities which will usually be of greatest importance with the extracellular constructs relate to the binding of the ligand. it has been discovered that domains D4 and D5 are not essential for ligand binding of a soluble extracellular region PDGF-R polypeptide.

Of the remaining domains, if domain D3 is separated from domains D1 and D2, the construct D1-D2 binds the ligand only at low affinity, but a D1-D2-D3 construct binds ligand at high affinity.

For example, A typical hPDGF-R nucleic acid sequence encodes a transitory amino terminal hydrophobic sequence, which is usually cleaved during the membrane translocation process. The classical function of a signal sequence is to direct the nascent polypeptide chain to membrane bound ribosomes, thereby leading to membrane translocation or cellular targeting.

However, since the signal sequence is typically removed in the translocation process, the signal sequence is usually absent in a mature polypeptide. Often a signal sequence will be attached upstream of a desired soluble peptide of this invention.

Solubility of a polypeptide depends upon the environment and the polypeptide. Many parameters affect polypeptide solubility, including the temperature, the electrolyte environment, the size and molecular characteristics of the polypeptide, and the nature of the solvent. Typically, the temperature at which the polypeptide is used ranges from about 4°C to about 65°C. Usually the temperature at use is greater than about 18°C and more usually greater than about 22°C. For diagnostic purposes, the temperature will usually be about room temperature or warmer, but less than the denaturation temperature of components in the assay. For therapeutic purposes, the temperature will usually be body temperature, typically about 37°C for humans, though under certain situations the temperature may be raised or lowered in situ or in vitro.

The electrolytes will usually approximate in situ physiological conditions, but may be modified to higher or lower ionic strength where advantageous. The actual ions may be modified to conform to standard buffers used in physiological or analytical contexts.

The size and structure of the polypeptide should be in a substantially stable and globular state, and usually not in a denatured state. The polypeptide may be associated with other polypeptides in a quaternary structure, e.g., to confer solubility.

The solvent will usually be a biologically compatible buffer, of a type used for preservation of biological activities, and will usually approximate a physiological solvent. On some occasions, a detergent will be added, typically a mild non-denaturing one.

Solubility is usually measured in Svedberg units, which are a measure of the sedimentation velocity of a molecule under particular conditions. The determination of the sedimentation velocity was classically performed in an analytical ultracentrifuge, but is typically now performed in a standard ultracentrifuge. See, Freifelder (1982) Physical Biochemistry (2d ed.), W.H. Freeman, and Cantor and Schimmel (1980) Biophysical Chemistry, parts 1-3, W.H. Freeman & Co., San Francisco, each of which is hereby incorporated herein by reference. As a crude determination, a sample containing a "soluble" polypeptide is spun in a standard full sized ultracentrifuge at about 50K rpm for about 10 minutes, and soluble molecules will remain in the supernatant. A soluble particle or polypeptide will typically be less than about 305, more typically less than about 15S, usually less than about l0S, more usually less than about 68, and, in particular embodiments, preferably less than about 4S, and more preferably less than about 3S.

This invention provides platelet-derived growth factor polypeptides and proteins having platelet-derived growth factor receptor ligand binding activity. The receptors of the present invention include PDGF receptor amino acid sequences Also provided are homologous sequences, allelic variations, induced such as those shown in Tables 6, 7, 8, 9, and 10. mutants, alternatively expressed variants, and proteins encoded by DNA which hybridize under high stringency conditions to PDGF receptor encoding nucleic acids retrieved from naturally occurring material.

The platelet-derived growth factor receptor peptides of the present invention will exhibit at least about 80% homology with naturally occurring domains of hPDGF receptor sequences in the domains D1, D2, D3, D4, and D5, typically at least about 85% homology with a natural form of a receptor sequence, more typically at least about 90% homology, usually at least about 95% homology, and more usually at least about 97% homology.

Homology, for polypeptides, is typically measured using sequence analysis software, see, e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wisconsin 53705. Protein analysis software matches similar sequences using measure of homology assigned to various substitutions, deletions, substitutions, and other modifications. Similar, or homologous, substitutions for LBR segments will be made in known sequences, thereby producing new binding molecules having modified affinity or specificity of ligand binding.

Various other software analysis programs can analyze the conformational structure of a polypeptide. Homologous conformation may also be achieved by appropriate insertion, deletion, substitution, or modification of amino acid sequences. Since the conformational structure of the domains and B—strand segments is only partially understood, the present invention also encompasses various modifications to the sequences disclosed and retaining these structural features.

In particular, ligand binding function is believed to be localized to the extracellular domain, particularly the LBR's, and the soluble forms will preferably retain this particular function. Soluble fragments of PDGF receptors will be useful in substituting for or for interfering with, e.g., blocking, by competing for PDGF binding, the functions of the natural receptor both in vitro and in vivo. Alternatively, soluble forms may interfere with the dimerization of PDGF receptor polypeptides, since the proteins may normally be in, or function in, a dimer form. Receptor dimerization may be essential for proper physiological signal transduction, and introduction of fragments may function to interrupt these processes by blocking their dimerization.

PDGF receptor polypeptides may be purified using techniques of classical protein chemistry, see, e.g., Deutscher (ed.) (1990) Guide to Purification; Methods in Enzymology, Vol. 182, which is hereby incorporated herein by reference.

Alternatively, a lectin affinity chromatography step may be used, or a highly specific ligand affinity chromatography procedure, e.g., one that utilizes a PDGF conjugated to biotin through cysteine residues of the protein mitogen. Purified PDGF receptor polypeptides may also be obtained by a method such as PDGF affinity chromatography using activated CH- Sepharose coupled to PDGF through primary amino groups as described in Imamura et al. l55:583-590. (1988) Biochem. Biophvs. Res.

Commun.

Depending on the availability of specific antibodies, specific PDGF receptor peptide constructs may also be purified using immuno-affinity chromatography. Antibodies prepared, as described below, may be immobilized to an inert substance to generate a highly specific immuno-affinity column. See, e.g., Harlow and Lane (1990) Monoclonal Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, which is hereby incorporated herein by reference.

Various cells or tissues may be selected as starting materials, usually selected on the basis of abundant expression of the desired receptor construct or polypeptide. High expression promoter sequences may be operably linked to a recombinant sequence, preferably an inducible promoter. The promoter is operably linked when it operates to promote the sequence. Appropriate cells that contain relatively large amounts of the receptor protein, as determined by high affinity binding of PDGF, can be transformed with variants of the PDGF receptor polypeptides. These may be used to replace the natural form of PDGF receptor by a construct with a deletion or insertion.

The ligand binding regions (LBR's) or other segments may be "swapped" between different new fusion constructs or fragments. Thus, new chimeric polypeptides exhibiting new combinations of segments can result from the structural linkage of different functional domains. Ligand binding regions which confer desired or modified specificities may be combined with other domains which have another function, e.g., each Ig-like domain could be substituted by a similar domain from other related polypeptides, or LBR's between different alleles or similar receptors may be combined.

The present invention also provides for fusion polypeptides between the receptor polypeptide domains and other homologous or heterologous proteins. Homologous proteins may be fusions between similar but different growth factor receptors resulting in, e.g., a hybrid protein exhibiting ligand specificity of one receptor with an intracellular domain of another, or a receptor which may have altered affinity or a broadened or narrowed specificity of binding. Likewise, heterologous fusions may be constructed which exhibit a combination of properties or activities of the derivative proteins. Typical examples are fusions of a reporter polypeptide, e.g., luciferase, with a domain of a receptor, e.g., a ligand binding domain from the extracellular region of a human platelet—derived growth factor receptor, so that the presence or location of a desired ligand may be easily determined. See, e.g., Dull et al., U.S. Patent No. 4,859,609, which is hereby incorporated herein by reference. other gene fusion partners include bacterial B-galactosidase, trpE, protein A, B-lactamase, a-amylase, alcohol dehydrogenase, and yeast a-mating factor. -816.

See, e.g., Godowski et al., (1988) Science 241: Additional sequences with various defined functions may be found by searching through the GenBankm (National Institutes of Health) sequence data bank. A heterologous fusion protein is one which includes sequences not naturally found in conjunction with one another. Thus, a heterologous fusion protein may be a fusion of two similar, and homologous, sequences.

Fusion proteins would typically be made by either recombinant nucleic acid methods with expression, or by synthetic polypeptide methods. Techniques for nucleic acid manipulation are described generally, for example, in Sambrook et al. volumes 1-3, Cold Spring Harbor Laboratory, which is hereby incorporated herein by reference. (1989) Molecular Cloning: A Laboratory Manual (2nd ed.) Techniques for synthesis of polypeptides are described, for example in Merrifield (1963) Q; Amer. Chem. 85:2149-2456: Atherton et al. (1989) Solid Soc.

Phase Peptide Synthesis: A Practical Approach, IRL Press, Oxford; and Merrifield (1986) Science 232:34l-347: each of which is hereby incorporated herein by reference.

The recombinant nucleic acid sequences used to produce fusion proteins of the present invention may be derived from natural or synthetic sequences. Many natural gene sequences are available from various cDNA or from genomic libraries using appropriate probes, see, e.g., GenBankm, National Institutes of Health.

Typical probes for isolating platelet-derived growth factor receptor genes may be selected from sequences of Tables 1 and 2, in accordance with standard procedures. Suitable synthetic DNA fragments may be prepared, e.g., by the phosphoramidite method described by Beaucage and Carruthers (1981) Tetra. Letts. 22:l859-1862. A double stranded fragment may then be obtained by either synthesizing the complementary strand and hybridizing the strands together under appropriate conditions or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.

III. Nucleic Acids The present invention provides nucleic acid sequences encoding various PDGF receptor sequences described above.

Tables 1 and 2, respectively set forth the corresponding cDNA sequences encoding human type B and type A PDGF receptor polypeptides.

Substantial homology in the nucleic acid context means either that the segments, or their complementary strands, when compared, are the same when properly aligned, with appropriate nucleotide insertions or deletions, in at least about 60% of the residues, typically at least about 70%, more typically at least about 80%, usually at least about 90%, and more usually at least about 95 to 98% of the nucleotides.

Appropriate nucleotide insertions or deletions include interdomain sequences, or those external to the cysteines within a domain, but the sequences within the paired cysteines (or their equivalents in the D4 domains) will often be very important to retain. Structural homology will exist when there is at least about 55% homology over a stretch of at least about nucleotides, typically at least about 65%, more typically at least about 75%, usually at least about 90%, and more usually at least about 95% or more.

Alternatively, substantial homology exists when the segments will hybridize under selective hybridization conditions, to a strand, or its complement, typically using a sequence of at least about 20 contiguous nucleotides derived from Table 1 or 2. However, larger segments would usually be preferred, e.g., at least about 30 contiguous nucleotides, more usually at least about 40, and preferably more than about 50.

Selectivity of hybridization exists when hybridization occurs which is more selective than total lack of specificity.

Kanehisa (1984) Nucleic Acids Res. 12:203—2l3, which is incorporated herein by reference.

See, stringent hybridization conditions will normally include salt concentrations of less than about 1 M, typically less than about 700 mM, more typically less than about 500 mM, usually less than about 400 mM, more usually less than about 300 mM, and preferably less than about 200 mM. Temperature conditions will typically be greater than about 20°C, more typically greater than about 25‘C, usually greater than about °C, more usually greater than about 37°C, and preferably in excess of about 40°C, depending upon the particular application. As other factors may significantly affect the stringency of hybridization, including, among others, base composition and size of the complementary strands, presence of organic solvents, and extent of base mismatching, the combination of parameters is more important than the absolute measure of any one.

Probes may be prepared based on the sequence of the PDGF receptor encoding sequences provided in Tables 1 and 2.

The probes may be used to isolate other PDGF receptor nucleic acid sequences by standard methods. e.g., Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, vols. 1-3, CSH Press, N.Y., which is hereby incorporated herein by reference. other similar nucleic acids may be selected for by using homologous nucleic acids.

See, Alternatively, nucleic acids encoding these same or similar receptor polypeptides may be synthesized or selected by making use of the redundancy in the genetic code. Various codon substitutions may be introduced, e.g., silent changes thereby providing various convenient restriction sites, or to optimize expression for a particular system, e.g., to match the optimum codon usage. Mutations may be introduced to modify the properties of the receptors, perhaps to change the ligand binding affinities, the inter— chain affinities, or the polypeptide degradation or turnover rate.

The DNA compositions of this invention may be derived from genomic DNA or CDNA, prepared by synthesis or may be a hybrid of the various combinations. Recombinant nucleic acids comprising sequences otherwise not naturally occurring in continuity are also provided by this invention. An isolated DNA sequence includes any sequence that has been obtained by primer or hybridization reactions or subjected to treatment with restriction enzymes or the like.

Synthetic oligonucleotides can be formulated by the triester method according to Matteucci et al. (1981) J. Am.

Chem. Soc. 103:3185 or by other methods such as commercial automated oligonucleotide synthesizers. oligonucleotides can be labeled by excess polynucleotide kinase (e.g., about 10 units to 0.1 nanomole substrate is used in connection with 50 mM Tris, pH 7.6, 5 mM dithiothreitol, 10 mM MgCl2, 1-2 mM ATP, 1.7 pmoles "P-ATP (2.9 mCi/mmole) 0.1 mM spermidine, 0.1 mM EDTA). Probes may also be prepared by nick translation, Klenow fill-in reaction, or other methods known in the art. e.g., Sambrook et al.

See, cDNA or genomic libraries of various types may be screened for new alleles or related sequences. The choice of CDNA libraries normally corresponds to a tissue source which is abundant in mRNA for the desired receptors. Phage libraries are normally preferred, but plasmid libraries may also be used.

Clones of a library are spread onto plates, transferred to a substrate for screening, denatured, and probed for the presence of desired sequences.

For example, with a plaque hybridization procedure, each plate containing bacteriophage plaques is replicated onto duplicate nitrocellulose filter papers (Millipore—HATF). The phage DNA is denatured with a buffer such as 500 mM NaOH, 1.5 M NaCl for about 1 minute, and neutralized with, e.g., 0.5 M Tris-Hcl, pH 7.5, 1.5 M Nacl (3 times for 10 minutes each).

The filters are then washed. After drying, the filters are typically baked, e.g., for 2 hours at 80°C in a vacuum oven.

The duplicate filters are prehybridized at 42°C for 4-24 hours with 10 ml per filter of DNA hybridization buffer (20-50% formamide, 5X SSC, pH 7.0, 5X Denhardt's solution (polyvinylpyrrolidone, plus Ficoll and bovine serum albumin; X = 0.02% of each), 50 mM sodium phosphate buffer at pH 7.0, 0.2% SDS, and 50 pg/ml denatured salmon sperm DNA).

Hybridization with an appropriate probe may be performed at 42°C for 16 hrs with 10 ml/filter of 1 x 10° cpm/ml of DNA hybridization buffer containing radioactively labeled probe.

The final concentration of formamide is varied according to the length of the probe and the degree of stringency desired. See, e.g., Wetmur and Davidson (1968) J. Mol. Biol. 31:349—370; and M. Kanehisa (1984) Nuc. Acids Res. 12:203-213, each of which is incorporated herein by reference, for a discussion of hybridization conditions and sequence homology.

An oligonucleotide probe based on the disclosed amino acid sequences may be used to site specifically mutate or generate recombinant fusion or deletion constructs. .See, e.g., Tables 11 and 12 for preferred oligonucleotide reagents.

Procedures such as those described by Kimbel et al. (1987) Methods in Enzymology 154:367, may be used. The sequences PA1 through PA9 correspond to seq. ID No. 6 through 14, respectively, and sequences PA101 through PA109 correspond to Seq. ID No. 15 through 23, respectively.

TABLE 11 HUMAN B-type PDGF-R HDTAGENESIS OLIGOMERS Domain 5 / 3'NonCoding ' CCA CAC TCC TTG CCC TTT AAG / TAGCTTCCTGTAGGGGGCTG 3‘ p H s L p F K / * itttittiit Domain 4 / 3'NonCoding ' TCC TTC GAC CTA CAG ATC BAT / TAGCTTCCTGTAGGGGGCTG 3' S F Q L Q I N / * **'k****'k-it Domain 3 / 3'NonCoding ' ATC ACC GTG GTT GAG AGC GGC / TAGCTTCCTGTAGGGGGCTG 3' I T V V E S G / * iiiiiiiiii Domain 2 3'NonCoding I ' TAC AGA CTC CAG GTG TCA TCC / TAGCTTCCTGTAGGGGGCTG 3' Y R L Q V 5 5 / i ********** Domain 1 / 3'NonCoding ' CTC TAC ATC TTT GTG CCA GAT CCC / TAGCTTCCTGTAGGGGGCTG 3' L Y I F V P D P / * *'k**'k*** Signal Sequence Domain 1 / Domain 2 ‘ CAG ATC TCT CAG GGC:CTG GTC / ACC GTG GGC TTC CTC CCT AAT CAT 3' Q I S Q G : L V / T V G F L P N D Signal Sequence Domain 1 / Domain 3 ' CAG ATC TCT CAG GGC:CTG GTC/ATC AAC GTC TCT GTG AAC GCA GTG CAG3' Q I S Q G : L V / I N V S V N A V Q Signal sequence Domain 1 / Domain 4 ‘ CAG ATC TCT CAG GGC:CTG GTC / TAC GTG CGG CTC CTG GGA GAG CTG 3' Q I S Q G : L V / Y V R L L G E V Signal Sequence Domain 1 / Domain 5 ' CAG ATC TCT CAG GGC : Q I S Q G CTG GTC / GTC CGA GTG are GAG CTA AGT 3' L v / v R v L w L A TABLE 12 PROPOSED HUMAN A-type PDGF-R HUTAGENESIS OLIGOHERS Domain 5 / 3'NonCoding PAlO1 5‘ GCT CCC ACC CTG CGT TCT GAA / TAACTGGCGGATTCGAGGGG 3' A p T L R 5 E / i itikttiiit Domain 4 / 3'NonCoding PAlO2 5' GAA CTG TTA ACT CAA GTT CCT / TAACTGGCGGATTCGAGGGG 3' E L L T Q V P / 9 tiiiiiitii Domain 1 / 3'NonCoding PAl05 5‘ ATT TAC ATC TAT GTG CCA GAC CCA / TAACTGGCGGATTCGAGGGG 3‘ I Y I Y V P D P / t ********** Signal Sequence : Domain 1 / Domain 2 PAlO6 5' AGC CTA ATC CTC TGC CAG CTT / GAT GTA GCC TTT GTA CCT CTA GGA 3‘ SLILC:QL/DVAFVPLG Signal Sequence : Domain 1 / Domain 3 PA107 5' AGC CTA ATC CTC TGC CAG CTT/GAG CTG GAT CTA GAA ATG GAA GCT CTT 3' S L I L C : Q L / E L D L E M E A L Signal Sequence : Domain 1 / Domain 4 PAl08 5' AGC CTA ATC CTC TGC CAG CTT / TTC ATT GAA ATC AAA CCC ACC TTC 3‘ S L I L C : Q L / F I E I K P T F Signal Sequence : Domain 1 / Domain 5 PAIO9 5' AGC CTA ATC CTC TGC CAG CTT / TCA TCC ATT CTG GAC TTG GTC 3' S L I L C : Q L / S S I L D L V In accordance with this invention any isolated DNA sequence which encodes substantially a PDGF-R complete structural sequence can be used as a probe. Alternatively, any DNA sequence that encodes a PDGF-R hydrophobic signal sequence and its translational start site may be used. An isolated partial DNA sequence which substantially encodes intact domains exhibiting PDGF-R activity (e.g., ligand or PDGF-R binding) is also part of this invention. Preferred probes are CDNA clones of PDGF receptor polypeptides.

The DNA sequences used in this invention will usually comprise intact domain structures, typically at least about 5 codons (15 nucleotides), more typically at least about 9 codons, usually at least about 13 codons, more usually at least about 18 codons, preferably at least about 25 codons and more preferably at least about 35 codons. One or more introns may also be present. This number of nucleotides is usually about the minimal length required for a successful probe that would hybridize specifically with a PDGF receptor sequence. For example, epitopes characteristic of a PDGF-R may be encoded in short peptides. Usually the wild-type sequence will be employed, in some instances one or more mutations may be introduced, such as deletions, substitutions, insertions, or inversions. These modifications may result in changes in the amino acid sequence, provide silent mutations, modify a restriction site, or provide specific mutations. The genomic sequence will usually not exceed about 200 kb, more usually not exceed about 100 kb, preferably not greater than about 0.5 kb.

Portions of the DNA sequence having at least about 10 nucleotides from a DNA sequence encoding an PDGF receptor peptide will typically be used, more typically at least about nucleotides, usually at least about 20 nucleotides, more usually at least about 25 nucleotides, and preferably at least about 30 nucleotides. The probes will typically be less than about 6 kb, usually fewer than about 3.0 kb, and preferably less than about 1 kb. The probes may also be used to determine whether mRNA encoding a specific PDGF-R is present in a cell or different tissues.

The natural or synthetic DNA fragments coding for a desired platelet-derived growth factor receptor fragment will usually be incorporated into DNA constructs capable of introduction to and expression in an in yigrg cell culture.

Often the DNA constructs will be suitable for replication in a unicellular host, such as yeast or bacteria, but may also be intended for introduction to, with and without integration within the genome, cultured mammalian, or plant or other eukaryotic cell lines. Human cells may be preferred hosts.

Higher eukaryote host cells will often be preferred because their glycosylation and protein processing patterns more likely simulate human processing. DNA constructs prepared for introduction into bacteria or yeast will typically include a replication system recognized by the host, the intended DNA fragment encoding the desired receptor polypeptide construct, transcriptional and translational initiation regulatory sequences operably linked to the polypeptide encoding segment, and transcriptional and translational termination regulatory sequences operably linked to the polypeptide encoding segment.

The transcriptional regulatory sequences will typically include a heterologous enhancer or promoter which is recognized by the host. The selection of an appropriate promoter will depend upon the host, but promoters such as the trp, lac, and phage promoters, tRNA promoters, and glycolytic enzyme promoters are known and available. e.g., Sambrook et al. (1989).

Conveniently available expression vectors which include the See, replication system and transcriptional and translational regulatory sequences together with the insertion site for the platelet-derived growth factor receptor DNA sequence may be employed. Examples of workable combinations of cell lines and expression vectors are described, e.g., in Sambrook et al. (1989); see also, Metzger et al. (1988) Nature 334:31-36.

Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, an enhancer and necessary processing information sites, e.g., ribosome—binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. Preferably, the enhancers or promoters will be those naturally associated with genes encoding the PDGF receptor polypeptides, although it will be understood that in many cases others will be equally or more appropriate. Other preferred expression control sequences are enhancers or promoters derived from viruses, such as SV40, Adenovirus, Bovine Papilloma Virus, and the like.

Similarly, preferred promoters are those found naturally in immunoglobulin—producing cells, sgg, e.g., U.S.

Patent No. 4,663,281, which is incorporated herein by reference, but SV40, polyoma virus, cytomegalovirus (human or murine) and the LTR from various retroviruses, e.g., murine leukemia virus, murine or Rous sarcoma virus and HIV, may be utilized, as well as promoters endogenous to PDGF—R genes.

See, Enhancers and Eukaryotic Gene Expression, (1983) Cold Spring Harbor Press, N.Y., which is incorporated herein by reference.

The vectors containing the DNA segments of interest, e.g., a PDGF receptor polypeptide gene or CDNA sequence, can be transferred into the host cell by well-known methods, which vary depending on the type of cellular host. calcium chloride transfection is commonly utilized for For example, prokaryotic cells, whereas calcium phosphate treatment may be used for other cellular hosts. See generally, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed.) CSH Press, which is incorporated herein by reference. The term "transformed cell" is meant to also include the progeny of a transformed cell.

As with the purified polypeptides, the nucleic acid segments associated with the ligand-binding segment, the extracellular domain and the intracellular domain are particularly useful. These gene segments will be used as probes for screening for new genes exhibiting similar biological activities, though the controlling elements of these genes may also be of importance.

IV. Methods for Making PDGF Receptor Polypeptide Constructs DNA sequences may also be used to express PDGF-R polypeptides. For example, a DNA sequence of from about 21 nucleotides (encoding about 7 amino acids) to about 2.1 kb— (about 700 amino acids) may be used to express a polypeptide having a PDGF receptor specific activity, typically ligand-binding. In particular, constructs retaining the ligand binding regions will be useful, as these constructs will possess binding activity.

In particular, various synthetic linkers and probes may be constructed to facilitate genetic engineering of the PDGF-R nucleic acid sequences. Polymerase chain reaction (PCR) techniques can be applied to producing large quantities of fragments or segments useful in the proper manipulation of the sequences encoding the constructs. e.g., Alternatively, nucleic acid synthesizers can produce sufficiently large quantities of fragments for hybridizing to any preselected sequence, e.g., from Table 1 or 2, or for manipulating the sequence to add or See, Innis et al. (1990) PCR Protocols, Academic Press. delete specific domains or segments. Particularly important segments will be the LBR's.

Large quantities of the receptor proteins may be prepared by expressing the whole receptor or parts of the receptor contained in the expression vehicles in compatible hosts such as E. coli, yeast, mammalian cells, insect cells, or frog oocytes. The expression vehicles may be introduced into the cells using methods well known in the art such as calcium phosphate precipitation (discussed below), lipofectin electroporation, or DEAE dextran transformation.

Usually the mammalian cell hosts will be immortalized cell lines. To study the characteristics of a PDGF—R and its corresponding ligand, it will be useful to transfect, or transform mammalian cells which lack or have low levels of a PDGF receptor. Preferably, a signal sequence can serve to direct the peptide to the cell membrane or for secretion.

Cells lacking significant amounts of PDGF receptors include Chinese hamster ovary (CHO) cells, most epithelial cell lines, and various human tumor cell lines.

Transformed or transfected cells can be selected which incorporate a DNA sequence which encodes a receptor that is functionally equivalent to a wild—type receptor thereby conferring a PDGF—sensitive mitogenic response. Such cells will enable the analysis of the binding properties of various added PDGF receptor polypeptides. Transfected cells may also be used to evaluate the effectiveness of a composition or drug as a PDGF antagonist or agonist. The level of receptor tyrosine kinase activity or the rate of nucleic acid synthesis can be determined by contacting transfected cells with drugs or ligands and comparing the effects of various ligand analogues against the controls. Although the most common procaryote cells used as hosts are strains of E. coli, other prokaryotes such as Bacillus subtilis or Pseudomonas may also be used. The DNA sequences of the present invention, including fragments or portions of the sequence encoding for receptor polypeptides comprising intact structural domains, a portion of the receptor, or a polypeptide having an PDGF-R activity, can be used to prepare an expression vehicle or construct for a PDGF-R polypeptide or polypeptide having a PDGF-R activity. the control sequence will be a eukaryotic promoter for Usually expression in a mammalian cell. In some vehicles the receptor's own control sequences may also be used. A common prokaryotic plasmid vector for transforming E. coli is pBR322 or its derivatives, e.g. the plasmid pkt279 (Clontech), see (1977) Qggg, 2:95. also contain prokaryotic promoters for transcription Bolavar et al. The prokaryotic vectors may initiation, optionally with an operator. Examples of most commonly used prokaryotic promoters include the beta—lactamase (penicillinase); lactose (lac) promoter, see Cheng et al. (1977) Nature, 198:1056; tryptophan promoter (trp), see Goeddell et al. (1980) 8: 457); PL promoter: and the N-gene ribosome binding site, see Shimatake et al.

Nucleic Acid Res., (1981) Nature, 292:128-; each of which is hereby incorporated herein by reference.

Promoters used in conjunction with yeast can be promoters derived from the enolase gene, see Holland et al. (1981) J. Biol. Chem., 256:1385 ; or the promoter for the synthesis of glycolytic enzymes such as 3-phosphoglycerate (1980) J. Biol. Chem., 255:.

Appropriate non-native mammalian promoters will include the early and late promoters from SV40, see Fiers et kinase, see Hitzeman et al. al. (1978) Nature, 273:1l3; or promoters derived from murine muloney leukemia virus, mouse mammary tumor virus, avian sarcoma viruses, adenovirus II, bovine papilloma virus, or polyoma. In addition, the construct may be joined to an amplifiable gene, e.g. dihydrofolate reductase (DHFR) so that multiple copies of the PDGF receptor gene may be made. See, e.g., Kaufman et al. (1985) M01. and Cell. Biol. 5:1750-1759; and Levinson et al. EPO publication nos. 0117059 and 0117060, each of which is incorporated hereby by reference.

Prokaryotes may be transformed by various methods, including using Caclz, see Cohen (1972) Proc. Nat'l Acad. Sci. ggg, 69:2110; or the Rbcl method, see Maniatis et al. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press. Yeast may be transformed, e.g., using a method described by Van Solingen et al. (1977) J. Bacteriol. 130:946; (1979) Proc. Nat'l Acad. Sci. USA 76:3829.

With respect to eukaryotes, mammalian cells may be transfected using a calcium phosphate precipitation method, see, e.g., Graham and van der Eb (1978) Virolo or Hsiao et al. , 52:546: or by lipofectin (BRL) or retroviral infection, see, e.g., Gilboa (1983) Experimental Manipulation of Gene Expression, Chap. 9, Academic 175. The actual expression vectors containing appropriate sequences may be prepared according to standard techniques involving ligation and restriction enzymes. e.g., Maniatis sgpga.

Press P.

See Commercially available restriction enzymes for cleaving specific sites of DNA may be obtained from New England BioLabs, Beverly, Massachusetts.

Particular cotransformations with other genes may be particularly useful. For example, it may be desired to co- express the nucleic acid with another processing enzyme. Such enzymes include signal peptidase, tertiary conformation conferring enzymes, or glycosylating enzymes. This expression method may provide processing functions which otherwise might be lacking in the expression host, e.g., mammalian-like glycosylation in a prokaryote expression system.

Alternatively, the host cell selected for expression may be chosen on the basis of the natural expression of those processing enzymes.

Cell clones are selected by using markers depending on the mode of the vector construction. The marker may be on the same or a different DNA molecule preferably the same DNA molecule. With mammalian cells the receptor gene itself may be the best marker. In prokaryotic hosts the transformant may be selected by resistance to ampicillin, tetracycline, or other antibiotics. Production of a particular product based on temperature sensitivity or compensation may serve as appropriate markers. Various methods may be used to harvest and purify the PDGF-R receptor protein or peptide fragment.

The peptide may be isolated from a lysate of the host. The peptide may be isolated from the cell supernatant if the peptide is secreted. The PDGF-R peptide is then further purified as discussed above using HPLC, electrophoresis, or affinity chromatography, e.g., immuno-affinity or ligand affinity.

Another method which can be used to isolate CDNA clones of PDGF-R related species involves the use of the polymerase chain reaction (PCR). e.g., Saiki et al. (1985) Science 230:1350. In this approach two oligonucleotides corresponding to distinct regions of the PDGF-R sequence are synthesized and then used in the PCR reaction, typically to amplify receptor-related mRNA transcripts from an mRNA source.

Annealing of the oligonucleotides and PCR reactions are The resulting amplified fragments are subcloned, and the resulting recombinant colonies are probed with ”P-labeled full-length PDGF-R CDNA. Clones which hybridize under low but not high stringency conditions represent PDGF-R related mRNA transcripts.

See, performed under conditions of reduced stringency.

This approach can also be used to isolate variant PDGF-R cDNA species which arise as a result of alternative splicing, see Frohman et al. (1988) Proc. Nat'l Acad. Sci. USA, 85:8998.

V. Antibodies Polyclonal and/or monoclonal antibodies to the various PDGF receptor constructs, receptor peptides, and peptide fragments may also be prepared. Peptide fragments may be prepared synthetically in a peptide synthesizer and coupled to a carrier molecule (i.e., keyhole limpet hemocyanin) and injected into rabbits over several months. The rabbit sera is tested for immunoreactivity to the PDGF receptor protein or fragment. Monoclonal antibodies may be made by injecting mice with PDGF-R protein, PDGF—R polypeptides, or mouse cells expressing high levels of the cloned PDGF receptor on its cell surface. Monoclonal antibodies will be screened by ELISA and tested for specific immunoreactivity with the PDGF receptor protein or polypeptides thereof. See, Harlow and Lane (1988) Antibodies: A Laboratory Manual, CSHarbor Press, which is hereby incorporated herein by reference. These antibodies will be useful in assays as well as pharmaceuticals.

Once a sufficient quantity of the desired PDGF receptor polypeptide construct has been obtained, the protein may be used for various purposes. A typical use is the production of antibodies specific for binding to epitopes characteristic of these receptors. These antibodies may be either polyclonal or monoclonal and may be produced by in vitro or in yiyg techniques.

For production of polyclonal antibodies, an appropriate target immune system is selected, typically a mouse or rabbit. The substantially purified antigen is presented to the immune system in a fashion determined by methods appropriate for the animal and other parameters well known to immunologists. Typical sites for injection are in the footpads, intramuscularly, intraperitoneally, or intradermally.

Of course, another species may be substituted for a mouse or rabbit, typically a mammal, but possibly a bird or other animal.

An immunological response is usually assayed with an immunoassay. Normally such immunoassays involve some purification of a source of antigen, for example, produced by the same cells and in the same fashion as the antigen was produced. The immunoassay may be a radioimmunoassay, an enzyme-linked assay (ELISA), a fluorescent assay, or any of many other choices, most of which are functionally equivalent but may exhibit particular advantages under specific conditions.

Monoclonal antibodies with affinities of at least about 106 M* preferably 10& 10”, or higher will be made by standard procedures as described, e.g., in Harlow and Lane, (1988) Antibodies: A Laboratory Manual, CSH Press: or Goding, (1986) Monoclonal Antibodies: Principles and Practice (2d ed) Academic Press, New York, which are hereby incorporated herein by reference. Briefly, appropriate animals will be selected and the desired immunization protocol followed. After the appropriate period of time, the spleens of such animals are excised and individual spleen cells fused, typically, to immortalized myeloma cells under appropriate selection conditions. Thereafter the cells are clonally separated and the supernatants of each clone are tested for their production of an appropriate antibody specific for the desired region of the antigen. other suitable techniques involve in yitrg exposure of lymphocytes to the antigenic polypeptides or alternatively to selection of libraries of antibodies in phage or similar vectors. ggg, Huse et al. "Generation of a Large Combinatorial Library of the Immunoglobulin Repertoire in Phage Lambda," Science 246:1275-1281 (1989), hereby incorporated herein by reference. The polypeptides and antibodies of the present invention may be used with or without modification.

Frequently, the polypeptides and antibodies will be labeled by joining, either covalently or non-covalently, a substance which provides for a detectable signal. A wide variety of labels and conjugation techniques are known and are reported extensively in both the scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescens, chemiluminescers, magnetic particles and the like. Patents, teaching the use of such labels include U.S. Patent Nos. 3,817,837? 3,850,752: 3,939,350: 3,996,345? ,277,437; 4,275,149: and 4,366,241. Also, recombinant immunoglobulins may be produced, sgg Cabilly, U.S. Patent No. 4,816,567.

Antibodies of particular interest are those raised against the ligand binding regions. These will include some antibodies which function as ligands. Or, antibodies may be used to select for compounds which could serve as ligands for modified receptors. See, e.g., Meyer (1990) Nature 347:424— 425; and Pain et al. (1990) Nature 347:444—447; each of which is hereby incorporated herein by reference.

VIII. Methods for Use The present invention provides platelet-derived growth factor receptor (PDGF-R) polypeptide purification methods as well as methods for synthesizing PDGF receptors within cells. Also provided are homogeneous receptors produced by these methods, nucleic acid sequences encoding the receptors or portions of the receptors, as well as expression vehicles containing these sequences, cells comprising the PDGF- receptors, and antibodies to the receptors. In particular, the present invention provides methods for assaying binding and other activities of receptor-like proteins having rearranged combinations of the domains.

The extracellular region of the human type B PDGF receptor protein has been used to successfully bind PDGF BB ligand in a receptor activation assay. PDGF BB ligand binding to NIH3T3 cell-associated PDGF receptors is measured. Ligand binding causes phosphorylation (activation) of the cell associated receptors. Receptor phosphorylation is followed in a multi-step process which first involves solubilization of NIH3T3 cells and separation of cell proteins by electrophoresis of cell extracts on sodium dodecyl sulfate polyacrylamide gels.

Gels are blotted onto nitrocellulose and treated with anti- phosphotyrosine monoclonal antibodies to aid in the detection of phosphorylated PDGF receptor. Monoclonal antibodies are visualized through autoradiography of antibody-associated 125-I protein A which has been introduced at the terminal stage of the assay.

If human type B receptor protein (at about a 60 fold molar excess to PDGF BB ligand) is preincubated with ligand for 1 hour prior to incubation with NIH3T3 cells, there is no cell- associated PDGF receptor phosphorylation. This indicates that the human type B PDGF receptor protein binds PDGF BB ligand in solution and prevents the ligand from activating cell- associated PDGF receptors. Thus, polypeptides which contain LBR's may be used to block normal PDGF responses.

The domain containing str :tures of the present invention will find use both as diagnostic and therapeutic reagents. The receptor polypeptides may be used as affinity reagents for detecting or binding ligand, as well as for interacting with receptor-like proteins, e.g., affecting receptor protein dimerization. The polypeptides will also be useful as reagents for detecting or purifying other proteins which associate with the receptors or fragments thereof.

The receptor polypeptides will also find use in generating other reagents, e.g., antibodies specific for binding epitopes peculiar to the modified receptors. In particular, antibodies raised against newly formed ligand binding determining segments may serve as ligands for the modified receptors. These techniques may provide for separating various functionalities of the receptors, thereby isolating each of the different effector functions from others, in response to PDGF binding.

The modified receptors of the present invention also provide methods for assaying ligands for them. For example, soluble ligand binding fragments will be useful as competing sites for ligand binding, a useful property in a ligand binding assay. In particular, the present invention provides an assay to screen for PDGF binding inhibition, allowing screening of large numbers of compounds. These compounds may be assayed in vitro, which allows testing of cytotoxic or membrane disruptive compounds. The present solid phase system allows reproducible, sensitive, specific, and readily automated assay procedures.

Polystyrene 96-well plates may be coated with the appropriate construct with LBR's to assay for ligand binding activity.

Moreover, modifications to the ligand binding domains will lead to binding region combinations with different ligand binding affinities. Thus, modulation of ligand effected response may be easily achieved by inclusion of the appropriate affinity modified analogue.

Solid phase assays using these modified receptors may also be developed, providing greater sensitivity or improved capacity over unmodified binding regions.

Diagnostic kits comprising these reagents are also provided. The kit typically comprise a compartmentalized enclosure, e.g., a plastic substrate having diagnostic reagents of the invention attached thereto. The package will typically also include various buffers, labeling reagents, and other reagents as appropriate for the diagnostic test to be performed. Instructions for use of the related reagents and interpretation of the results will be provided.

In particular, the important functional segment of the extracellular domain will usually be attached to a plastic or other solid phase substrate. The binding regions will usually be selected for a combination of the affinity and ligand binding spectrum of the modified binding segments.

Appropriate ligands will often be introduced to determine the ligand binding activity and affinity. Different LBR combinations will be used, and can be used to test for differently modified, e.g., labeled, ligands.

In addition, the peptides will be useful for therapeutic administration. The quantities of reagents necessary for effective therapy will depend upon many different factors, including means of administration, target site, physiological state of the patient, and other medicants administered. Thus, treatment dosages should be titrated to optimize safety and efficacy. Typically, dosages used in vitro may provide useful guidance in the amounts useful for in situ administration of these reagents. Animal testing of effective doses for treatment of particular disorders will provide further predictive indication of human dosage. Various considerations are described, e.g., in Gilman et al. (eds), (1990) Goodman and Gilman's: The Pharmacological Basis of Therapeutics, 8th ed., Pergamon Press; and Remington's Pharmaceutical Sciences, (1985) 7th ed., Mack Publishing Co., Easton, Penn.; each of which is hereby incorporated by reference. Methods for administration are discussed therein, e.g., for oral, intravenous, intraperitoneal, or intramuscular administration, transdermal diffusion, and others.

Pharmaceutically acceptable carriers will include water, saline, buffers, and other compounds described, e.g., in the Merck Index, Merck & Co., Rahway, New Jersey. Because of the high affinity binding between PDGF and its receptors, low dosages of these reagents would be initially expected to be effective. Thus, dosage ranges would ordinarily be expected to be in amounts lower than 1 mM concentrations, typically less than about 10 uM concentrations, usually less than about nM, preferably less than about 10 pM (picomolar), and most preferably less than about 1 fM (femtomolar), with an appropriate carrier.

The pharmaceutical compositions will be administered by parenteral, topical, oral or local administration, such as by aerosol or transdermally, for prophylactic and/or therapeutic treatment. The pharmaceutical compositions can be administered in a variety of unit dosage forms depending upon the method of administration. For example, unit dosage forms suitable for oral administration include powder, tablets, pills, capsules and dragees.

Preferably, the pharmaceutical compositions are administered intravenously. Thus, this invention provides compositions for intravenous administration which comprise a solution of the compound dissolved or suspended in an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers may be used, e.g., water, buffered water, 0.4% saline, and the like. sterilized by conventional, well known sterilization These compositions may be techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.

For solid compositions, conventional nontoxic solid carriers may be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. For oral administration, a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95% of active ingredient, preferably about 20% (see, Remington's, 599.13)- For aerosol administration, the compounds are preferably supplied in finely divided form along with a surfactant and propellant. The surfactant must, of course, be nontoxic, and preferably soluble in the propellant.

Representative of such agents are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride such as, for example, ethylene glycol, glycerol, erythritol, arabitol, mannitol, sorbitol, the hexitol anhydrides derived from sorbitol, and the polyoxyethylene and polyoxypropylene derivatives of these esters. Mixed esters, such as mixed or natural glycerides may be employed. The surfactant may constitute 0.1%-20% by weight of the composition, preferably 0.25-5%. propellant.

The balance of the composition is ordinarily Liquefied propellants are typically gases at ambient conditions, and are condensed under pressure. Among suitable liquefied propellants are the lower alkanes containing up to 5 carbons, such as butane and propane: and preferably fluorinated or fluorochlorinated alkanes. Mixtures of the above may also be employed. In producing the aerosol, a container equipped with a suitable valve is filled with the appropriate propellant, containing the finely divided compounds and surfactant. The ingredients are thus maintained at an elevated pressure until released by action of the valve.

The compositions containing the compounds can be administered for prophylactic and/or therapeutic treatments.

In therapeutic applications, compositions are administered to a patient already suffering from a disease, as described above, in an amount sufficient to cure or at least partially arrest the symptoms of the disease and its complications. An amount adequate to accomplish this is defined as "therapeutically effective dose." Amounts effective for this use will depend on the severity of the disease and the weight and general state of the patient.

In prophylactic applications, compositions containing the compounds of the invention are administered to a patient susceptible to or otherwise at risk of a particular disease.

Such an amount is defined to be a "prophylactically effective dose." In this use, the precise amounts again depend on the patient's state of health and weight.

The invention will better be understood by reference to the following illustrative examples. The following examples are offered by way of illustration and not by way of limitation.

EXPERIMENTAL In general, standard techniques of recombinant DNA technology are described in various publications, e.g., Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory; Ausubel et al. (1987) Current Protocols in Molecular Biology, vols. 1 and 2 and supplements: and Wu and Grossman (eds.) (1987) Methods in Enzymology, Vol. 53 (Recombinant DNA Part D); each of which is incorporated herein by reference.

I. Human Extracellular Region Equivalent techniques for construction, expression, and determination of the physiological effect of truncation or deletion analogues of the soluble extracellular receptor fragments from the human receptor may be performed using the nucleic acid, polypeptide, and other reagents provided herein.

A. Type B Segments Constructs of type B receptor polypeptides were made as follows: The 3.9 kb EcoRI-Hind III CDNA fragment of the human type B hPDGF-R was subcloned into the EcoRI-Hind III site of M13 Mp18 to produce a vector Mp18PR. For techniques, see Maniatis et al. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., which is incorporated herein by reference. Verification of subcloning was performed by restriction enzyme digestion analysis and dideoxy chain termination sequencing, as described by Sanger et al. (1977) Proc. Nat'l Acad. Sci. USA 74:5463. Oligonucleotide directed in vitro mutagenesis was performed according to the method described by Kunkel et al. (1987) Methods in Enzvmol., 1S4:367.

The strategy for oligonucleotide directed in vitro deletion mutagenesis of Mp18PR is outlined in Fig. 1.

In brief, a series of oligonucleotides were designed to create a nested set of soluble type B hPDGF receptor extracellular regions by deletion mutagenesis. These domains are designated Domain 1 through Domain 5 (D1-D5), suitable for expression in an appropriate eukaryotic expression system. A description of the mutagenic oligonucleotides aligned with the corresponding regions of the human PDGF receptor are listed in Table 11.

Table 13. throughout.

The resulting constructs are labeled as indicated in The antisense strand was used for mutagenesis Mutagenesis of PA1, PA2, PA3, PA4, and PAS, utilized Mp18PR as the template and mutagenesis of PA6, PA7, PA8, and PA9, utilized MP 18 PA1 as the template. PA1, a 41 bp oligomer, introduced a TAG stop codon after Lysinewg (K ) of D5 and removed the transmembrane (TM) as well as entire intracellular kinase domain (K), producing an Mp18 PA1 (see Fig. 1).

PA1 codes for 530“ 148“ precursor proteins.

TABLE 13 HUMAN TYPE B PDGF-R EXPRESSION CONSTRUCTS Soluble Membrane Bound pBJPR pBJPA1 pBJPA2 pBJPA3 pBJPA4 pBJPA5 pBJPA6 pBJPA7 pBJPA8 pBJPA9 ———__—__..—_————————————-a-———————----_—_——_—-——-———.-.--..———--.——._...._ The human PDGF receptor constructs were subsequently subcloned into the EcoRI-Hind III site of pBJl a derivation of pCDL-sRa296, as described in Takabe et al. (1988) Molec. Cell Biol; 8:466, and co—transfected with psV2NEO, as described by Southern and Berg (1982) J. Mol. Appl. Gen., 1: 327, See Figs. 2 and 3. into Chinese hamster ovary cells (CHO).

Function of the constructs was demonstrated as follows: A sample of 0.33 nM PDGF BB ligand is preincubated for 1 hr at 4°C under the following conditions: . a polyclonal antibody to human PDGF (this antibody recognizes human PDGF AA, PDGF BB and PDGF AB); . 18 nM (60 fold molar excess to PDGF BB) human type B PDGF receptor; phosphate buffered saline solution that the receptor and antibody are in; or . no additions but the ligand itself.

In a duplicate set of experiments, 0.33 nM PDGF AA is incubated with three of the above preincubation conditions, e.g., 2, 3, not appreciably recognize PDGF AA but this ligand will still activate cell-associated human type A PDGF receptor from NIH3T3 and 4 above. The human type B PDGF receptor does cells and so is a control for human type B PDGF receptor specificity and PDGF BB—dependent activation versus non- specific general cellular effect, e.g., cytotoxicity.

The preincubated materials were in a final volume of 0.5 ml. They were placed in one well each of a six well tissue culture dish containing a confluent layer of serum starved (quiescent) NIH3T3 cells which were chilled to 4°C. The cells and incubation mixtures were agitated, e.g., rocked, at 4°C for 2 h. They were then washed twice with 4°C phosphate buffered saline. Forty ul of 125 mM Tris(hydroxymethyl)amino methane (Tris), pH 6.8, 20% (v/v) glycerol, 2% (w/v) sodium dodecyl sulfate (SDS), 2% (v/v) 2-mercaptoethanol, and 0.001% bromphenol blue, (known as SDS sample buffer), was added per microtiter well followed by 40 ul of 100 mM Tris, pH 8.0, 30 mM sodium pyrosphoshate, 50 mM sodium fluoride, 5 mM ethylenediaminetetraacetic acid (EDTA), 5 mM ethylenebis(oxyethylenenitrilio)tetraacetic acid, 1% (w/v) SDS, 100 mM dithiothreitol, 2 mM phenylmethylsulfonylfluoride (PMSF), and 200 0M sodium vanadate was added to the cells. The cells were solubilized and 40 pl additional SDS sample buffer was added to the solubilizate. This material was boiled 5 minutes and loaded onto a single gel sample well of a 7.5% sodium dodecyl sulfate polyacrylamide gel. Cellular proteins were separated by electrophoresis.

The separated proteins were transferred to nitrocellulose by electrotransfer and the resulting "Western blot" was incubated with 3 changes of 0.5% (w/v) sodium chloride, 5 mg/ml bovine serum albumin, 50 mM Tris, pH 7.5, (designated blocking buffer) for 20 minutes each at room A 1/1000 dilution of PY20 (a commercially available monoclonal antibody to phosphotyrosine [ICN]) in blocking buffer was incubated with the blot overnight at 4°C.

The blot was washed 3 times for 20 minutes each at room The blot was incubated with 4 uci/40 ml of l”I—Protein A [Amersham] in blocking buffer for 1 hour at room temperature and washed 3 times for 20 minutes each at room temperature in blocking buffer. temperature. temperature in blocking buffer.

The blot was exposed to X-ray film for 48 h with one intensifying screen at —70°C and developed with standard reagents.

Figure 4 shows the results of the autoradiogram with the conditions mentioned above plus the additional condition of no added ligand (no PDGF). This added condition defines the level of cell-associated receptor activation (e.g., phosphorylation) in the absence of any added ligand. Both the antibody and the human type B PDGF receptor neutralized the activation of cell-associated PDGF receptor by PDGF BB. This is apparently due to direct binding and sequestration of the ligand making it unavailable for PDGF receptor activation. p185 shows the receptor position.

B. Type A Sequence Similar manipulations using the mutagenic oligonucleotides of Table 12 are used to construct the type A constructs listed in Table 15. Note that the type A constructs have not actually been produced, but would readily be produced by these methods. Similar assays are used to test the function of the constructs.

TABLE 15 SUGGESTED HUMAN TYPE A PDGF-R EXPRESSION CONSTRUCTS type A Soluble Membrane Bound pARSR pARSA1 pARSA2 pARSA3 pARSA4 pARSA5 pARSA6 pARSA7 pARSA8 pARS/ 71 C. PDGF Plate Assay Polystyrene microtiter plates (Immulon, Dynatech Laboratories) were coated with the extracellular region fragment of the type B human PDGF receptor (described above) by incubating approximately 10-100 ng of this protein per well in 100 pl of 25 mM Tris, 75 mM NaCl, pH 7.75 for 12 to 18 h at 4°C. The protein was expressed in transfected CHO cells and collected in serum-free media (Gibco MEMa) at a concentration of 0.2 - 1 pg/ml, with a total protein concentration of 150 - 300 pg/ml.

The human PDGF type B receptor extracellular region fragment was concentrated and partially purified by passing the media over wheat germ-agglutinin-sepharose at 4°C (at 48 ml/h) in the presence of 1 mM PMSF. After extensive washing, the protein was eluted in 0.3 M N-acetyl-glucosamine, 25 mM Hepes, 100 mM NaCl, 1 mM PMSF, pH 7.4. to Sephacryl S-200 HR (Pharmacia) equilibrated in 0.15 M ammonium bicarbonate pH 7.9.

This fraction was then applied The fractions containing receptor (3 - 10 ng/pl) were detected by SDS-PAGE and Western blotting with a polyclonal rabbit antibody, made by standard methods, against a Domain 1 (D1) segment from the receptor external region. These fractions (3 - 10 ng/pl) were used to coat the microtiter wells as described above. The wells were then drained, rinsed once with 200 pl each of 0.5% gelatin (Bio-Rad, EIA grade), 25 mM Hepes, 100 mM NaCl, pH 7.4, and incubated for 1-2 h at 24°C with 150 pl of this same solution. The wells were drained and rinsed twice with 0.3% gelatin, 25 mM Hepes, 100 mM NaCl, pH 7.4 (150 pl each). 90 pl of the 0.3% gelatin solution was put in each well (wells used to test nonspecific binding received just 80 pl and then 10 pl of 0.01 mg/ml non- labeled PDGF in the 0.3% gelatin solution). PDGF BB (Amgen) was iodinated at 4°C to 52,000 CPM/ng with di-iodo Bolton- Hunter reagent (Amersham) and approximately 40,000 CPM was added per well in 10 pl, containing 0.024% BSA, 0.4% gelatin, mM Hepes, 80 mM NaCl, 70 mM acetic acid, pH 7.4. The plate was incubated for 2-3 h at 24°C, after which wells were washed three times with 150 pl each with 0.3% gelatin, 25 mM Hepes, mM NaCl, pH 7.4. The bound radioactivity remaining was solubilized from the wells in 200 pl 1% SDS, 0.5% BSA, and counted in a gamma-counter. The nonspecific binding was determined in the presence of a 150-fold excess of unlabeled PDGF BB (Amgen) and was about 7% of the total bound 1251-PDGF. similar assays will be possible using type A receptor fragments. However, the type A receptor fragments are more sensitive to the presence of other proteins than the type B fragments, and appear to require a different well coating reagent from the gelatin. Hemoglobin is substituted for gelatin in the buffers at about the same concentrations. Other blocking proteins will be useful selected from, e.g., the Sigma Chemical Company. Titrations to optimize the protein type and concentration will be performed to find proteins which do not affect the receptor protein binding.

The present assays require less than 5 ng/well of receptor soluble form, which was expressed in transfected CHO cells, and partially purified by affinity and gel chromatography. Using iodinated PDGF-BB, the specific binding of less than 10 pg of ligand can be detected in an assay volume of 100 pg/well. At 4°C, the binding of 125I-PDGF as to immobilized receptor is saturable and of high affinity. The Kd by Scatchard analysis was about 1 nM with 1.8 x 10” sites per well. The nonspecific binding, determined in the presence of a 100-fold excess of cold PDGF BB, was usually only about 5-10% of the total binding. The binding was also specific for the isoform of the ligand, insofar as excess cold PDGF AA did not inhibit ”5I-PDGF BB binding. Furthermore, the external region of the type B PDGF receptor in solution competes with its immobilized form for binding iodinated PDGF BB (lcw = 5nM).

The ”5I—PDGF BB bound after 4 h at 4°C is only slowly dissociable in binding buffer (tln > 6 h), but is completely displaced by the addition of a 150-fold excess of unlabeled PDGF BB (tin < 1 h).

These studies were made possible by the availability of growth factor preparations devoid of contamination with other growth factors and by the use of a receptor expression system in which all of the measured PDGF responses could be attributed to this single transfected receptor CDNA.

All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

"SEQUENCE LISTING" (1) GENERAL INFORMATION: (i) APPLICANT: Wolf, David Tomlinson, James E.

Fretto, Larry J.

Giese, Neill A.

Escobedo, Jaime A.

Williams, Lewis T. (ii) TITLE OF INVENTION: DOMAINS OF EXTRACELLULAR REGION OF HUMAN PLATELET-DERIVED GROWTH FACTOR RECEPTOR POLYPEPTIDES (iii) NUMBER OF SEQUENCES: 23 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: TOWNSEND and TOWNSEND (B) STREET: Stauart Street Tower, 20th Floor \ One Market Plaza (C) CITY: San Francisco (D) STATE: California (E) COUNTRY: US (F) ZIP: 94105 (v) COMPUTER READABLE FORM: (A) MDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (c) opsnxrxus 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: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Ching, Edwin P. (3) REGISTRATION NUMBER: 34,090 (C) REFERENCE/DOCKET NUMBER: 12418-14 (ix) TELECOMUNICATION INFORMATION: (A) TELEPHONE: (415) 326-2400 (B) TELEFAX: (415) 326-2422 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 5427 base pairs (8) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLDGY: linear (ii) MOLECULE TYPE: CDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo Sapiens (B) STRAIN: lambda gtlo (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: l87..3504 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: GGAGGGGGTG ACTGTCCAGA GCCTGGAACT GTGCCCACAC CAGAAGCCAT CAGCAGCAAG 180 GACACC ATG CGG CTT CCG GGT GCG ATG CCA GCT CTG GCC CTC AAA GGC 228 Net Arg Leu Pro Gly Ala Met Pro Ala Leu Ala Leu Lys Gly 1 5 10 GAG CTG CTG TTG CTG TCT CTC CTG TTA CTT CTG GAA CCA CAG ATC TCT 276 Gln Leu Leu Leu Len Ser Leu Leu Leu Leu Leu Glu Pro Gln Ile Ser 30 CAG GGC CTG GTC GTC ACA CCC CCG GGG CCA GAG CTT GTC CTC AAT GTC 324 Gln Gly Leu Val Val Thr Pro Pro Gly Pro Glu Leu Val Len Asn Val 45 TCC AGC ACC TTC GTT CTG ACC TGC TCG GGT TCA GCT CCG GTG GTG TGG 372 Ser Ser Thr Phe Val Leu Thr Cys Ser Gly Ser Ala Pro Val Val Trp 50 55 60 GAA CGG ATG TCC CAG GAG CCC CCA CAG GAA ATG GCC AAG GCC CAG GAT 420 Gln Arq Met Ser Gln Glu Pro Pro Gln Glu Met Ala Lys Ala Gln Asp 65 70 75 GGC ACC TTC TCC AGC GTG CTC ACA CTG ACC AAC CTC ACT GGG CTA GAC 468 Gly Thr Phe Ser Ser Val Leu Thr Leu Thr Asn Leu Thr Gly Leu Asp 80 85 90 ACG GGA GAA TAC TTT TGC ACC CAC AAT GAC TCC CGT GGA CTG GAG ACC 516 Thr Gly Glu Tyr Phe Cys Thr His Asn Asp Ser Arg Gly Leu Glu Thr 105 110 GAT GAG CGG AAA CGG CTC TAC ATC TTT GTG CCA GAT CCC ACC GTG GGC 564 Asp Glu Arg Lys Arg Leu Tyr Ile Phe Val Pro Asp Pro Thr Val Gly 125 TTC CTC CCT AAT GAT GCC GAG GAA CTA TTC ATC TTT CTC ACG GAA ATA 612 Phe Leu Pro Asn Asp Ala Glu Glu Leu Phe Ile Phe Leu Thr Glu Ile 130 135 140 ACT GAG ATC ACC ATT CCA TGC CGA GTA ACA GAC CCA CAG CTG GTG GTG 660 Thr Glu Ile Thr Ile Pro Cys Arg Val Thr Asp Pro Gln Leu Val Val 145 150 155 ACA CTG CAC GAG AAG AAA GGG GAC GTT GCA CTG CCT GTC CCC TAT GAT 708 Thr Leu His Glu Lys Lys Gly Asp Val Ala Leu Pro Val Pro Tyr Asp 160 165 170 CAC CAA CGT GGC TTT TCT GGT ATC TTT GAG GAC AGA AGC TAC ATC TGC 756 His Gln Arg Gly Phe Ser Gly Ile Phe Glu Asp Arg Ser Tyr Ile Cys 185 190 AAA ACC ACC ATT GGG GAC AGG GAG GTG GAT TCT GAT GCC TAC TAT GTC 804 Lys Thr Thr Ile Gly Asp Arg Glu Val Asp Ser Asp Ala Tyr Tyr Val 205 TAC AGA CTC CAG GTG TCA TCC ATC AAC GTC TCT GTG AAC GCA GTG CAG 852 Tyr Arg Leu Gln Val Ser Ser Ile Asn Val Ser Val Asn Ala Val Gln 210 215 220 ACT GTG GTC CGC CAG GGT GAG AAC ATC ACC CTC ATG TGC ATT GTG ATC 900 Thr Val Val Arg Gln Gly Glu Asn Ile Thr Leu Met Cys Ile Val Ile 225 230 235 GGG AAT GAT GTG GTC AAC TTC GAG TGG ACA TAC CCC CGC AAA GAA AGT 948 Gly Asn Asp val val Asn Phe Glu Trp Thr Tyr Pro Arg Lys Glu Ser 240 245 250 GGG CGG CTG GTG GAG CCG GTG ACT GAC TTC CTC TTG GAT ATG CCT TAC 996 Gly Arg Leu Val Glu Pro Val Thr Asp Phe Leu Leu Asp Met Pro Tyr GAA Glu CTG Leu ACA Thr 335 TTC Phe CTG Leu CTG Leu TTC Phe GTC val 415 GAA Glu ATC Ile ccc Pro AAC Asn CTG Leu 495 TCC Ser GTG Val ACC Thr AAG Lys GGA Gly 320 CTG Leu TCC Ser GTT Val CAT His 400 CCT Pro CAG Gln rec; Trp ACG Tm: GTG Val 480 CGT Arg AAC ASH TTG Leu CTC Leu M’-'9 TAC Tyr GCC Ala 305 GAG Glu CAG Gln GAC Asp ACG Thr CGC Arq 385 GAG Glu GTC Val ACA Thr TCT Ser CTG 465 ACG Thr CTG Leu GCT Ala CCC Pro ACC Thr ACC Thr 290 ATC Ile GTG Val GTA Val AAC Asn CGC Arq 370 GTG Val GAT Asp CGA Arg GTC Val GCC Ala 450 TAC Tyr CAG Gln GTG Val Phe 530 ATC Ile Ile 275 TGC Cys AAC Asn GGC Gly GTG Val ccc Arg 355 AAC Asn AAG Lys GCT Ala GTG Val CGC Arg 435 ‘rec Cys ma Trp CAC His GGC Gly S15 AAG Lys ATC Ile AAT Asn ATC Ile ACA Thr TTC Phe 3 4 0 ACC Thr GTG Val GTG Val GAG Glu CTG Leu 420 TGT Cys AGA Arg AAC Asn GAG Glu GTG Val 500 CAG Gln GTG Val TCC Ser GTG Val ACC Tm: CTA Leu 3 25 GAG Glu CTG Leu TCG Ser GCA Ala GTC Val 405 GAG Glu CGT Arg GAC Asp AGT Ser GAG Glu 485 GAT Asp GAC Asp GTG Val ACG Thr GTG Val 310 GCC Ala GGC Gly GAG Glu GAG Glu 390 CAG Gln CTA Leu GGC Gly CTC Leu TCC Ser 470 GAG Glu CGG Arg ACG Thr GTG Val Ile_ GAG Glu 295 GT1‘ Val TAC Tyr GAG Asp ACC Thr 3 7 5 GCT Ala CTC Leu AGT Ser CGG Arg Lys 455 GAA Glu CAG Gln CCA Pro CAG Gln ATC Ile 535 ATC Ile Ser 280 AGT Ser GAG Glu GCT Ala CCA Pro TCC Ser 360 CGG Arg GGC Gly TCC Ser GAG Glu GGC Gly 440 AGG Arg GAG Glu GAG Glu CTG Leu GAG Glu 520 TCA Ser CTC Leu GTG Val AGC Ser GAG Glu CCG Pro 345 AGC Ser TAT Tyr CAC His 'I‘1‘C Phe AGC Ser 425 ATG Met 'rc;'r Cys GAG Glu TCG Ser 505 GTC Val GCC Ala ATC Ile AAT Asn GGC Gly CTG Leu 330 CCC Pro GCT Ala GTG Val TAC Tyr CAG Gln 410 CAG His CCG Pro CCA Pro AGC Ser GAG Glu 490 GTG Val ATC Ile ATC Ile ATG Met GAC Asp TY! 315 CAT His ACT Tm: GGC Gly TCA Ser ACC Thr 395 CTA Leu CCT Pro CAG Gln CGT Arg CAG Gln 475 GTG Val csc Arq GTG Val CTG Leu CAT His 300 GTG Val CGG Arg GTC Val GAA Glu GAG Glu 380 ATG Met CAG Gln GAC Asp CCG Pro GAG Glu 460 CTG Leu GTG Val TGC Cys GTG Val GCC Ala 540 TGG Trp Asp 285 CAG Gln CGG Arg AGC Ser CTG Leu ATC Ile 365 CTG Leu CGG Arg ATC Ile AGT Ser AAC Asn 445 CTG Leu GAG Glu AGC S82‘ ACG Thr CCA Pro 525 CTG Leu CAG Gln GAT Asp CTC Leu CGG Arg TGG 350 GCC ACA Thr GCC Ala AAT Asn GGG Gly 430 ATC Ile CCG Pro ACT Thr ACA Thr CTG Leu 510 CAG His GTG Val AAG Lys GAC Asp 575 TCC Ser GGC Gly AGC Ser ACA Thr ATG Met 655 "rec Cys GGA Gly CAC His GCT Ala GAG Glu 735 TAT Tyr GAG Glu CCT Pro AGC Ser GAG Glu 815 AAC Asn GGC Gly ACG Thr TCT Ser CAT His GCC Ala 640 AGT Ser ACC Thr GAC Asp CAC His CTG 720 AGC GTG val TCC Ser GAG Glu Tyr 800 GTG Val CAT His TGG TIP TCT Ser 625 CGC Arg CAC His CTG Leu TCC Ser 705 CCC Pro GAC Asp CCC Pro TCC Ser AGG Arg 785 ATG Met CTG Leu CTC Leu GAG Glu GAG Glu GCC Ala 610 CAG Gln AGC Ser GGA Gly GTG Val 690 GAC Asp GTT Val GGT Gly ATG Met AAC Asn 770 ACC Thr GAC Asp GCC Ala ATC Ile TAC Tyr CTG Leu 595 GCC Ala AGT Ser GGA Gly 675 GAC Asp AAG Lys GGC Gly CTG 755 TAC Tyr TGC Cys CTC Leu TCC Ser CYS 835 ATC Ile 580 CCG Pro ACG Thr GAG Glu CCC PIG 660 CCC Pro TAC Tyr CGC Arg CTC Leu TAC Tyr 740 GAC Asp ATG Met CGA Arg GTG Val AAG Lys 820 GAA Glu TAC Tyr CGG Arg CAG Gln ATG Met AAG Lys 645 CAC His ATC Ile CTG Leu CGC Arg CCC Pro 725 ATG Met ATG Met GCC Ala GCA Ala GGC Gly 805 AAC Asn GGC Gly GTG Val GAC Asp GTG Val Lys 630 CTG Leu TAT Tyr CAC His CCG PIC 710 CTG Leu GAC Asp CCT Pro ACT Thr 790 TTC Phe TGC Cys AAG Lys GAC Asp CAG Gln GTG val 615 GT6 Val GCC Ala AAC Asn ATC Ile CGC Arg 695 CCC Pro CCC Pro ATG Met GGA Gly TYI 775 TTG Leu AGC Ser GTC Val CTG Leu CCC Pro Leu 600 GAG Glu GCC Ala GTG Val ATC Ile 680 AAC Asn AGC Ser AGC Ser AGC Ser GAC Asp 760 GAT Asp ATC Ile TAC Tyr CAC His GTC Val 840 ATG not 585 GT6 val GCC Ala GTC Val ATG Met GTC Val 665 ACT Tnr GCG Ala CAT His AAG Lys 745 GTC Val AAC Asn AAC Asn CAG Gln AGA Arg 825 AAG Lys CAG Gln CTG Leu ACA Thr AAG Lys TCG Ser 650 AAC Asn GAG Glu CAC His GAG Glu GTG Val 730 GAC Asp TAC Tyr GAG Glu GTG Val 810 GAC Asp ATC Ile CTG Leu cm my GCT Ala ATG Met 635 GAG Glu CTG Leu TAC Tyr ACC Thr CTC Leu 715 Tcc Ser GAG Glu TAT Tyr GTT val TCT Ser 795 GCC Ala CTG Leu TGT Cys CCC Pro CGC Arg CAT His 620 CTG Leu TGC Cys TTC Phe 700 TAC Tyr TTG Leu TCG Ser GCA Ala CCC Pro 780 CCA Pro AAT Asn GCG Ala GAC Asp TAT Tyr ACC Thr 605 GGT Gly AAG Lys GGG Gly CGC Arg 685 CTG Leu AGC Ser ACC Thr GTG Val GAC Asp 765 TCT Ser GTG val GGC Gly GCT Ala Phe 845 GAC Asp 590 CTC Leu CTG Leu TCC Ser ATC Ile GCC Ala 670 TAC Tyr CAG Gln AAT ASH GGG Gly GAC Asp 750 ATC Ile GCC Ala CTA Leu ATG Met AGG Arg 830 GGC Gly ACC Thr CTC Leu GAG Glu 895 GAG Glu GCC Ala GAG Glu GAG Glu GAG Glu 975 CGC Arg GTC Val ATC Ile GAG Glu TCC Ser GAG Glu GAA Glu TAC Tyr 880 ATC Ile CAG Gln CAT His AAG Lys AGA Arg 960 GAG Glu TTG Len CTC Leu CCC Pro cor Gly 1040 TCA Ser CCA PIC CAG Gln GAT AGC CGCTGCCAGC ACCCAGCATC TCCTGGCCTG GCCTGGCCGG GCTTCCTGTC AGCCAGGCTG CCCTTATCAG CTGTCCCCTT CTGGAAGCTT TCTGCTCCTG ACGTGTTGTG CCCCAAACCC TTG 865 ACC Thr TTC Phe TTC Phe GCC Ala Phe 945 CTG Leu CCT Pro TAT Tyr CTG Leu 1025 TCC Ser ACC Thr GAG Glu TTG Leu TTC Phe 1105 CCT Pro TTA Len ACC Thr CTG Leu ACC Thr TTG Leu TAC Tyr AAT Asn 915 TCC Ser 930 GAC Asp GAG Glu ATT Ile TTG Leu GGC Gly CTG Leu AGG Arg GGG Gly TTC Phe 995 ACT GCC Thr Ala 1010 CCT PIG GAC Asp CCC Pro AGC Ser ATC Ile TCC Ser CCC Pro AAG Lys AGC Ser GGT Gly 900 GCC Ala GAG Glu csc Arg GAA Glu AGT Ser 980 CAT His GTG Val CCC PIC CTA Leu was Trp GAC Asp 835 GGC Gly ATC Ile ATC Ile CCC Pro GGT Gly 965 GAC Asp GGC Gly CAG Gln AAA Lys GCC Ala ATG net 870 GTG Val ACC Thr TAT Tyr CCC Pro 950 TAC Tyr CAC His CTC Leu CCC Pro CCT Pro 1030 AGC Ser Cys Asp Ser CAG CTT GAG CTC Gln Leu Glu Leu 1075 GAT TCG GGG TGC Pro Asp Ser Gly Cys GCT Ala TGG Trp CCT Pro CGG Arg GAG Glu 935 TTC Phe AAA Lys CCA PIG CGA Arg AAT ASH GAG Glu TCC Ser CCC Pro CAG Gln CCT Pro CCG Pro GAG Glu TCC Ser TTC Phe TAC Tyr CCA Pro 905 GGT Gly 920 TAC Tyr ATC Ile ATG Hat TCC Ser CAG Gln AAG Lys AAG Lys GCC Ala ATG Ile 985 TCT CCC Ser Pro 1000 GAG GGT Glu Gly GTT Val GCT Ala ACC Thr CTG Leu CTG Leu GTG Val 1080 AGC Ser GGG Gly 890 GAG Glu CGC Arg CAG Gln CTG Leu TYI 970 CTT Leu CTG Leu GAC Asp GAC Asp AAT Asn ATC Ile 875 ATC Ile CTG Leu ATG Met LY3 CY GTG Val 955 CAG Gln CGG Arg GAC Asp AAC Asn GAG Glu TTC Phe AAC ASH CTG Leu CTC Leu CCC Pro ATG Met GCC Ala CAG Gln 925 TGC TGG s Trp 940 CTG Leu CTT Leu CAG Gln GTG Val TCC Ser CAG Gln ACC Thr AGC Ser AGC Ser TGG Trp AAC Asn 910 CCT Pro GAA Glu CTC Leu GAT Asp GCC Ala 990 TCC Ser GAC TAT Asp Tyr 1020 GGC CCA Gly Pro GAA Glu Glu Pro Gln 1065 GAG CCG GAG Glu Pro Glu GCG CCT GGG GCG Ala Pro Arg Ala GTG Val AAC Asn GAC Asp GAA Glu CCG Pro GAG Glu ATC Ile CTG Leu ACC Thr CCA Pro 1070 CTG Leu GAA Glu 1100 GCA Ala CTG TAGGGGGCTG GCCCCTACCC TGCCCTGCCT GAAGCTCCCC GAG Glu TGGGAAAGTT AGACCGAATC CTGTTCAGCC GGAGCTAGGG GCAGTGTTGC CCCTGGCCCG AGTACAGGAC AGCAAGTGCC CCTCAGTCTT AGAATGTAAA TGCATTGGAC CCCTTCAAAA TCTAGAGTAT TTCCATCCTG GACTCAGGAA GGCCTTGACT ATGGGAAGAC AAAGGGACAA CGAGGTCTGC AAGCTTCAGA TCACCCACCA CCCAGACCTA GAGTCAGGGT GCTCAACCCC CCAGAGTTGG TAGGTTTACA TTTGTATGCT TAAACCTGGT AGGCTTGATG CCTCCCTGGG AGCTACCCCT AAGAGACCCT CTCATCCAGA AGCTGGTCTG ACCCCCAGCC TGTGTCCCTG AATCCATCCA TGTGCCAGTG CTGCTATGAG AATGAATAAG CCAGGTGGTT GGGGTCAGCT CCATGCCCCT TAGAGTGACA CACGGGACCT AGAGGGCAAA GTCGAAGACA TGGGTACCCC GCTGCCCCAT GCAGTGACAT TGTAGCCAAG TCTGGGCACC TCCAAGGAGG AATATTTTTA GTTAAGTTTT GCTTCTCACT ACCCAGAATC AAGATTCTTG CAAGGAATCA AGCCTCCCTG AGAAAGCCAG GGGCCATTAG TGCAGCCCTT TCCTTCAGGC CCAGAGTCTA TGGAGTGGCC GCTTTGGAGG TCGGACTTAT GCACATTTGT GGGCTCCTGG TCCCCAGGCC GCCGGTGTCC CTTTCACTAC TGAGATCACC GAATGGACAG AAGAAGGATG CCCTGAGGCA CTCATTGTCC ACGCCCCCGC AACCCTGCAT GAGAGTGGGT GGACTCACGT TCTATCTGTG CAC (2) INFORMATION FOR SEQ ID NO:2: TAGGATTCTC GAGTTACTGA TAGCTCTCTC GCTGCTGGCT TCTCCTCCCT GCAGCCTAAT GCCCAGGGCA CCATCAGTCC GAAGGCCAGA ACGTGTGTGT AATCCCTCAC TAACTCTGAG CCAGATGAAG GAGATTCCAG CCCAGCAAGT TGGAAAGCCC CCACGATGAC TCCTGCAGCC TGAGGACAGT TGAGAGGTGG GCGCTCCATG CCAGCCCAGT ACGGGGAGGG TGCAGGTTGG TCTCAATACG TAACTCACAT TACTTPTTTT TCCCTGGCTG GGTGGTAAAT CTCGCACTTT GAGCTAGGGC ATGATGCCAG TAATGCTGGA CTTGGAGCAC TGGGGCTTTT CGGGCCCCGC GCCAGATATG CCTCTCTGGG TGCCTTGCCA CAAGGCCATA ATCACACATC CTCAAGAACA CCAGCAGCTG CTCCGGGGGT CACCACTCCA TATGTCTTGT GCGCTTTGGA GGGGTATGGT GGGCATTGGA TTGGGAAGGG CACCTTACTT GTACCAAAGA TTATACAGCA TAAGGGAAAG SEQUENCE CHARACTERISTICS: (A) LENGTH: 1106 amino acids (8) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: ACAGGTGGGG TAACTTTTTT TATCCACCCA CTAGCCTTGA TCCCTGCGTT GGCTGAGCCA ACGCAGCCAT TCTTTATCAC ATCTGTGATG GCCCTGGCTC CCTCAGTTTC GCACTAACAT TACCCTAAAC ACACTCTGGG CAGCTGCACA CCCCAGGGAC ATCCTGGGCA GCACCTGTGC AAAAGACAAG GGTTTGCCCC TTTGTCACTG GGTGCCAGGG GGTGCAGGAA CCCTGGGATC TATAATCACC GAAATGCTAT ATTTTAATAT 3964 4024 4084 4144 4204 4264 4324 4384 4444 4504 4564 4624 4684 4744 4804 4864 4924 4984 5044 5104 5164 5224 5284 5344 5404 S427 Val 225 Arg Tyr Ala 3 05 Glu Gln Asp Val Phe Ser Ser Tyr Lys Asn 130 Thr Glu Gly Ile Gln 210 An; Val Val Ser Thr 290 Ile Val Val Asn Arg Val Gln Ser Phe Arg 115 Asp Ile Lys Phe Gly 195 Val Gln val Glu Ile 275 Cys Asn Gly val Arg 355 Cys 100 Ala Pro Lys Ser 180 Asp Ser Gly Asn Pro 260 Asn Ile Thr Phe 340 Thr Pro Thr Pro Leu Th: Cys Gly 165 Gly Arg Ser Glu Phe 24 5 Val His Val Thr Leu Pro Cys Pro Thr His Ile Glu Arg 150 Asp Ile Glu Ile Asn 230 Glu Tm: Ile Thr val 3 10 Gln Ala Gly Gly Ser Gln Asn Phe Leu 13 5 Val Val Phe Val Asn Ile TIP Asp Pro Glu 295 Thr 375 Gly Glu Thr Asp Val 120 Phe Thr Ala Glu Asp 200 Val Thr Thr Phe Ser 280 Ser Glu Ala Pro Ser 3 60 Art; Glu Ser Met Asn Ser 105 Pro Asp 185 Ser Tyr Leu 265 Ala Val Ser Glu Pro 345 Ser Val Pro Lys Thr Gly Gln 155 Val Ser CYS 235 Arg Asp Tyr 315 His Thr Gly val 60 Ala Thr Thr 14 0 Ala 220 Ile Lys Met Glu His 300 Val Arg Val Glu Glu Asn 4 5 Val Glu Val 125 Glu Ile Tyr 205 Val Val Glu Pro Asp 285 Gln Ile 365 Val T1’? Asp Asp Thr 110 Gly Ile Val Asp Cys 190 Val Gln Ile Ser Tyr 270 S81‘ Arg T1‘? 350 Ala Ser Glu Gly Thr Asp Phe Thr Thr His 175 Lys Tyr Thr Gly Gly 255 His Gly Thr 335 Ser Arg Thr Glu Leu 160 Gln Thr Arg Val Asn 240 Arg Ile Thr Lys Gly 320 Leu Lys Ser Val Th: Ser Leu Ala Pro Thr 545 Arg His TIP Gly Ser 625 Arq His Lys Leu Ser 705 Pro Asp Ala 450 Tyr Gln val Phe 530 Ile Tyr Glu Glu Ala 610 Gln Ser Len Gly Val 690 Asp Val Gly Val Arg 435 Cys Gly TIP His 515 Lys Ile Glu Tyr Leu 595 Phe Ala Ser Gly Gly Asp Lys Gly Leu 755 Leu 420 Arg Asn Glu Val 500 Gln Val Ser Ile Ile 580 Pro Gly Thr Glu Pro Tyr 740 Glu Arg Asp Ser Glu 485 Asp Arg 565 Tyr Arg Gln Met Lys 645 Arg Pro 725 Met Ser 470 Glu Arg Thr Ile 550 TIP Val Asp Val Lys Ser Arg Lys 455 Glu Gln Pro Gln Ile 535 Ile Lys Asp Gln Val 615 Val Ala Asn Ile Arg 695 Pro Pro Met Glu Gly 440 Arg Glu 520 Val Pro Leu 600 Glu Ala Leu Val Ile 680 Asn Ser Ser Asp 7 Ser 425 Met Cys Glu Phe Ser 505 Val Ala Ile Ile Met 585 Val Ala val Met Val 665 Thr Lys Ala His Lys His Pro Pro Ser Glu 490 Val Ile Ile Met Glu 570 Gln Len Thr Lys Ser 650 Asn Glu His Glu Val 730 Asp Asn Tyr Pro Gln Arg Gln 475 Val Leu 555 ' Gly Ala Met Thr Leu 715 Ser Glu Val Ala 540 TIP Phe 700 Thr 605 Gly Lys Lys Gly Arg 685 Ser Thr Val Asp Gly 430 Ile Pro Thr Thr Leu 510 His Val Lys Ala 670 Tyr Gln Asn Gly Asp 750 Glu Ile Pro Asn Leu 495 Arg Ser Val Lys Asp 575 Ser Gly Ser Thr Met 655 Gly His Ala Glu 735 Gln TIP Thr Val 480 Pro 560 Gly Thr Ser His Ala 640 Ser Thr Asp His Leu 720 Ser Val Met Asp Leu Val Gly Phe Ser Tyr Gln Val Ala Asn Gly Met Glu Phe 805 810 815 Leu Ala Ser Lys Asn Cys Val His Arg Asp Leu Ala Ala Arg Asn Val 820 825 830 Leu Ile Cys Glu Gly Lys Leu Val Lys Ile Cys Asp Phe Gly Leu Ala 835 840 845 Arg Asp Ile Met Arg Asp Ser Asn Tyr Ile Ser Lys Gly Ser Thr Phe 850 855 860 Leu Pro Leu Lys Trp Met Ala Pro Glu Ser Ile Phe Asn Ser Leu Tyr 865 870 875 880 Thr Thr Leu Ser Asp Val Trp Ser Phe Gly Ile Leu Leu Trp Glu Ile 885 890 895 Phe Thr Leu Gly Gly Thr Pro Tyr Pro Glu Leu Pro Met Asn Glu Gln 900 905 910 Phe Tyr Asn Ala Ile Lys Arg Gly Tyr Arg Met Ala Gln Pro Ala His 915 920 925 Ala Ser Asp Glu Ile Tyr Glu Ile Met Gln Lys Cys Trp Glu Glu Lys 930 935 940 ?he Glu Ile Arq Pro Pro Phe Ser Gln Leu Val Leu Leu Leu Glu Arg 945 950 955 960 Leu Leu Gly Glu Gly Tyr Lys Lys Lys Tyr Gln Gln Val Asp Glu Glu 965 970 975 Phe Leu Arg Ser Asp His Pro Ala Ile Leu Arg Ser Gln Ala Arg Leu 980 985 990 Pro Gly Phe His Gly Leu Arq Ser Pro Leu Asp Thr Ser Ser Val Leu 995 1000 1005 Tyr Thr Ala Val Gln Pro Asn Glu Gly Asp Asn Asp Tyr Ile Ile Pro 1010 1015 1020 Leu Pro Asp Pro Lys Pro Glu Val Ala Asp Glu Gly Pro Leu Glu Gly 1025 1030 1035 1040 Ser Pro Ser Leu Ala Ser Ser Thr Leu Asn Glu Val Asn Thr Ser Ser 1045 1050 1055 Thr Ile Ser Cys Asp Ser Pro Leu Glu Pro Gln Asp Glu Pro Glu Pro 1060 1065 1070 Glu Pro Gln Leu Glu Leu Gln Val Glu Pro Glu Pro Glu Leu Glu Gln 1075 1080 1085 Leu Pro Asp Ser G1y Cys Pro Ala Pro Arg Ala Glu Ala Glu Asp Ser 1090 1095 1100 Phe Leu 1105 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4100 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo Sapiens (B) STRAIN: lambda gtlo (ix) FEATURE: (A) NAME/KEY: CD5 (3) LOCATION: l29..3395 (Xi) SEQUENCE DESCRIPTION: SEQ ID N0:3: TTGGAGCTAC AGGGAGAGAA ACAGAGGAGG AGACTGCAAG AGATCATTGG AGGCCGTGGG 60 CACGCTCTTT ACTCCATGTG TGGGACATTC ATTGCGGAAT AACATCGGAG GAGAAGTTTC 120 CCAGAGCT ATG GGG ACT TCC CAT CCG GCG TTC CTG GTC TTA GGC TGT CTT 170 Met Gly Thr Ser His Pro Ala Phe Leu Val Leu Gly Cys Leu l0 CTC ACA GGG CTG AGC CTA ATC CTC TGC CAG CTT TCA TTA CCC TCT ATC 218 Leu Thr Gly Leu Ser Leu Ile Leu Cys Gln Leu Ser Leu Pro Ser Ile 20 25 30 CTT CCA AAT GAA AAT GAA AAG GTT GTG CAG CTG AAT TCA TCC TTT TCT 266 Leu Pro Asn Glu Asn Glu Lys Val Val Gln Leu Asn Ser Ser Phe Ser 40 45 CTG AGA TGC TTT GGG GAG AGT GAA GTG AGC TGG CAG TAC CCC ATG TCT 314 Leu Arg Cys Phe Gly Glu Ser Glu val Ser Trp Gln Tyr Pro Met Ser 50 55 60 GAA GAA GAG AGC TCC GAT GTG GAA ATC AGA AAT GAA GAA AAC AAC AGC 362 Glu Glu Glu Ser Ser Asp Val Glu Ile Arg Asn Glu Glu Asn Asn Ser 75 GGC CTT TTT GTG ACG GTC TTG GAA GTG AGC AGT GCC TCG GCG GCC CAC 410 Gly Leu Phe Val Thr val Leu Glu Val Ser Ser Ala Ser Ala Ala His 90 ACA GGG TTG TAC ACT TGC TAT TAC AAC CAC ACT CAG ACA GAA GAG AAT 458 Tnr Gly Leu Tyr Thr cys Tyr Tyr Asn His Thr Gln Thr Glu Glu Asn 95 100 105 110 GAG CTT GAA GGC AGG CAC ATT TAC ATC TAT GTG CCA GAC CCA GAT GTA 506 Glu Leu Glu Gly Arg His Ile Tyr Ile Tyr Val Pro Asp Pro Asp Val 115 120 125 GCC TTT GTA CCT CTA GGA ATG ACG GAT TAT TTA GTC ATC GTG GAG GAT 554 Ala Phe Val Pro Leu Gly Met Thr Asp Tyr Leu val Ile Val Glu Asp 130 135 140 GAT GAT TCT GCC ATT ATA CCT TGT CGC ACA ACT GAT CCC GAG ACT CCT 602 Asp Asp Ser Ala Ile Ile Pro Cys Arg Thr Thr Asp Pro Glu Thr Pro 155 GTA ACC TTA CAC AAC AGT GAG GGG GTG GTA CCT GCC TCC TAC GAC AGC 650 val Thr Leu His Asn Ser Glu Gly Val Val Pro Ala Ser Tyr Asp Ser 170 AGA CAG GGC TTT AAT GGG ACC TTC ACT GTA GGG CCC TAT ATC TGT GAG 698 Arg Gln Gly Phe Asn Gly Thr Phe Thr Val Gly Pro Tyr Ile Cys Glu 175 180 185 190 GCC ACC GTC AAA GGA AAG AAG TTC CAG ACC ATC CCA TTT AAT GTT TAT 746 Ala Thr Val Lys Gly Lys Lys Phe Gln Thr Ile Pro Phe Asn Val Tyr 195 200 205 ACC Thr AAC Asn GGC Gly 255 TTG Leu GAT Asp AAG Lys CCC Pro Phe 335 GTG Val CGT Arg GAA Glu TCA Ser 415 CAG Gln TGG Trp ACT Thr CGA Arg Val Ty AAT A511 240 GTG Val TAC Tyr ACC Tnr 320 GTT Val AAC Asn GAA Glu GCT Ala GAT Asp 400 TCC Ser ACG Thr ATG Met ATT Ile GAC Asp 480 r 225 GAG Glu GGC Gly TAC Tyr GAA Glu GTC Val 305 TTC Pne GTA Val AAT Asn AAG Lys AAG Lys 385 GCT Ala ATT Ile GTG Val ATA Ile TTG Leu 465 AGG Arg AAG Lys GTG Val ATC Ile ACT Thr TGT 290 ACT AGC Ser GAG Glu CTG Leu ATT Ile 370 GAA Glu GTG Val CTG Leu AGG Arg TGC Cys 450 GCC Ala AGT Ser TCA Ser GTT Val ACA Thr TTG Leu 275 GCT Ala ATT Ile CAG Gln GTG Val ACT Thr 355 CAG Gln GAA Glu AAG Lys GAC Asp TGC Cys 435 AAC Asn ACC Thr ass Gly GAC Asp ATG Met 260 ACG Thr GCC Ala TCT Ser TTG Leu CGG Arg 340 CTG Leu GAA Glu GAC Asp AGC Ser TTG Leu 420 ACA Th: GAT Asp AAT Asn GTG Val GAA Glu Leu 245 GTC Val CGC Arg GTC Val GAA Glu 325 GCC Ala ATT Ile ATA Ile AGT Ser Tyr 405 GTC Val GCT Ala ATT Ile GTC Val GAG Glu 4 ACG Thr 230 GAA Glu CCC Pro CAG Gln CAT His 310 GCT Ala TAC Tyr GAA Glu AGG Arg GGC Gly 390 ACT Thr GAT Asp GAA Glu AAG Lys TCA Ser 470 GGC Gly ATT Ile TGG Tr? GAA Glu GAG Glu GCT Ala 295 GAG Glu GTC Val CCA Pro AAT Asn TAT 375 CAT His GAT Asp GGC Gly Lys 455 AAC Asn CGT Arg GTG Val ACT Thr ATC Ile GCC Ala 280 ACC Thr AAC Asn CCT PIO CTC Leu 360 CGA Arq TAT Tyr GAA Glu CAC His ACG Thr 440 TGT Cys ATC Ile GTG Val GTC Val TAC Tyr Lys 265 ACG Thr GGT Gly CTG Leu CCC Pro 345 ACT Thr AGC Ser ACT Thr CTG Leu CAT His 425 CCG Pro AAT Asn ATC Ile ACT Thr ACC Thr CCT Pro 250 GTC val GTG val GAG Glu TTC Phe CAT His 330 AGG Arg GAG Glu ATT Ile TTA 410 GGC Gly AAT Asn ACG Thr TTC Phe 490 TGT Cys 235 GGA Gly CCA Pro GTC Val ATT Ile 315 GAA Glu ATA Ile ATC Ile TTA Len GTA Val 395 ACT Thr TCA Ser CCT Pro GAA Glu GAG Glu 475 GCC Ala GCT Ala GAA Glu TCC Ser GAC Asp LYS 300 GAA Glu GTC Val TCC Ser ACC Thr AAG Lys 330 GCT Ala CAA Gln ACT Thr GAT Asp ACT Thr 460 ATC Ile GTT Val GTG Val ATC Ile AGT Ser 285 GAA Glu ATC Ile TGG Tr? ACT Thr 365 CTG Leu CAA Gln GTT val GGG Gly ATT Ile 445 TCC Ser CAC His GTG Val Lys 270 GGA Gly ATG Met CAT His CTG Leu 350 GAT Asp ATC Ile AAT Asn CCT Pro GGA Gly 430 GAG Glu TGG Trp TCC Ser GAG Glu AAC Asn GTG Val ATT Ile TGG T1-'P GTG Val 575 GAT Asp GTG Val CAA Gln TTG 655 TAC Tyr CAT His AAA Lys AGC Ser AAG Lys 735 GTT val TCA Ser CGA Arg GCT Ala GTC Val AGG Arg 560 GAC Asp GGA Gly GTT Val GTT Val GCT Ala 640 AAC ASH ATC Ile AAG Lys GAG Glu TAT Tyr 720 CAG Gln TCT Ser TAT Tyr GAG Glu GCT Ala CTG 545 GTC Val CCG PEO CTA Leu GAA Glu GCA Ala 625 CTC Leu ATT Ile ATC Ile AAT Asn CTG Leu 705 GTT Val GCT Ala AAG Lys CTG Leu GCA Ala 530 GT1‘ Val ATT Ile ATG Met GTG Val GGA Gly 610 GTG Val ATG Met GTA Val ACA Thr AGG Arg 6 9 0 GAT Asp ATT Ile GAT Asp TAT Tyr AAG Lys 77 0 AAG Lys 515 GTC Val GTC Val GAA Glu CAG Gln Leu 595 ACA Thr AAG Lys TCT Ser AAC Asn GAG Glu 675 GAT Asp ATC Ile TTA Leu ACT Thr TCC Ser 755 CTG Leu CTG Leu ATT Ile TCA Ser CTG Leu 580 GGT Gly GCC Ala ATG Met GAA Glu TTG Leu 660 TAT Tyr AGC Ser TCT Ser ACA Thr 740 GAC Asp TCT Ser GTG Val GTG val TGG TIP ATC Ile 565 CCT Pro CGG Arg TAT Tyr CTA Leu CTG 64 5 CTG Leu TGC Cys TTC Phe GGA Gly Phe 725 CAG Gln ATC Ile ATG Met GCT Ala CTG Leu Lys 550 AGC Ser TAT Tyr GTC Val GGA Gly Lys 630 AAG Lys GGA Gly '1'rc Phe CTG Leu TTG Leu 7 10 GAA Glu TAT Tyr CAG Gln TTA Leu CCC PIG TTG Leu 535 CAG Gln CCA Pro GAC Asp TTG Leu TTA 615 CCC ATA Ile GCC Ala TAT Tyr AGC Ser 695 AAC Asn AAC Asn GTC Val AGA Arg GAC Asp 77 5 ACC Thr 2 0 GTG Val GAT Asp TCA Ser Gly 600 AGC Ser ACG Thr ATG Met TGC Cys GGA Gly 680 CAC His CCT Pro AAT Asn CCC PIC TCA S3!‘ 760 TCA Ser CTG Len ATT Ile CCG Pro GGA Gly CGG Arg GCC Ala ACT Thr ACC Thr 665 GAT Asp CAC His GCT Ala GGT Gly ATG Met 7 4 5 CTC Leu GAA Glu CGT Arg GTG val AGG Arg CAT His 570 TGG Trp GGA my TCC Ser AGA Arq CAC His 650 AAG Lys TTG Leu CCA PTO GAT Asp GAC Asp 7 3 0 CTA Leu TAT Tyr GTC Val TCT Ser ATC Ile TAT Tyr 55 GAA Glu GAG Glu GCG Ala TCC Ser 635 CTG Leu TCA Ser GTC Val GAG Glu GAA Glu 715 TAC Tyr GAA Glu GAT Asp GAA Glu ATC Ile 540 GAA Glu TAT Tyr CCT PIC 62 O AGT Ser GGG Gly GGC Gly AAC ASH AAG Lys 700 AGC Ser ATG Met AGG Arg CGT Arg AAC Asn 780 CTC Leu 525 TCA Ser ATT Ile ATT Ile CCA Pro GGG Gly 605 GTC Val GAA Glu CCA Pro CCC Pro TAT Tyr 68 5 CCA Pro ACA Thr GAC Asp CCA Pro 7 65 CTC Leu ACG Thr CGC Arg TAT Tyr AGA Art; 590 AAG Lys ATG Met CAT His ATT Ile 670 TTG Leu AAG Lys CGG Arg ATG Met GAG Glu 750 GCC Ala CTT Leu Thr Tyr Gln Val GTC Val 815 CAC His CGT Arg GAT Asp ATT Ile GTG Val AAG Lys ATC Ile TCG S81’ AAC Asn TAT Tyr GTG Val 850 GCT Ala CCT PIC GAG Glu 865 AGC Ser rec T1-'P TCT Ser 880 TAT Tyr GGC Gly CCT Pro 895 TAC Tyr CCC Pro GGC Gly AGT Ser GGG Gly TAC Tyr CGG Arg GAG Glu ATC Ile ATG Met GTG Val 930 TAC Tyr CAC His 945 Phe Leu AAA Lys AAG Lys 960 AGT Ser TAT Tyr CCT Pro 975 GCT Ala GTG Val GCA Ala GTC Val ACC Thr TAC Tyr AAA Lys CTG Leu GAT Asp GAG Glu CAG Gln CCT Pro GAC Asp ATT GAC Ile Asp 1025 AGA Arg CAC His 1040 AGC TCG Ser Ser AGC AGT TCC ACC Ser Ser Ser Thr 1055 GAC ATG ATG GAC Asp Met Met Asp CTG Leu Cys 835 TCG Ser ATC Ile ATT Ile ATG Met ATG Met 915 AGT Ser GAA Glu CGC Arg AAC Asn 995 AGA Arg CCT Pro CAG Gln TTC Phe GAC Asp Arg Gly Met Glu GCT Ala 820 GCT Ala CGC Arg AAC Asn GAC Asp GGC Gly Phe Leu GGC Gly AGT Ser ACC Thr 855 GAC Asp AAC Asn 870 Phe Leu CTG Leu CTC Leu 885 TGG TI‘? GAG Glu ATG Met 900 GT6 Val GAT Asp TCT Ser GCC Ala AAG Lys CCT Pro GAC Asp TGC Cys ms Trp AAC Asn AGT Ser 935 GAG Glu ATT Ile GTG val 950 GAG Glu AAA Lys ATT Ile 965 CAC His CTG Leu ATG Met 930 CGT Arg GTG Val GAC Asp GAG Glu GAA Glu GAC Asp AAG Lys CTG Leu AGC Ser GCT Ala GAC Asp 1015 GTC Val CCT Pro GAG GAG Glu Glu 1030 ACC Thr TCT Ser 1045 GAA GAG Glu Glu ATC AAG AGA GAG Ile Lys Arg Glu 1060 ATC GGC ATA GAC Ile Gly Ile Asp Phe Ala Ser Lys GTT Val CTC Leu 825 CTG Leu Ala Gln GCC Ala 840 AGA GAC ATC Ile ATG Met CCC Pro GTG Val AAG Lys 860 Phe Leu TAC Tyr ACC Thr ACA Thr CTG Leu 875 AGT Ser ATC Ile wcc Ser 890 Phe Leu Gly ACT Thr TTC Phe 905 TAC Tyr AAT Asn AAG Lys CAC His 920 GCT Ala ACC Thr AGT Ser GAA Glu GAG Glu CCG Pro GAG Glu AAG Lys AGA Arg 940 AAT Asn CTG Leu CTG Leu CCT Pro 955 GGA Gly GAC Asp TTC Phe CTG Leu 970 AAG Lys AGT Ser TCA Ser GAC Asp 985 AAT Asn GCA Ala TAC Tyr CTG Leu 1000 AAG Lys GAC Asp TGG Tr? GAG Glu AGT Ser GGC Gly TAC Tyr ATC Ile ATT Ile Asn Cys GGA Gly Lys CAT His 845 GAT Asp TGG Trp ATG Met GAT Asp GTC Val GGC Gly ACC Thr ATC Ile AAG Lys 910 GTC val 925 TAC Tyr CCC Pro TCC Ser CAA Gln TAT Tyr GAC Asp CAT His ATT Ile GGT Gly 990 GGT GGT Gly Gly 1005 CCT Pro CTG Leu GAG Glu GAC Asp CTG Leu GGC AAG Gly Lys 1035 AG? Ser GCC Ala ATT Ile 1050 GAG ACG Glu Thr GAC Asp GAG ACC ATT GAA Glu Thr Ile Glu 1065 TCT Ser TCA GAC CTG GTG Ser Asp Leu Val AGG Arg AAC Asn GGT Gly TCC Ser GAC Asp ATC Ile 1070 GAA Glu GAC Asp Ser Phe Leu ACCTCTGGAT GGTTGATGTT TGAATGGGAT TCTCAGTGGT GAACTTTCTG AACTTCAGCA CTATCTTCTT CCTGATGTCA AAAAGGTACT ATAATTAACC ATATTGTAAT TGTAA CCCGTTCAGA TAAAGAGAAG ATTTTGAAAT GTGTGAAGTT CTTCAAGGAC TTGTAATTAT TGGACTTCTG GCTGCTGTTG GGTACTATAG AACCTTGTTT CTATGTTTAT AAACCACTTT TTCCCAGCCA GAACTTTGTC TGGAGATAGA ATTGGTGAGA GTAAATAACT AAGAGACCAC AACTTTTTAA CATTTTGCTA AATAGATTTG AATACTACTA (2) INFORMATION FOR SEQ ID NO:4: ATTGCAATGC AGGGCCTCGG AGTGTTGCCT TGGATAAGGG GTCCAACAGA CTAACCACGG TCAATCCATC AGAAGTGCAT TCTTTTTTAG GGTCATTTAG CTGTTATCAG (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1089 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (Xi) SEQUENCE DESCRIPTION: SEQ ID Met Gly Thr Gly Leu Ser Glu Asn Asn Phe Gly Glu 65 Ser Ser Phe Val Thr Tyr Thr Glu Gly Arg val Pro 130 Ser Ala Ile Ser His Leu Glu Lys Glu Ser Asp Val Val Leu Cys Tyr 100 His Ile Gly Met Ile Pro Leu Val Glu Glu 70 Pro Ala Phe Leu Val Cys Gln val Gln val 55 Ile Arg Val Ser Asn His Ile Tyr Asp Tyr Arg Thr Val Leu Ser Glu Glu Ala 90 Ser Gln Thr Pro Asp val Ile Pro 155 GGAGGTTGAG AGGAGGACTT GGAGCCTTTC CTTGCAATGC AATAATAGGC CACAATTTAT CTGTGTTTAG CATGTACTTC GAAAAACCAT TGTTAAAGAG AAGCCTGACA TAATGCTAAA NO:4: PIG Ser TAAATATGAA CTCAGTAGCA CACAGAAGGT ACTGCGACAG ATTGTATTAA CCTCTTGAAA TTTTGACCTT ATAAAGAATA ACTCATTTTC TGTGTAATAA Ser Phe Pro Met Asn Asn Ala Ala Glu Glu Arg Glu Glu Leu 80 Thr 95 Pro Asp val 140 Glu Thr Ala Phe Asp Asp Val Thr 3615 3675 3735 3795 3855 3915 3975 4035 4095 4100 Val Lys Tyr 225 Glu Gly Tyr Glu Val 305 Phe Val Asn Lys Lys 385 Ala Ile Val Ile Leu 465 Art; Ile Lys Ala 2 10 Lys Val Ile Thr Cys 290 Thr Ile 370 G111 Val Leu Arg cys 450 Ala Ser Ala Leu Ala Val Gly 195 Thr Ser Val Thr Leu 275 Ala Ile Gln Val Thr 355 Gln Glu Lys Asp Cys Lys Asn Thr val Lys 515 Lys Ser Gly Asp Met 2 60 Thr Arg 3 40 Glu Asp Ser Leu 4 2 0 Thr Asp Tyr 405 Val Ala Ile Glu 485 T1-"P Thr 230 Gln Glu Pro Gln His 310 Ala Tyr Glu Arg Gly 390 Thr Asp Glu Lys Ser 470 Gly Leu Ala Gln Asp 215 Ile TIP Glu Glu Ala 295 Glu Val Tyr 37 5 His Phe Asp Gly Lys 4 5 5 Asn Arg Ala Pro Leu Gln Th: 2 00 Val Thr I la Ala 28 0 Thr Lys Asn Pro Leu Thr 440 Cys Ile Val Lys Thr 520 Ile Lys 265 Thr Pro 3 4 5 Thr His 425 Pro Asn Ile Thr Asn 505 Len Pro Met Thr Pro 250 Val Val Glu Phe His 33 0 Art; Glu Lys Ile Leu Asn Thr Phe 4 9 0 Leu AI9 Cys 235 Gly Pro Lys Val Ile 315 Glu Val 395 Thr Ser Pro Glu Glu Ala TY!‘ Asn Ala 220 Ala Glu Ser ASP Lys 300 Glu Val Ser Thr Lys 380 Ala Gln Thr Asp Thr 460 Ile Lys Gly Glu Ile Glu Val 205 Len Val Val Ile Ser 285 Glu Ile Lys TIP Thr 365 Gln val Gly Ile 445 Ser His Val Ala Leu 525 S81‘ Tyr Lys Phe Lys Lys 27 0 Gly Met Lys His Leu 3 5 0 Asp Ile Asn Pro Gly Glu TIP Ser Glu Glu 510 Thr Ala Thr Asn Gly 255 Asp Lys Pro P112 3 35 Lys Val Arg Glu Ser Gln TIP Thr Arg Glu 495 Asn T‘-'P Leu Val Asn 240 Lys Val Tyr Lys Thr 320 Val Asn Glu Ala Asp 400 Ser Thr Met Ile Asp 480 Thr Arg Ala Glu 865 TY!‘ Met Val Gly 610 Val Met val Thr Arg 690 Asp Lys 770 Asn Val Asp Ile Val 850 Ser Gly Gly Val 930 Gln Leu 595 Thr Lys Ser Asn Glu 675 Thr Ser 755 Lys Ser Ala Leu Cys 835 Ser Ile Ile Met Met Leu 580 Gly Ala Met Glu Leu 660 Tyr Ser Phe Ser Thr 740 Asp Ser Glu Arg Ala 820 Asp Met 900 Phe Gly Phe 725 Gln Ile Met Gly Gly 805 Ala Phe Gly Asp Leu 885 Val Tyr Val Gly Lys 630 Lys Leu 7 10 Glu Leu 790 Met Arg Gly A511 870 T1’? Asp Leu Leu 6 15 Pro Ser 695 Asn Asn val Arg Asp 775 Thr Glu Asn Leu Thr 855 Leu Glu Ser Ser 935 Ser Gly 600 Ser Thr Met Cys Gly 680 His Pro Asn Pro Ser Phe val Ala Phe Tyr Ile Thr His 920 Arg 585 Ser Thr 665 Asp Leu 825 Thr Phe Phe 9 0 5 TTP Gly Ser Arg His Lys TY!‘ Ser 890 Glu Ala Gln Ser 63 5 Ser Val Glu Glu 715 Tyr Glu Asp Lys Leu 795 Ser Ala Ile Val Leu 875 Leu Asn ' Phe Phe Pro 62 0 Ser Gly Gly Asn Lys 700 Ser Met Art; Art; Asn 7 8 0 Lys Gln Met Lys 8 60 Ser Gly Lys Arg 940 Pro Gly 605 Val Glu Tyr 685 Pro Thr Asp Lys Pro 765 Ser Asn Gly His 845 TIP Asp Gly Ile Val Arg 590 Lys Met Lys His Ile 670 Lys Arq Met Glu Ala Phe cys Lys 8 3 0 Asp Met Val Thr Lys Asp Val Lys Gln Leu 655 Tyr His Lys Ser Lys 73 5 Val Ser Ser Thr Val 8 15 Ile Ser Ala Trp Pro 895 Ser Gly val Val Ala 640 Asn Ile Lys Tyr 72 O Gln Ser Tyr Asp Tyr 800 His Val Asn Pro Ser 880 Tyr Gly Ser Tyr Glu Lys Ile His Leu Asp Phe Leu Lys Ser Asp His Pro Ala 965 970 975 Val Ala Arg Met Arg Val Asp Ser Asp Asn Ala Tyr Ile Gly Val Thr 980 985 990 Tyr Lys Asn Glu Glu Asp Lys Leu Lys Asp Trp Glu Gly Gly Leu Asp 995 1000 1005 Glu Gln Arq Leu Ser Ala Asp Ser Gly Tyr Ile Ile Pro Len Pro Asp 1010 1015 1020 Ile Asp Pro Val Pro Glu Glu Glu Asp Leu Gly Lys Arg Asn Arq His 1025 1030 1035 1040 Ser Ser Gln Thr Ser Glu Glu Ser Ala Ile Glu Thr Gly Ser Ser Ser 1045 1050 1055 Ser Tnr Phe Ile Lys Arg Glu Asp Glu Thr Ile Glu Asp Ile Asp Met 1060 1065 1070 Net Asp Asp Ile Gly Ile Asp Ser Ser Asp Leu Val Glu Asp Ser Phe 1075 1080 1085 (2) INFORMATION FOR SEQ ID N0:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6375 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (0) TOPOIDGY: linear (ii) MOLECULE TYPE: com to mRNA (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: no (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (B) STRAIN: lambda qtlo (ix) FEATURE: (A) NAME/KEY: COS (B) LOCATION: l29..3395 (D) OTBER INFORMATION: /note= "nucleotide number 1 of this sequence is identical to the nucleotide number 1 of the previous 4100 long sequence" (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: TTGGAGCTAC AGGGAGAGAA ACAGAGGAGG AGACTGCAAG AGATCATTGG AGGCCGTGGG 60 CACGCTCTTT ACTCCATGTG TGGGACATTC ATTGCGGAAT AACATCGGAG GAGAAGTTTC 120 CCAGAGCTAT GGGGACTTCC CATCCGGCGT TCCTGGTCTT AGGCTGTCTT CTCACAGGGC 180 TGAGCCTAAT CCTCTGCCAG CTTTCATTAC CCTCTATCCT TCCAAATGAA AATGAAAAGG 240 TTGTGCAGCT GAATTCATCC TTTTCTCTGA GATGCTTTGG GGAGAGTGAA GTGAGCTGGC 300 AGTACCCCAT GTCTGAAGAA GAGAGCTCCG ATGTGGAAAT CAGAAATGAA GAAAACAACA 360 GCGGCCTTTT TGTGACGGTC TTGGAAGTGA GCAGTGCCTC GGCGGCCCAC ACAGGGTTGT 420 TCATCGTGGA CTGTAACCTT TTAATGGGAC TCCAGACCAT TGGAAGCTCT TTAACAATGA TCACAATGCT CCGAGGCCAC AGGTCAAAGA AACCCACCTT AGGTGCGGGC AAAATCTCAC AATTAAAGCT ATGAAGATGC TGGACTTGGT AAGGCACGCC ATGAAACTTC CCCGAGACAG CCGTGCGATG CTCCCACCCT TGATCATCTC GCTGGAGGGT TGCAGCTGCC TCTTGGGGTC CCCAACCTGT AACAAGCTCT TAAACTTGCT TCTATGGAGA CAGAGAAGCC GGAGCTATGT ATACTACACA AGAGATCACT TCAAAAACCT TCACCTATCA GGATGATGAT ACACAACAGT CTTCACTGTA CCCATTTAAT TAAAACCGTG GGTGGTTGAC GGAAGAAATC GGTGAAAGAC AATGAAGAAA CAGCCAGTTG CTACCCACCT TGAGATCACC GATCCGTGCT TGTGAAGAGC CGATGATCAC GCTTCCTGAT CTGGACTATT GAGTACCGTG CCTGGCTAAG GCGTTCTGAA ACTTATTGTC CATTGAATCA TTATGACTCA TGGAGCGTTT CATGAAAGTT CATGTCTGAA GGGAGCCTGC TTTGGTCAAC AAAGAAAGAG TATTTTATCT GTATGTCCCC CTATGATCGT CCTTTCAGAT AGTTGCCCGA TCTGCCATTA GAGGGGGTGG GGGCCCTATA GTTTATGCTT TATAAGTCAG CTTCAATGGA AAAGTCCCAT AGTGGAGATT GTCACTATTT GAAGCTGTCA CCCAGGATAT ACTGATGTGG AAGGAAGAAG TATACTTTTG CATGGCTCAA ATTGAGTGGA TTGGCCAACA GAGGGCCGTG AATCTCCTTG CTCACGGTGG CTGGTTGTCA ATCAGCCCAG AGATGGGAGT GGGAAGGTGG GCAGTGAAGA CTGAAGATAA ACCAAGTCAG TATTTGCATA CTGGATATCT TTTGAAAACA ATGCTAGAAA CCAGCCTCAT GATAACTCAG GGAATGGAGT TACCTTGTCG TACCTGCCTC TCTGTGAGGC TAAAAGCAAC GGGAAACGAT CTTACCCTGG CCATCAAATT ACGAATGTGC CTGTCCATGA ACCTGCATGA CCTGGCTGAA AAAAGATTCA ACAGTGGCCA AACTGTTAAC CTGGGGGACA TGATATGCAA ATGTCTCAAA TGACTTTCGC GAGCTGAGAA CTGCTGCAGT TTTGGAAACA ATGGACATGA TTCCAAGAGA TTGAAGGAAC TGCTAAAACC TGACTCACCT GCCCCATTTA AGAATAGGGA TTGGATTGAA ATGGTGACTA GGAAAGAGGT ATAAGAAGAA AAGGCCTTAC TTTTGGCTTC CACAACTGAT CTACGACAGC CACCGTCAAA ATCAGAGCTG TGTGGTCACC AGAAGTGAAA GGTGTACACT TGCCCGCCAG GAAAGGTTTC AGTCAAACAT AAACAATCTG GGAAATAAGG TTATACTATT TCAAGTTCCT GACGGTGAGG AGATATTAAG CATCATCACG CAAAGTGGAG CCGAGAGCTG CCTGGTGCTG GAAACCGAGG ATATATTTAT TGGACTAGTG AGCCTATGGA CACGGCCAGA GGGGCCACAT CATCATCACA TAGCTTCCTG CCCTGCTGAT CATGGACATG TTCTAAATAT ATCTATGTTA TTTATTGGAT AAAAAATTGT CCCGAGACTC AGACAGGGCT GGAAAGAAGT GATCTAGAAA TGTGCTGTTT GGCAAAGGCA TTGACGGTCC GCTACCAGGG ATTGAAATCA TTTGTTGTAG ACTCTGATTG TATCGAAGCA GTAGCTCAAA TCATCCATTC TGCACAGCTG AAATGTAATA GAGATCCACT GAGACCATCG AAGCTGGTGG TTGGTGATTG TATGAAATTC GTGGACCCGA CTTGGTCGGG TTAAGCCGGT TCCAGTGAAA TTGAACATTG GAGTATTGCT AGCCACCACC GAAAGCACAC AAGCAGGCTG TCCGACATCC GACTCAGAAG TTGTTGAGCT GTCCACCGTG 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 CCGTGAAGTG TCTGGTCTTA GCATGATGGT CTGACCACGC AGAAGAGACC ATAAAAAGAG CACGCATGCG ACAAGCTGAA ACATCATTCC ACAGACACAG CCTTCATCAA TAGACTCTTC TCCACTTCTG TTGAGAGGAG CTTTCTAAAT AATGCCTCAG TAGGCCACAG TTTATACTGC TTTAGATTGT ACTTCCCTCT ACCATTTTTG AAGAGATAAA TGACAACTCA CTAAATGTGT CTTACTAAGT CTATAAAGTA AAAGTAGTGT TCGTATTAAA TTAAGTCCTA TATATTACCC TTTTTTTTTT ATCTATGAAC CTTACCCCAA TGGAAGTGCA ACCTGGGTTT GATGGCTCCT TGGCATTCTG GGATTCTACT TACCAGTGAA CTCCTTTTAC TTATGAAAAA TGTGGACTCA GGACTGGGAG TCTGCCTGAC CTCGCAGACC GAGAGAGGAC AGACCTGGTG GGGCCACCTC GACTTGGTTG ATGAATGAAT TAGCATCTCA AAGGTGAACT GACAGAACTT ATTAACTATC TGAAACCTGA ACCTTAAAAG GAATAATAAT TTTTCATATT AATAATGTAA AGGTGATGAG TGGTAATAGC TGTCCAGGAA AAACAATTAA AAAGTTCTCA AATGGAAAAT TCTTCTTGCC CTGAAAAGGG AGAGAAAGAG TTAGCCTGAT CCATCCTTGA GAGAGCATCT CTCTGGGAGA TTCTACAATA GTCTACGAGA CACCTGAGTG ATTCACCTGG GACAATGCAT GGTGGTCTGG ATTGACCCTG TCTGAAGAGA GAGACCATTG GAAGACAGCT TGGATCCCGT ATGTTTAAAG GGGATATTTT GTGGTGTGTG TTCTGCTTCA CAGCATTGTA TTCTTTGGAC TGTCAGCTGC GTACTGGTAC TAACCAACCT GTAATCTATG CATGATTTCC TTTGACAGTT TTTAGTGAAT GTCAGAATTT CTGCCCTCTG ATGTAGAGGC ATAATGATCA TGATGAAAGC TCACAAAGGA TTTGAAACTC CCTCAGTTCT GATTCTGAAG TTGACAACCT TCTTTTCCCT AGATCAAGAG TCATGGTGAA AGATTGTGGA ACTTCCTGAA ACATTGGTGT ATGAGCAGAG TCCCTGAGGA GTGCCATTGA AAGACATCGA TCCTGTAACT TCAGAAAACC AGAAGTTCCC GAAATGAACT AAGTTTGGAG AGGACATTGG ATTATGTAAA TTCTGAAGAG TGTTGAACTT TATAGCATTT TGTTTAATAG TTTATAATAC CTCCACACAA TTTGACATTT TAAATTTAGT TTAACTGTAC AAATAATGGG ATAAACCTGT GCGCANAAAG TTTGGCGACC TGCCCAGACA GAGACCATAA CAAATGTGTG TATGAAGTCT CTACACCACA TGGTGGCACC TGGGTACCGG ATGCTGGAAC GAATCTGCTG GAGTGACCAT CACCTACAAA ACTGAGCGCT GGAGGACCTG GACGGGTTCC CATGATGGAC GGCGGATTCG ACTTTATTGC AGCCAAGGGC TTGTCAGTGT ATAGATGGAT TGAGAGTCCA TAACTCTAAC ACCACTCAAT TTTAAAGAAG TGCTATCTTT ATTTGGGTCA TACTACTGTT AGCACAATTT ATATTAAATA TGAGCATAGA TGAATAGGTT ATTAGAAACA GCTGAACATA ACTGGATTTG CCAATATATG TCAGCCTCCT AGATATTCTT TGGCAGCCAG GAGGGAAACC CTGAGTGATG CCTTACCCCG ATGGCCAAGC AGTGAGCCGG CCTGGACAAT CCTGCTGTGG AACGAGGAAG GACAGTGGCT GGCAAGAGGA AGCAGTTCCA GACATCGGCA AGGGGTTCCT AATGCGGAGG CTCGGGGAGC TGCCTCTTGC AAGGGAATAA ACAGACACAA CACGGCTGTG CCATCCATGT TGCATGAAAA TTTAGTGTTA TTTAGAAGCC ATCAGTAATG AAAAACAATC ACATGTTTCT GAACAAAGTA CCCCAATCCA AACAAAACTC ACTTCTCATG CAGAAGTTNT TATTTTTTGA TCTTTCACCC TAGTGGAGGC GTAGACTAGT AGAGTCTGTA 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 4380 4440 4500 4560 4620 4680 4740 CAGGAAGTTG CGTCCTTCGG ATCGTTAACG ACCAAAACTG TGAAAGGGGC TGTTTTTAAT TTPPTTGTAA GATAGGTGTG TAACGACTAT GTCTACTACG TAGAGTTACG GGTCTAACCG TATGCAAACA ATGTCGGAAT TACTCCGGCC CTTAAACATA AAGGAAAACC AAAAATACTA TCACTACAGG CCCTGTATTC ACCTCTTATG ATTGACACTA TAAAATCTTT CACACTCTTT AAGTACAAAA TTAAATTTCC CCATGGGAAA ATGATAAATT CTGGAATTAA TCCAAAAGGT AGAGGGCAGA ACGTTAAGTC AATTCAAAAT ACAGTTTGTC GGTATACTTA AAACCTTCTA TTCGGGGTTG GTTAATTTTT CAAAGATCTT AAAACAACCA TACTTTGAAG TACTGACGTA GGAGGACGTT CTGTTAGTTT AATTTTACAC TATTTTACTA ACGGTTTTGT TTAGGGGTGT ATTATTTTTC TTGATTGAAC ACAAAATTTT TTAAG CAAATAATTT TAGGAACCGA ATTGAAAGGT TTTCATTTCT AGAGCGGAGG ACAAGGTTCA GACTATTAAA CAACCACACC CTTTGTACCC CGTCTTCGTT AAAGAATAGG CTTTTGGACT AGTGTCGAGT CGAAACGTAA AGTCAGGTCG AACACCCACA TCAGAGGTCT CGGCCGGACT CAGACGGTTA CAATATGTAG AAATACTGTT CCGTGTAATT TTTCTATGAA TATCCCAGAT GCACATTTAC (2) INFORMATION FOR SEQ ID NO:6: GAACTTTGGA AGTCCAATCA CAGAATCGAC ACGATGAAGG GTGAGGTATG GAGACACATT CTCCAATCTA CAAGTAACCG GACACTAATG ATTATTTCAT TTGAAAAAGT GATCCAAGAC TCGTAAGACA AACTATAACG TCAAAGGTCA CACACACAAA TCTTTTAAAC CTTTGTGATA GACATGTTTT TTATACATAT CGACATAGTG GACAACGTGA TGTACAAGGG GGTTATGTTT TTCTAGAAAT (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 base (B) TYPE: nucleic acxd pairs (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: N0 (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: ACAGGGTTCT CTGTAAATTA TCCGACTCTT GTGACATACC GGGCGGTTCC GGTCGAGTCA CCCTCCTACT TAAGAAACGT ACGTTAGTGA GAACTGATGG ATCATTCACG ATCTCGGTTA AATAGCGAGT ACACTCGGAA GGATTGTTTA AGTCGTTTAA GGTTAGAAAG AACACTGAAA ACCAGGATAA ATACATAAAG ACGGAAGCAA AAACTTACAG TTTTGTTACC TACATAATGC ATAAAGTTAT TAAGTTGGTG CGGTAGATCG TCGATTTCAA CCCTCTAACT TTTCCGTACA CAAAACCACC TAACAGTGTA TATGACGAAT CACGATAGCC ATGACCACAT CTTCTGACTC ATCTGAACTT GAGAGGGAAC CGTACTGTAG CGAGGGTGGA GGTCTAAACA GATGAAAGAT AATTTGCTAA AAACACTTCT ATATATCTGA ATATAAAAAA GTTTTAAATA ACACCACTTA TTACGGGGAC TTACTATATA 5040 5100 5160 5220 5280 5340 5400 5460 5520 5580 5640 5700 5760 5820 5880 5940 6000 6060 6120 6180 6240 6300 6360 63 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOIDGY: linear (ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Home Sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: TCCTTCGACC TACAGATCAA TTAGCTTCCT GTAGGGGGCT G 41 (2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 base pairs (8) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo Sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: ATCACCGTGG TTGAGAGCGG CTAGCTTCCT GTAGGGGGCT G 41 (2) INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 base pairs B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo Sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: TACAGACTCC AGGTGTCATC CTAGCTTCCT GTAGGGGGCT G 41 (2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 44 base pairs (ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo Sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l0: CTCTACATCT TTGTGCCAGA TCCCTAGCTT CCTGTAGGGG GCTG (2) INFORMATION FOR SEQ ID NO:ll: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 base pairs (3) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLDGY: linear (ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo Sapiens (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll: CAGATCTCTC AGGGCCTGGT CACCGTGGGC TTCCTCCCTA ATCAT (2) INFORMATION FOR SEQ ID NO:12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 48 base pairs (8) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo Sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: CAGATCTCTC AGGGCCTGGT CATCAACGTC TCTGTGAACG CAGTGCAG (2) INFORMATION FOR SEQ ID NO:13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 base pairs (3) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: CAGATCTCTC AGGGCCTGGT CTACGTGCGG CTCCTGGGAG AGCTG (2) INFORMATION FOR SEQ ID NO:14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTN: 42 base pairs (8) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: No (vi) ORIGINAL souncz: (A) ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: CAGATCTCTC AGGGCCTGGT CGTCCGAGTG CTGGAGCTAA GT (2) INFORMATION FOR SEQ ID NO:l5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: CDNA to mRNA (iii) HYPOTHETICAL: YES (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo Sapiens (B) STRAIN: lambda gt10 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: GCTCCCACCC TGCGTTCTGA ATAACTGGCG GATTCGAGGG G (2) INFORMATION FOR sno ID NO:16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 base pairs (8) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLDGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: YES (iv) ANTI-SENSE: N0 (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo Sapiens GAACTGTTAA CTCAAGTTCC TTAACTGGCG GATTCGAGGG G 41 (2) INFORMATION FOR SEQ ID NO:l7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 base pairs (3) TYPE: nucleic acid (c) STRANDEDNESS: single (D) TOPOIOGY: linear (ii) MOLECULE TYPE: CDNA to mRNA (iii) HYPOTHETICAL: YES (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo Sapiens (B) STRAIN: lambda qtlo (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: ATTTCTGTCC ATGAGAAAGG TTAACTGGCG GATTCGAGGG G 41 (2) INFORMATION FOR SEQ ID NO:l8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 base pairs (8) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: CDNA to mRNA (iii) HYPOTHETICAL: YES (iv) ANTI-SENSE: no (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo Sapiens (B) STRAIN: lambda gtlo (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: TATGCTTTAA AAGCAACATC ATAACTGGCG GATTCGAGGG G 4l (2) INFORMATION FOR SEQ ID NO:19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 44 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOIDGY: linear (ii) MOLECULE TYPE: CDNA to mRNA (iii) HYPOTHETICAL: YES (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo Sapiens (B) STRAIN: lambda gtlo (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 base pairs (8) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: CDNA to mRNA (iii) HYPOTHETICAL: YES (iv) ANTI-SENSE: NO (vi) oamxmu. somzca: (A) ORGANISM: Homo Sapiens (B) STRAIN: lambda gtlo (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: AGCCTAATCC TCTGCCAGCT TGATGTAGCC TTTGTACCTC TAGGA (2) INFORMATION FOR SEQ ID NO:2l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 48 base pairs (8) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: CDNA to mRNA (iii) HYPOTHETICAL: YES (iv) ANTI-SENSE: no (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo Sapiens (B) STRAIN: lambda gtlo (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2l: AGCCTAATCC TCTGCCAGCT TGAGCTGGAT CTAGAAATGG AAGCTCTT (2) INFORMATION FOR szo ID NO:22: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: CDNA to mRNA (iii) HYPOTHETICAL: YES (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo Sapiens (B) STRAIN: lambda gtlo (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: AGCCTAATCC TCTGCCAGCT TTTCATTGAA ATCAAACCCA CCTTC (2) INFORMATION FOR SEQ ID NO:23: (8) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: CDNA to mRNA (iii) HYPOTHETICAL: YES (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo Sapiens (B) STRAIN: lambda gt10 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: AGCCTAATCC TCTGCCAGCT TTCATCCATT CTGGACTTGG TC

Claims

1. A type B or a type A human platelet—derived growth factor receptor (hPDGF—R) fragment consisting of one or two said domains selected from the group consisting of one D2 and D3, fragment having platelet-derived growth factor receptor said fragment binds a extracellular domains, or two of only D1, said ligand binding activity, wherein platelet—derived growth factor ligand with a K” of less than lOuM.

2. A type B or a type A hPDGF-R fragment wherein said fragment consists of extracellular domains D1 and D2, and wherein said fragment has platelet-derived growth factor andf binds a. platelet- of less than 10uM. receptor ligand binding activity, derived growth factor ligand with a Kb

3. A type B or a type A hPDGF-R fragment, wherein said fragment consists of extracellular domains D1, D2 and D3, and wherein said fragment having platelet—derived growth ligand binding activity, and binds a factor receptor platelet—derived growth factor ligand with a Kn of less than 10pM.

4. A hPDGF-R fragment of claim 1, 2 or 3, wherein said fragment exhibits an affinity of 5 nM.

5. A hPDGF-R fragment of claim 1 or claim 3, wherein said fragment comprises at least about 15 contiguous amino acids from a domain D3 intra—cysteine region.

6. A hPDGF-R fragment as claimed in any preceding claim wherein said fragment is soluble.

7. A hPDGF—R fragment of claim 1 or claim 3, wherein at least one of said domains is a domain D3.

8. A hPDGF—R fragment of claim 1, 2 or 3 wherein said type B hPDGF—R fragment is a contiguous sequence within Table 1, from position 1 (Leu) to position 282 (Gly) or wherein said type A hPDGF-R fragment is a contiguous sequence within Table 2 from position 1(Gly) to position 290 (Gly).

9. A type A or B hPDGF—R fragment, wherein said fragment is selected from the group of formulae consisting Of: a) X1-Dm—X1; Vb) Xl—Dm-Xl—Dn-X1; C) X1-Dm-Xl-Dn-X1—Dp—Xl; and wherein: X1 is, if present, a spacer segment located before Dn, and Dp is, if or after a D domain; and each of Dm, independently of one another, and D3 wherein said fragment present, selected from the group consisting of D1, D2, has platelet—derived growth factor receptor ligand binding activity, and binds a platelet-derived growth factor with a Kn or less than 10uM.

10. A hPDGF-R fragment of claim 1, 2, 3 or 9 wherein said fragment is pure.

11. A nucleic acid sequence encoding a hPDGF—R fragment of claim 1, 2, 3 or 9.

12. A nucleic acid of claim 11, wherein said encoding sequence is operably linked to a promoter.

13. A cell comprising a hPDGF-R fragment of claim 1, 2, 3 or 9.

14. A mammalian cell comprising a nucleic acid of claim 11.

15. A cell comprising both a nucleic acid of claim 11 and a protein expression product of said nucleic acid.

16. A method for measuring the PDGF ligand binding activity of a biological sample comprising the steps of: a) contacting an aliquot of said sample to a PDGF ligand in the presence of a hPDGF-R fragment of claim 1 or claim 2 in a first analysis; b) contacting an aliquot of said sample to a PDGP ligand in the absence of said hPDGF—R fragment in a second analysis; and c) comparing the amount of said PDGF ligand binding in the two analyses. of claim 16, wherein said hPDGF—R fragment cell.

17. A method is attached to a

18. A method of claim 16, wherein said hPDGF—R fragment is attached to a solid substrate. wherein said solid substrate

19. A method of claim 18, is a microtiter dish.

20. A method for measuring the PDGF ligand content of a biological sample comprising the steps of: a) contacting an aliquot of said sample to a ligand binding region (LBR) in the presence of a hPDGF-R fragment of claim 1 or claim 2, in a first analysis; b) contacting an aliquot of said sample to a LBR said PDGF-R fragment in a second in the absence of .analysis; and c) comparing the amount of binding in the two analyses. - 103 *

21. A method of claim 20, wherein said contacting steps are performed simultaneously. F. R. KELLY & co., AGENTS FOR THE APPLICANTS,