W. J. (1979) Biochemistry 18, 5294-5299. 19. Nistdr, M., Wedell, B., Betsholtz, C., Bywater, M., Pettersson, M.,. Westermark, B. & Mark, J. (1987) Cancer Res.
Proc. Natl. Acad. Sci. USA Vol. 86, pp. 4917-4921, July 1989 Biochemistry
cDNA cloning and expression of the human A-type platelet-derived growth factor (PDGF) receptor establishes structural similarity to the B-type PDGF receptor LENA CLAESSONWELSH*, ANDERS ERIKSSON*, BENGT WESTERMARKt, AND CARL-HENRIK HELDIN* *Ludwig Institute for Cancer Research, Biomedical Center, Box 595, S-751 23 Uppsala, Sweden; and tDepartment of Pathology, University Hospital, S-751 85 Uppsala, Sweden
Communicated by Viktor Mutt, April 17, 1989 (received for review March 3, 1989)
rum reactive with PDGF receptors, and the expressed protein bound all three isoforms of PDGF with high affinities.A
The primary structure of the human A-ype ABSTRACT receptor for platelet-derived growth factor (PDGF) has been determined. A 6.5-kilobase (kb) transcript was identified through low-stringency hybridization with a probe derived from the B-type PDGF receptor cDNA. The sequence of a cDNA clone corresponding to the 6.5-kb transcript contains an open reading frame that predicts a 1089-amino acid growth factor receptor-like molecule, which displays 44% overall amino acid similariy with the PDGF B-type receptor. The two receptors have a similar domain organization, with five immunoglobulin-like domains extracellularly and an intracellular split protein tyrosine kinase domain. Transfection of the new cDNA into COS cells led to the expression of a protein specifically recognized by an antiserum previously shown to react with the PDGF A-type receptor. The expressed protein was shown to display high-affinity binding of all three 125Ilabeled dimeric forms ofPDGF A and B chains in a manner that is characteristic for the PDGF A-type receptor.
MATERIALS AND METHODS Isolation and Characterization of cDNA Clones. mRNA isolated using the guanidinium thiocyanate protocol (18) from U-343 MGa 31L clonal human malignant glioma cells (19) and from AG 1518 human foreskin fibroblasts (Human Genetic Mutant Cell Repository, Camden, NJ) was used for oligo(dT)-primed cDNA synthesis using the cDNA synthesis kit from Amersham (for U-343 MGa 31L glioma mRNA) or from Pharmacia (for AG 1518 mRNA), and the cDNA was cloned into Agtl0 (20). A 1200-base-pair (bp) Pst I fragment derived from the human PDGF B-type receptor cDNA (13) was used for hybridization of the U-343 MGa 31L cDNA library under low stringency [40%o formamide in 5 x SSC (1 x SSC is 15 mM sodium citrate, pH 7.0/150 mM NaCl)/lOx Denhardt's solution (21)/0.1% SDS/salmon sperm DNA (0.1 mg/ml), at 370C]. A partial clone of the PDGF A-type receptor (phPDGFRA1), the sequence of which covered areas homologous to the tyrosine kinase domain in the B-type receptor, was thus isolated and used for high-stringency screening of the AG 1518 fibroblast cDNA library. Nucleotide sequence analysis of positive clones was carried out by subcloning into M13 vectors followed by primed DNA synthesis on singlestranded DNA templates in the presence of dideoxynucleoside triphosphates using Sequenase (United States Biochemical). Cell Culture and Transfection. COS-1 cells (American Type Culture Collection) were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10%1o fetal calf serum (Flow Laboratories), 100 units of penicillin, and 50 gg of streptomycin per ml. The fragment from the A-type PDGF receptor cDNA phPDGFRA15 covering the translated part of the cDNA (see Fig. lA) was cloned into the simian virus 40-based expression vector pSV7d (22). Transfection of this construct (pSV7dl5.1+5) into COS cells was performed by the calcium phosphate precipitation method (23) to yield transient expression. Cells were examined for expression 2 days after transfection. Radioactive Labeling and Immunoprecipitation. Transfected and control COS cells and U-343 MGa 31L human glioma cells were labeled in methionine- and cysteine-free DMEM, containing 10o fetal calf serum and [35S]methionine and [35S]cysteine each at 200 ,Ci/ml (1 Ci = 37 GBq; Amersham) for 3 hr at 370C. Labeled cells were lysed and prepared for immunoprecipitation by enrichment of the glycoprotein fraction by affinity chromatography on Lens culinaris lectin-Sepharose 4B columns as described (17).
Platelet-derived growth factor (PDGF) is a major mitogen in serum for connective tissue- and glia-derived cells (reviewed in refs. 1 and 2). PDGF is found as dimers of homologous polypeptide chains, denoted A and B (3-5). All three possible isoforms, PDGF-AA, -AB, and -BB, have been identified and purified from platelets and tumor cell lines (6-8). The PDGF isoforms have recently been shown to bind with different affinities to two distinct types of cell-surface receptors for PDGF (9, 10). The PDGF B-type receptor, which binds PDGF-BB with high affinity, and PDGF-AB with lower affinity, but not PDGF-AA, has been purified and characterized structurally through cDNA cloning (11-13). It is a 1074-amino acid transmembrane glycoprotein endowed with a ligand-activated protein tyrosine kinase. The extracellular part can be divided into five immunoglobulin-like domains, based mainly on the spacing of the cysteine residues. Binding of PDGF-BB and -AB activates the tyrosine kinase domain, leading to autophosphorylation, as well as to phosphorylation of cellular substrates (14). A sequence of =100 amino acid residues without similarity to kinases is located within the tyrosine kinase domain and has been implicated in the determination of substrate specificity (15). The type A PDGF receptor binds all three PDGF isoforms with high affinity (9, 10, 16). We previously demonstrated that the PDGF A-type receptor is a Mr 170,000 glycoprotein, structurally related to the PDGF B-type receptor (17). We now report the cloning and sequencing of a PDGF A-type receptor cDNA; expression of the cDNA in COS cells gave rise to a protein that was immunoprecipitated by an antise-
Abbreviations: BSA, bovine serum albumin; PDGF, platelet-derived
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
growth factor.
MThe sequence reported in this paper has been deposited in the GenBank data base (accession no. M22734).
4917
4918
Biochemistry: Claesson-Welsh et al.
Proc. Natl. Acad. Sci. USA 86 (1989)
Antibodies. The rabbit antiserum PDGFR-1 was raised against highly purified porcine PDGF receptor preparations (24) and has previously been shown to react with both the PDGF A- and B-type receptors (17). The rabbit antiserum PDGFR-3 was raised against a synthetic peptide based on the sequence of the murine PDGF B-type receptor amino acids 981-994 and reacts specifically with the human B-type PDGF receptor (17). Scatchard Analysis. Confluent transfected COS cells in 12-well dishes were washed once with cold phosphatebuffered saline (PBS) containing 1 mg of bovine serum albumin (BSA) per ml. Cells were incubated in 0.5 ml of PBS/BSA per well on ice for 2 hr in the presence of constant amounts of 1251-labeled PDGF isoforms and increasing amounts of corresponding unlabeled ligands. The cells were then washed five times with cold PBS/BSA and lysed in 1% Triton X-100/10%o (vol/vol) glycerol/20 mM Hepes, pH 7.4; solubilized radioactivity was determined in a -y-counter. Calculations of Kd values were performed with the RBINDING A
0
2
computer program (E. J. J. van Zoelen, personal communication). PDGF-AA and PDGF-BB were recombinant human forms purified from a Saccharomyces cerevisiae expression system (16). PDGF-AB was purified by immobilized metal ion affinity chromatography (6) from PDGF purified from human platelets as described (25). PDGF isoforms were "2I-labeled by using the Bolton and Hunter protocol (26) to specific activities of 41,000 (PDGF-AA), 64,000 (PDGF-AB), and 39,000 (PDGF-BB) cpm/ng.
RESULTS Isolation of a PDGF A-Type Receptor cDNA Clone. Human glioma U-343 MGa 31L cells have previously been shown to express only PDGF A-type receptors (17). When mRNA from these cells was probed with a fragment corresponding to the intracellular part of the B-type receptor under lowstringency conditions, a 6.5-kb transcript could be visualized (data not shown). Because crossreactive antiserum and pep4
*
6
kb
A
mRNA
I
1
phPDGFRA1 phPDGFRA15
pSV7dl5.1+5
B
SSGGAGCSACACGGAGAGAAACAGAGCAGCACACSOCAAGACASCASSGCAGGCCOTGCCCACGCSCSSSACSCCASCSCSGCGACASTCASSGCGGAASAACASCCCACCAGAAGSST C
120
Out Gly Thr SOr .is Pro Ala Ph. Lou Val Lou Gly Cys Lou Lou Thr Gly Lou SOr Lou II1 Lou Cys Gin Lou SOr Lou Pro CCAGAGCT ATG GGG ACT TCC CAT CCG GCO TTC CTG OTC TTA GGC TSC C?? CC ACA GGG CTG AGC CTA ATC CTC TOC CAG CT? TCA TSA CCC
212
SOr II Lou Pro Asn Glu Asn Glu Lys Vol Vol Gin Lou Asn SOr SOr Pho SOr Lou Ar; [EFijPh. Gly Glu SOr Glu Val SOr ?rp G0n SCT ASC CT? CCA AA? GAA AAS GAA AAG GT? CTS CAG CTG ALT SCA SCC 7?? SCS CTG AGA |SCCTT? GGG GAG AGT GAA 7TG0 AGC TGG CAG
302
V
Tyr Pro Oet SOr Glu Glu Glu SOr O*r Asp Val Glu I1* Arg Asn Glu Glu Asn Asan Sr Gly Lou Ph. Vol Thr Val Lou Glu Vol SOr TAC CCC ASG TCT CAA GAA GAG AGC TCC GAT GTG GAA AC AGA AA? GAA GAA AAC AAC AGC GGC CT? T7T GTG ACG GTC TTG GAA GTG AGC
FyjTyr
SOr Ala SOr Ala Ala Nis ?hr 0ly Lou Tyr Thr Tyr Aso Nis Shr G0n Thr Glu Glu Asn Glu Lou Glu Gly Arg His II. Tyr AG? 0CC TCC CCG GCC CAC ACA 00G T7G ?AC AC? !GcSTAS TAC AAC CAC ACT CAG ACA GAA GAG AA? GAG CT? GAA CGC AGO CAC AT? ?AC Il1 Tyr Vol Pro Asp Pro Asp Val Ala Ph. Vol Pro Lou 0ly Out Thr Asp Tyr Lou Val Ile Vol Clu Asp Asp Asp SOr Ala Ilu I1u 0G7 CCA GAC CCA GAT GA 0CC 7?? CTA CC? CA CGA ATS ACC GA? TAT TSA GTC ATC GG GAG OAT GA? GA? ?C? GCC AT? AA
ASC TAT
Cys|Arg Thr Thr Asp Pro 0lu Thr Pro Val Thr Lou .is Asn SOr 0lu 0ly Vol Val Pro Ala S*r Tyr Asp SOr Arg 01n CCTGS CGC ACA ACT GAT CCC GAG AC? CC? OTA ACC TA CAC AAC AG? GAG GGG G07 CSA CC? 0CC 7CC TAC GAC AGC AGA CAG Asn Gly Thr Ph. Thr Vol 0ly Pro Tyr l |ecy.|Glu Ala Thr Val Lys Gly Lys Lys Ph. GCn ?hr I1. Pro Ph. Asn Vol Tyr AAT 0GG ACC ??C AC? CTA GGG CCC TA? ATC TJjGAG GCC ACC GC AAA CGA AAG AAG TTC CAG ACC AC CCA 7?? AA? G0S TAT Lys Ala ?hr SOr Glu Lou Asp Lou 0lu Out Glu Ala Lou Lys Shr Val Tyr Lys SOr 0ly Glu Thr I1 Val Vol ?hr FCyS Ala AAA GCA ACA ?CA GAG CSG GA? CA GAA ATG CAA OCT CT? AAA ACC 070 TA? AAG TCA 0GG GAA ACG AS? CTS OTC ACC |GS|GCT Pro
Gly Ph. GGC 7?? Ala Lou GC? 7?A Vol Ph.
0T? 7T7
Asn Asn 0lu Val Val Asp Lou 01n Trp Thr Tyr Pro Gly Glu Vol Lys 0ly Lys 0ly II1 Thr Iut Lou Glu Glu I1e Lys Val Pro S*r AAC AA? GAG 070 OTT GAC C?? CAA 0G0 AC? ?AC CC? CGA GAA 0G7 AAA GGC AAA GCC ATC ACA ATS CCGAA GAA ATC AAA OTC CCA 7CC Il Lys Lou Val Tyr Thr Lou 7hr Val Pro Glu Ala Thr Vol Lys Asp SOr 0ly Asp Tyr Glu ATC AAA 77G G0G TAC ACT STG ACG GTC CCC GAG OCC ACC 0T7 AAA GAC AG? CGA GA? 7AC GAA
IE~ysAla Ala Ar; G0n Ala Thr Arg
20
58
302 110 402 140 072 170 002 200 752
230 042 200 032 239
Glu OCT GCC COC CAG OCT ACC AGO GAG
1022
l is Glu Lys 0ly Ph. I1 Glu Ile Lys Pro Thr Ph. SOr G0n Lou 0lu Ala Vol Asn Val Lys Glu Out Lys Lys Vol Thr Il- SOr Vol OTC AAA GAA ATG AAG AAA OTC AC? AS? SC? GTC CAT GAO AAA GOT STC AT CAA ATC AAA CCC ACC TTC ACC CAG TSG GAA OCT GTC AAC
1112
Lou 0is Glu Val Lys .is Ph. Vol Vol Glu Val Ar; Ala Tyr Pro Pro Pro Ar; Ile SOr Srp Lou Lys Asn Asn Luu Thr Luu I11 Glu CTS CAT GAA GTC ALA CAT 7T7 0T7 GTA GAG G0G COG 0CC SAC CCA CC? CCC AGO ATA ?CC 700 CTS AAA AAC AAT CTG ACT CC AT? GAA
Asn Lsu Thr Glu Ile Thr Thr Asp Val Glu Lys Il* G0n Glu II* Ar; Tyr Arq SOr Lys Lou Lys Lou II Arg Ala Lys 0lu Glu Asp AAS CTC ACT GAO ATC ACC ACT GA? GSG GAA AAG AT? CAG GAA ATA AGO TA? CGA AGC AAA TTA AAG CTG ATC COT OCT AAG GAA GAA GAC
SOr 0ly 0is Tyr Thr IIl Val Ala Gln Asa 0lu Asp Ala Val Lys SOr Tyr Thr Ph. 0lu AG? GGC CAT TAT ACT ATT CTA OC? CAA AS CAA GA? OCT G0G AAG AGC TAT ACT 77? CAA Asp GAC
Lou Lou Thr G0n Vol Pro SOr SOr Il. CG STA AC? CAA OTT CC? TCA 7CC AS?
Lou CTG
Luu Vol Asp Asp 0is Nis 0ly SOr Thr 0ly 0ly lin Thr Vol Ar;[gis Thr Ala 0lu 0ly Thr Pro Lou Pro Asp Ilb Glu ?rp Out ACA OCT GAA GGC ACC CCC CT? CCT GA? A7? GAG 0G0 ATG 77G GTC OAT GA? CAC CA? GCC ?CA AC? 0C0 CGA CAG ACG 070 A00GC
Ilfe-Cya1Lys
ATAITGCAAAA
F;-IAsn
Asp Ile Lys Lys Asa 0lu Thr Our Trp Thr IXl Lou Ala Ass Asn Val OSr Asa Il. Il. Thr 01u I1- 0is SOr OAT ASS AAG AAAITGJAAT AA? OAA ACT TCC T00 AC? A7? SSG 0CC AAC AAL OTC TCA AAC ATC ATC ACC GAG ASC CAC 7CC
Asp Arg SOr Thr Val CCA GAC AGO AG? ACC 0TG
Arg
Ar;|CysjLou J CTG
0lu Gly Ar; Vol Thr Ph- Ala Lys Vol 0lu 0lu Thr I1 Ala Val GAO CGC COT GTG AC? SSC 0CC AAA 0TG GAG GAG ACC ATC 0CC 0T7 CGA
Ala Lys Asn Lou Lou 0ly OC? AAG AA? CTC CT? GGA
Ala 0lu Asn Ar; Glu Lou Lys Leu Val Ala Pro Thr Lou Ar; SOr 0, Vo Ala Ala Ala Val Lou Vol Lou Luu Val Ile Vol OCT GCA GTC CTG GTG CG 7TG GTG AT? GTG OCT GAO AAC COA GAG CC AAG CG GTG OCT CCC ACC CTG COT CTC ACG ACT CAGLG
I1 Ile SOr Lou II1 Val Luu Vol Val X1l Trp Lys 01n Lys Pro Ar; Tyr 0lu II- Arg Trp Ar; Vol Ile 0lu S*r Xl. ATC ATC 7CA C?? ASS OTC CTG OTT CTC ASS TOG AAA CAG AAA CCG AGO TAT GAA AT? CCC TOG AGO GTC ATT GAA TCA ATC
0ly 0is 0lu
Tyr IIs Tyr GCA CAT CAA TAT AT? TAT
ELou Gl0
Vol ASp Pro Out G0n Lou Pro Tyr Asp Sur Ar; Trp Glu Ph. Pro Ar; Asp Gly 0TG CAC CCC ATG CAG CTS CC? TA? GAC TCA AGA TOG GAG TTT CCA AGA OAT CGA
SOr Gly Ala Ph. Gly Lys Vol Vol Glu Gly Thr Ala Tyr 77TG 0G0 TCT CGA GCO 7?? 0GG AAG G07 07T GAA CGA ACA 0CC TAT Lys Pro Thr Ala Ar; SOr S-r Glu Lys 01n Ala Luu Out Sr Lou CTA AAA CCC ACG GCC AOA 7CC ALT CLA AAA CLA GC? CTC ATG SCT As, Lou Lou Gly Ala Cys Thr Lys SOr 0ly Pro IIl Tyr I1l I1l AAC 77G CT? CGA 0CC TGC ACC AAG TCA GGC CCC AT? TAC ATC ASC
Gly Lou SOr Ar; SOr GCA TTA ACC CGG 7CC Glu Lou Lys Ile Out GAA CTG AAG ATA ATC
Lou Val LQ CA GTG CT?
SOr
Pro
Asp
ACC CCA Gly Ao s
OAT
G00 Lys Val Ala Vol CAA CCT OTC ATG AAA G07 GCA GTG Thr Nis Lou 0ly Pro 0is Lou AsL
G0n
Pro Vol Met
-;
COG OTC
LyS not
ATC I1l Val AAG
0GG CCA CAT 7TG AAC AT? CTA Thr Glu Tyr Cys Ph. Tyr Gly Asp Luu Val Asn Tyr Lou His Lys ACA GAO TAT TOC SSC TAT CCA LA? SSC OTC AAC TA? 7T7 CAT AAG ACT CAC CG
Asn Ar LAsp Sr Ph- Luu SOr Nis 0is Pro Glu Lys Pro Lys Lys Glu Lou Asp I1 Ph. Gly Lou Asn Pro Ala Asp 01. $sr Thr Arg LATA AOGJ AT AGC TTC CTG ACC CAC CAC CCA GAG AAG CCA AAG AAA GAG CTC GAT ATC 77? GA STG AAC CCT OCT OAT CAA ACC ACA COG .
SOr Tyr Val I11 Lou S*r Phu 0lu As Loua 0ly Asp Tyr Out Asp Out Lys G0n Ala Asp Thr Thr ACC TAT 0TT AT? TTA TC? 777 CAA LAC AAT GOT GAC TAC ATO GAC ATS AAG CAG OCT GAT ACT ACA Lys 0lu Vol SOr Lys Tyr SOr Asp II1 01n Arg SOr Lou Tyr Asp Arg Pro Ala SOr Tyr Lys Lys AAA GAG 07? SCT AAA TA? SCC GAC ATC CAG AOA TCA CTC TAT GA? COT CCA 0CC TCA TAT LAG AOG
01n Tyr Vol
Pro Out Luu 0lu Ar; CAC TAT GTC CCC ATS CA GAA AGO
Lys SOr Not Luu Asp SOr Glu Val AAA ?CT ATG TTA GAC TCA CAA CTC
FIG. 1. (Figure continues on the opposite page.)
320
350 1202 300 1202 752 410 1302 440 1472
470 1562
500 1052 030 1742 560 10832
1022 028 2012
050 2102 000
2192 710 2202 740 2372 778
2462
Proc. NatH. Acad. Sci. USA 86 (1989)
Biochemistry: Claesson-Welsh et al.
Lys Asn Lou Lou Sor Asp Asp Asn Sor Glu Gly rLsu Shr Lou Lou Asp Lou Lou Sor Ph. Thr Tyr Gln Val Ala Arg Gly MNt Glu Ph. AAA AAC CTC CTT TCA GAT SAT AAC TCA GAA GGC LCTT ACT TTA TSG GAT TTG STG AGC TTC ACC TAT CAA 0T? GCC CGA GGA ATG GAG TTT
2552
Lou Ala Sor Lys Asn Cys Val His Arg Asp Lou Ala Ala Arg Asn Val Lou Lou Ala Gln Gly Lys Il* Vol Lys I1l Cys Asp Ph. Gly STG GCT SCA AAA AAS TGT GTC CAC CGT GAT CTG GCT OCT CGC AAC OTT CTC CTG GCA CAA GOA AAA AT? GTG AAG ATC TOT GAC TTT GGC
2642
Lou Ala Arg Asp II1 MNt His Asp Str Asn S2r Val Str Lys Gly Str Shr Ph. Lou Pro Vol Lys Trp Mot Ala Pro Olu Sor I1- PhCTG GCC AGA GAC ATC ATG CAT GAT SCG AAC TAT GTSG CG AAA GGC AG? ACC VTT CTG CCC GTG AAG SGG ATG SC? CCT GAG AGC ATC TTT
2732
Asp Asn Lou Tyr Thr Thr Lou Sor Asp Val Srp S~r Tyr Gly II1 Lou Lou Srp Glu Ile Ph* Ser Lou Gly Gly Shr Pro Tyr Pro Gly GAC AAC CTC SAC ACC ACA CTG AGT GAT GTC SGG SCT TAT GGC ATT CSG CSC TGG GAG ASC STT SCC CTT GGS GGC ACC CCS SAC CCC CGC
898 2822
g00
030 868
Mo*t Met Val Asp S-r Thr Ph* Tyr Asn Lys Ile Lys S-r Gly Tyr Arg Moat Ala Lys Pro Asp Nlis Ala Thr S~r Glu Val Tyr Glu I1* ATG ASG GTG GAT TCT ACT TTC TAC AAT AAG ASC AAG, AGT GGG SAC CGG ATG GCC AAG CCT GAC CAC GCT ACC AGT GAA GTC TAC GAG ATC
921
i La. S-r Glu Ile Val Glu Asn Lou L-u Pro Gly Gln Tyr Noet Val Lys Cys Trp Asa S*r Glu Pro Glu Lys Arg Pro S-r Ph.ly ASG GTG AAA SGC TGG AAC AGT GAG CCG GAG AAG AGA CCC SCC T TT TAC CAC CTG AGT GAG ATT GSG GAG AAS CTG CTG CCS GGA CAA TAT
3002
Lys Lys S-r Tyr Glu Lys I1* His Lou Asp Ph- Lou Lys S-r Asp His Pro Ala Val Ala Arg Mo-t Arg Val Asp S*r Asp Asn Ala Tyr AAA AAG AGT TAT GAA AAA ATT CAC CTG GAC TTC CTG AAG AGT GAC CAT CCT GCT GTG GCA CGC ATG COT GTG GAC SCA GAC AAT GCA TAC
958
30922
I1e Gly Val Shr Tyr Lys Asn Glu Glu Asp Lys Lou Lys Asp Srp Glu Gly Gly Lou Asp Glu Gln Arg Lou S-r Ala Asp Sor Gly Tyr ATS GGT GTC ACC TAC AAA AAC GAG GAA GAC AAG CTG AAG GAC SGG GAG GGS GGT CTG GAT GAG CAG AGA CTG AGC OCT GAC AGT GGC TAC
10103182
I1e Ile Pro Lou Pro Asp Ile Asp Pro Val Pro Glu Glu Glu Asp Lou Gly Lys Arg Asn Arg *is S-r S-r Gln Thr S-r Glu Glu S*r ATC ATT CCT CTG CCT GAC ATT GAC CCT GTC CCT GAG GAG GAG GAC CTG GGC AAG, AGO AAC AGA CAC AGC SCG CAG ACC SCS GAA GAG AGT
1048 3272
Ala Ile Glu Thr Gly S*r S-r S-r Sor Thr Phe II1 Lys Arg Glu Asp Glu Thr Ile Glu Asp I1- Asp I-t It-t Asp Asp I1 Gly I1* OCC ATT GAG ACG GOT TCC AGC AGT SCC ACC TSC ASC AAG AGA GAG GAC GAG ACC ASS GAA GAC ASC GAC ASG ASG GAC GAC ATC GGC ATA
Asp S-r Ser Asp L-u Val Glu Asp Ser Ph- Lou GAC TCT TCA GAC CTG GTG GAA GAC AGC TTC CSG
107061009
CCACAGAAGGTGAACTTTCTGCTTCAAGGACASTGGTGAGAGTCCAA
3471 3591 3711
CAGACACAATTTATACTGCGACAGAACTTCAGCATTGTAATTATGTAKATAACTCTSAACCACGGCTGTGTTTAGATTGTATTAACTATCTTCTTTGGACTTCTGAAGAGACCACTCAAT C
3831
TAACSGGCGGASTCGAGGGGTSCCTTCCACTTCTGGGGCCACCTCTGGATCCCGTTCAGAAAACCACTTTATTGCA
ATGCGGAGGSSGAGAGGAGGACTTGGTTGATGTTTAJAAGAGAAGTT CCCAGCCALAGGGCCSCGGGGAG.CCSSTCTAAATATGAASGAATGGGATATSSTGAAATGAACSSSCTCAGTGTS GCCT CTSGCAATGC CTCAGTAGCATCTCAGTGGTGTGTGALAGTTSGGAGATAGATGCATAAGGGAATAASTAGG
CAT CCASGTACTT CCCTCTTGAAACCTGATGTCAGCTGCTGTTGAACTSSSSAAAGAAGTGCATGAAAAAC
CATTSSTGACCSTAAAAGCTACTGGTACTASAGCASSSCA
TT
TSAGTGTTAAAGAGATAAAGAATAATAATTAACCAAC CTSGTTTAASAGASTTGGGTCATTTAGAAGCCTGACALACTCATTTTCASASSGTAASTCTASGSTA~TAATACSACTACTGTTA T CAGTI, GCST-CaXTGTCJ.TAOAACATSA?-!T rrCTCCACACAJAAG _ACAA.TTT.AAAAACAASCCTTACTAAGTAGGTGATGAGSSTGACAGTSTTTGACATSSASASTAAATAA
CATGSSTCSCTATAAAGTASGGTAATAGCSSSAGTGAASTAAASSSAGTTGAGCASAGACALACAAAGSAAAAGSACTGTSGSCCAGGAAGT CAGAATSTTTAACSGTACTGAASAGGTTC
3951
4071
4191 4311
CC CAATC CATCGTATTAAAAAACAASTAACSG CCCTCSGAJAASAASGGGASSAGAAACAAACAAALACSCSSALAGSC CSAAAAGTSCSCAASGTAGAGGCASAAACCSGSGCSGAAA
CSSTCT CATGTASASSAC CCAASGGAAA.ASASAASGASTCAGCG CAAAGACTGGATTTGCACAAGSTTTTTTTTTTTSSCSSTCSSGC CSGASGAAAG CTTTGGCGACC CCAAAAGA STTTTGAAT CTATGAAC CTGAAALAGGGTCACAAAGGASGCCCACACASTCAGC CSTCCTTCTTTCACCCCTTACCC CAAAGAGAAAGAGTTTGAAACT CCAGAC CASAALAGASASTCSTTAG SGCAGCCTG GAAGSGCASTACCCTCASTCCT CAG TT CTCAAATGTG TGSGGCAGC CAGGTAGACTAGSAC CTGGG TTT CCATC CSTGACATT CTCAAGTATGAACSCTGACGGAAAC CAGA GT CTGSATTTSSTCSAAACSTC CCTGG CTCSTTCTCAT CGCCCCAGTTT CGGAAACACTGACTTAGGTTT CACGAAGTSGCCCASCGGAAACAAASALATSTCAACTTTGGAACAGCGSTTCTTAAG
STCGTGCGSC CTSTCGCASCATAAASSTACGJAACCCGAAGTCCAATCACSGTAAATTACGGTAGASCGASCGSSAACGCTGGAATTAJAASSGAAAGGTCAGAAT CGACTC CGACTCTTTCGA
STTTCAAAC CAAAACTGT CCAAAAGGSTTTCATTT CTACGASCAACCGTGACATACCCC CSCSAACTSGAAAGOGGGCAGAGGGCAGAAGAGCGGAGGGTGAGGTASGGGG CGGTT CCSSSC
CGTACASGTTTSTJAASACGSSJAAGTCACAAGGSSCAGAGACACASTGGTCGAGTCACAAAACCACCTTSSSSGSAAJAASTCAAAASGACTASTAALACTCCAATCTACCCTCCSACTTAAC
4671
4791 4911 5031 5151 5271
ATAGCCGTCTACTACGAAAC CTTCTACGTCTTCGTTASTATTTCASGAACSGATGGATGACCACASSAGAGTSACGTSTCGGGGTTGAAAGALATAGGTSGAAAAAGTATCATS CACGCTTC
5391 5511
SGACTCGGTCTAACCGGTSAATTSTTCTTSTGGACTGATCCAAGACATCSCGGSTAJATCSGAACSSTATGCAAACACAAAGATCSSAGTGSCGAGSSCGSALAGACAAASAGCGAGTGAGA
5631
GGG.CAACATGTCGGCAATAAAACALACCACGA'AACGTAAAACTATAACGACACT CGGAACGTACSGTAGSACSCCGGCCSACSSSGAAGAGSCAGGTCGTCAAAGGSCAGGATSGTTTACGAG
5751
GGTGGACTTAAACATASACTGACGSAAACACC CACACACACACAAAAGTCGTTTAAGGSCTAAACAAAGGAAAACCGGAGGACGTSSCAGAGGTCTTCTTTTAAACGGSSAGAAAGGATG
5871
AGTGSAGASAGGSGTGACAGTTSGTCCAACCACACCCAAGTALAC CGTAAGAALACGSTASGACGAASSAACGACSATGGTASACTTACTTTGTACCCGACACTAASGACGSSAGTGACACG
AA.AGATAAAAASACTACTGTTAGSTTCGGCCGGACTCTTTGTGATAAACACTGAAAAASSSGCSAASCACSACAGGAASSSSACACCAGACOGTTACACATCTSSSACCACCATAAAAAC
5991
ACSSTCSCC CTGTATT CTATTTTACTACAASASGTAGTTATACATASASACATAAAGASASAT CSGAAC CT CTTASGACGGSSSTGTAJAASACTGTT CGACASAGSGACGGAAG CAAATAS
6111
AAAAAAASTGACACTATTAGGGGTGSCCGTGTAATTGACAACGTCAAAACTTACAGGTSSSAAATASAAAATCTTSASTASSSSSCSTTCSASGAATGTACAAGGGSTSTGTTACCACAC
6231
CACTSACACACSCTTTTTGATTGAACTASCC CAGASGGSTASGSTSTACASAASGCTSACGGGGOACAAGTACAAAAACAAAATTTTGCACASSTACSTCTAGAAATATAAAGTTATSSAC
6351
TATASASTAAASTSCCTTAAG
6375
tide mapping indicated that the two PDGF receptors are structurally related (17), it was conceivable that the 6.5kilobase (kb) transcript encoded the PDGF A-type receptor. A partial cDNA (phPDGFRA1) was isolated when the B-type receptor tyrosine kinase fragment was used as a probe to screen 1 x 106 clones of an unamplified AgtlO U-343 MGa 31L cDNA library under low-stringency hybridization conditions. The most 5' fragment of phPDGFRA1 was used to screen under high-stringency conditions 1 x 106 clones of an AG 1518 human foreskin fibroblast cDNA library. A 6.4-kbp clone (phPDGFRA15) was isolated and shown to cover and extend the first cDNA clone. The organization and sequence of phPDGFRA15 are shown in Fig. 1. Nucleotide Sequence and Deduced Amino Acid Sequence. The size of the insert in phPDGFRA15 is 6375 bp (Fig. 1). There is a 3267-bp open reading frame, which is flanked by 128-bp 5' and 2980-bp 3' untranslated sequences. The 3' untranslated region contains several ATTTA sequences; this motif has been implicated in posttranscriptional regulation of, for example, the nuclear protooncogene c-fos (28) and the granulocyte/macrophage colony-stimulating factor (29). A poly(A) addition signal is located close to the 3' end of phPDGFRA15, but the poly(A) tail is missing. The nucleotides surrounding the proposed initiating codon are largely in agreement with the Kozak rules for initiation (30). The characteristics of the amino acid sequence immediately following this methionine are those of a cleavable signal sequence. The mature amino terminus of the deduced polypeptide can tentatively be assigned to Gln-24, which
Mr x
10-3
4919
FIG. 1. (A) PDGF A-type reIsolated cDNA ceptor clones. clones are shown against a schematic outline of the predicted receptor mRNA with translated (an) and untranslated (-) regions indicated. The cDNA fragment used for insertion into an vector to expression (B) pSV7dl5.1+5 is shown.yield Nucleotide sequence and deduced amino acid sequence of the human PDGF A-type receptor cDNA. The complete sequence of phPDGFRA15 is shown, with nucleotides and deduced amino acids numbered on the right. Amino acids are numbered from the initiating methionine. Arrowhead indicates putative start of mature protein. Potential sites of N-linked glycosylation are overlined and cysteine residues are boxed in the extracellular part of the mature protein. The transmembrane region is boxed and the borders of the two tyrosine kinase segments are indicated by brackets. The amino acids thought to be involved in nucleotide binding (a Gly-Xaa-GlyXaa-Xaa-Gly sequence and as well as Tyr-849 [hoLys-627) automologous to the major phosphorylation site in pp60VSrC (27)] are underlined. In the 3' untranslated region, the AT'TA motifs and the poly(A) addition signal are underlined.
a b c d e
f g h
200-
^4 __
170
--140 120
92
69
pSV7dl5.1+5 contr. COS COS
U-343MGa31L
FIG. 2. Immunoprecipitation from metabolically labeled COS cells transfected with pSV7dl5.1+5 (lanes a-c), control COS cells (lanes d-), and U-343 MGa 31L human glioma cells (lanes g-i). Antisera used were PDGF B-type receptor-specific antiserum PDGFR-3 (lanes a, d, and g), PDGF A- and B-type receptor-crossreactive antiserum PDGFR1 (lanes b, e, and h), nonimmune serum (lanes c, f, and i). Molecular weights of the precursor (140,000) and mature (170,000) forms of the PDGF A-type receptor and a Mr 120,000 species reactive with the PDGFR-1 antiserum in transfected COS cells are indicated, as well as the relative migration positions of molecular weight standards run in parallel (myosine, 200,000; phosphorylase b, 92,500; BSA, 69,000).
4920
Biochemistry: Claesson-Welsh
Proc. Natl. Acad. Sci. USA 86 (1989)
et al.
0.1-
A
NGTSHPAFLVLGCLLTGLSLLCQLSLPSIL-PN3N3KVVQL3rsLv'G3V
1'
::.:
-
..:.:....:.:: 1:1:* :.:.:***:
1* NRLPGARPALALKGKLLLLSLLLLLFPQI8QG1VTPPGP3LVL!ISiTFVLT&G3APV
K
56'
Kd
O
0.05
0.2 nM
=
TYNSQT3N3LZGR
SWQYPNSEtLSSDV3IRN33NNSGLFVVL3V88SAAHTGLY1
61 " Vg-RMSQZPPQKR---- AAQDGTF8SVLTLTSLGLDTGtY RGLYTD-URK ::.::::::: -: ::-- --: 116'61. H!YIYVPDPDVAFVPLGMTDYLVIVEDDDSAI I flTTDPETPVTLHNs3G-VVASYD
115'" RLYI FVPDPTVGFLPNDAKELFI FLTSITtITI PIC VTDPQLVVTLHKKKGDVALPVPYD
0~~~~~~
0-
174' SRQGFNGTFTVGPY
\
0.3-
B
175'
GTVKGFQTIPFNVYALKAT8ELDLIM3ALKTVYK8GZTIVV
*HQRGFSGI'FtDRSY ITTIGDR3VDSDAYYVYRLQVSS-!UIVNAVQTVV3QGKPL
234 ' T =VFNN3VVDLQWTYPGKKGKGITNL3ZIKVP-SIKLVYTLTVPKATVKDGY : :. ::
.... . .. ...
234*
0.2X
..
.:
.:
..
T.DS.T.
293' RQATR3VKENKKVTISVBKlGFI*IKPTFSQLEAVNLHEVKHFWVVIVAYPPPRTISWLKN
:.: .:.......::::ss:... .'.........:
\°
n
.
I VIGNDVVNrWTYPR3KSGRLV3PVTDFLLDNPYHIR8ILHIP8A3LED8GTY1l!
K,=
0.1 nM
294'
TiSVND.QD£KAIM! SSGYVRLLGZVGTLQFAKLHRSRTLQVVF3AYPPPTVLVFKD
C
353' NLTLIE-NLTSITT'DVKKIQLIRYRSKLKLIRAREZDSGHYTIVAQNKDAVKSYTFKLLT
0
m
0.1
X
354* PZLGDS8AGEIALSTRMJETRYVSELTLVRVKVAEAGHYTNEAFr3DAZVQLsFQLQI AXGTPLPDI3O IDI
412' QVP8SILDLVDDHUGSTGGQTV 414" NVPVRVL3L8E8HPD8-G
Sea. 0.1
C
470' -INI ITZI---HSRDRSTVE GRVTFAV3TIAVII3 CA 473"
ESQLETNY=YNKQKFKVVSTLRLRNVDRPLSVU
526 ' TVAAVLVLLVIVIISLIVL
533" Kd
0.05
=
0.5 nM
G3GMPQPNZ18 IDLK
QTV
8tILANIL-RELPTLLGNSSEZ
NLLGAENLKLLVAPTLR
F
rLRNAVGQDTQZVIVVPHSLpFrV
IQPRYLIRWRVIFSISPWIISYI YVDPQLPYDSR
LALWLVTIISLIILIKMPE
PRYIRWKVIFSVSSDG8BYIYVDPNQLPYDST
5866' WFPRDGLVLGRLG8SGAFGKVVGTAYGLSRSQPVNKVAVKNLKPTARSSZKQALR8EL
593' W3LPRDQLVLGR G8GAFGQVVEATAlGLSSQATRKVAVKNLKS8TASS3KOALRSUL 646 '
0
5
10
15
Bound, fmol per well
1ITHLGPELNIVNLLGACTKSGPIYI ITSYCFYGDLVNYLHKt43srLSBBPRPVK-KK
653'" KIRSLGPHLNVVNLLGACTKGGPIYI ITEYCRYGDLVDYLHRNYITFLQHBSDKRRPPS
705' LDIFGLN-PADESTRSYVILSFKNNGDYXDRKQADTTQYVPlMLEREVSItKYSDIQRSLYD
713 * AZLYSNALPVGLPLPSHVSLTGKSDGGYKDNSKDESVDYVPNLDRKGDVKYADIESSNYl
FIG. 3. Scatchard analyses of 125I-labeled PDGF-AA (A), -AB (B), and -BB (C) binding to COS cells transfected with the type-A receptor construct (pSV7dl5.1+5).
764' RPASYKKKSRLDSKVKNLLSDDN8KfTLLDLLSFTYQVARGMEFLASKNCVHRDLAARN
based on statistical analyses is a potential cleavage site for the
832' VLICEGRLVKICDFGLARDIRRDSNYISKGSTFLPLKWMPZSIFNSLYSTLSDVWSFGI
signal peptidase (31). The calculated molecular weight of the polypeptide after cleavage of the signal sequence is 120,215, comprising 1066 amino acids. The deduced polypeptide is divided into two parts by a stretch of 25 hydrophobic amino acids, most probably constituting the transmembrane segment. The transmembrane segment is flanked by positively charged residues (-Lys-Gln-Lys-Pro-Arg-) on the carboxylterminal side. The NH2-terminal extracellular domain contains 10 characteristically distributed cysteine residues and 8 signals for N-linked glycosylation (Fig. 1B). The cytoplasmic domain contains a classical tyrosine kinase sequence including a potential ATP binding site. As for other members of the PDGF receptor family, the tyrosine kinase domain in the deduced phPDGFRA15 sequence is split into two segments by insertion of a sequence of =100 amino acid residues. Transient Expression in COS Cells. The part of phPDGFRA15 covering the translated area (bp 1-3442) (see Fig. 1) was cloned into a simian virus 40-based expression vector and
884' LLEI FSLGGTPYPGEMVDSTFYNKIKSGYRRAKPDHATSEVYEIRVKCWNSKPEKRPSF
transfected into COS cells. The transfected cells were labeled with [35S]methionine and [35S]cysteine and examined for expression of the PDGF A-type receptor by immunoprecipitation (Fig. 2). No reactivity was seen with the B-type specific antiserum PDGFR-3 (lane a), whereas PDGFR-1, which recognizes both A- and B-type receptors, precipitated components of Mr 140,000 and 170,000 (lane b). Metabolically labeled untransfected COS cells were found to be negative (lanes d-f). The data are consistent with the conclusion that the transfected COS cells express the A-type PDGF receptor. When metabolically labeled U-343 MGa 31L glioma cells were analyzed, for comparison, PDGFR-3 was again negative (lane g). PDGFR-1 recognized the PDGF A-type receptor precursor (Mr, 140,000) and mature (Mr, 170,000) forms (lane h). PDGFR-1 also precipitated a major component of Mr 120,000 from the transfected COS cells (Fig. 2, lane b), which was absent both in the U-343 MGa glioma cell-derived samples and in the control COS cells. The relation between
773" APYDNYVPSAPERTCRATLINZSP- SYRtDLVGFSYQVANGMEFLASKNCVHRDLAARN 824' VLLAQGKIVKICDFGLARDINHDSNYVSKGSTFLPVKMRAPESI FDNLYTTLSDVWSYGI
892" LLWEIFTLGGTPYPKLPMINQFYNAIKRGYRRAQPANASDKIYKINQKCVIKKFEIRPPF 944' YHLSEIVENLLPGQYKKSYEKIBLDFLKSDHPAVARRRVDSDNAYIGVTYKNKKDKLKDW
952" SQLVLLLZRLLGUGYKKKYQQVDzzrLRSDHPAILRSQ-ARLPGFHGL--RSPLDTSSVL 1004' ZGGLDEQRLSADSGYIIPLPDIDP-VPZEKDLGKRNRRSSQTS3KSAIETGS8SSTFIKR
.~.. s:::::.: :....... :::::..:
1009' YTAV--QPNZGDNDYS IPLPDPKPKVADBGPLGSP8 LA88SLNKVNTSSTX8CD8PLKP 1063' KDZTIDIDDMDDIGIDSSDLVKDSFL 1067' QDEPEPKPQLELQVZPEPZLEQLPDSGCPAPRAPAKDSFL
FIG. 4. Comparison of the amino acid sequences (single-letter code) of the human A-type (') and B-type (") PDGF receptors. Identical amino acids are indicated by two dots, and conserved exchanges are shown by one dot. The comparison includes the signal sequences and extends through the molecules. Arrowheads indicate starts of mature proteins. Potential sites ofN-linked glycosylation are indicated (-). The cysteine residues are boxed in the extracellular parts ofthe mature proteins. The transmembrane segments are boxed and the borders of the two tyrosine kinase segments are indicated.
this component and the PDGF A-type receptor remains to be determined. Scatchard Analysis of Ligand Binding. The transfected COS cells were tested for ligand binding by using 125I-labeled PDGF isoforms. PDGF-AA, -AB, and -BB all bound with high affinities to cells transfected with the expression vector containing phPDGFRA15 but not to control COS cells. Scatchard analysis revealed that PDGF-AB and PDGF-AA bound to the receptor with the highest affinities (Kd = 0.1 and 0.2 nM, respectively) (Fig. 3 A and B), whereas PDGF-BB bound with slightly lower affinity (Kd = 0.5 nM) (Fig. 3C).
DISCUSSION The primary structure of the PDGF A-type receptor demonstrates that it belongs to a subfamily among the protein tyrosine kinases. Other members of this family include the
Biochemistry: Claesson-Welsh et al. PDGF B-type receptor (11-13), the receptor for colonystimulating factor 1 (32), and the c-kit product (33), the latter being a receptor without an as yet identified ligand. Each of these receptors has a characteristic spacing of the cysteine residues in the extracellular domain and an inserted sequence in the tyrosine kinase domain, splitting it into two parts. The two PDGF receptors are more related to each other than to the other two members of the subfamily; the primary structures are compared in Fig. 4. The overall similarity between the two PDGF receptor molecules is 44%. In the extracellular parts, 30%o of the amino acids are identical. PDGF-BB binds to both receptors with similar affinities (16). The primary structures of the two receptors give no clue to where the ligand binding site is located; there are no contiguous conserved stretches between the two molecules longer than five amino acid residues. In the intracellular part, the tyrosine kinase domains are highly conserved (87% and 74% similarities between the first and second segments of the tyrosine kinase domains, respectively). It is noteworthy that the segments between the transmembrane and the first tyrosine kinase domains are as much as 83% identical. Alterations in the corresponding region in the insulin receptor have been shown to decrease the biological activity, possibly due to interference with phosphorylation of substrates (34). The inserted sequence in the tyrosine kinase domain in the PDGF receptor family has also been implicated in interactions between substrate and receptor; removal of the insert sequence from the PDGF B-type receptor alters the pattern of phosphorylated substrates (ref. 15; L. Severinsson, B. Ek, L.C.-W., and C.H.H., unpublished data). The similarity between the insert sequences in the A- and B-type PDGF receptors is 35%. The carboxyl-terminal tails of the PDGF A- and B-type receptors contain a number of tyrosine residues, four in the B-type and six in the A-type receptor, of which three are located at identical positions. It is possible that some of these residues serve as targets for autophosphorylation of the receptors. The A-type receptor has a shorter carboxyl-terminal tail than the B-type receptor, but the last five amino acid residues in each receptor are identical. During the preparation of this manuscript data on the primary structure of a cDNA molecule encoding the PDGF A-type receptor (denoted type a PDGF receptor), isolated by the use of a colony-stimulating factor 1 receptor-derived tyrosine kinase probe, was reported (35). The presented deduced amino acid sequence is identical to the one obtained
by us.
It is conceivable that the functional effects of PDGF in a
given situation are dependent on the particular combination of PDGF isoform and receptor type. Our present knowledge concerning the unique functions transmitted by each receptor is incomplete. Interpretations have thus far been complicated by the differences in numbers of each receptor expressed on the same cells and the fact that comparisons have been made between different cell types expressing only one of the two receptors. The availability of complete cDNAs for both the A- and B-type receptors makes it possible to introduce these individually or in combination into the same cellular background to delineate the unique signal pathway connected with each receptor.
Expert technical assistance was provided by Anita Moren and Charlotte Sandstrom. Linda Baltell assisted in the preparation of this manuscript. We thank Monica Nister for the U-343 MGa 31L cells and for valuable contributions during the initiation of this work. 1. Heldin, C.-H., Wasteson, A. & Westermark, B. (1985) Mol. Cell. Endocrinol. 39, 169-187. 2. Ross, R., Raines, E. W. & Bowen-Pope, D. F. (1986) Cell 46, 155-169.
Proc. Natl. Acad. Sci. USA 86 (1989)
4921
3. Johnsson, A., Heldin, C.-H., Wasteson, A., Westermark, B., Deuel, T. F., Huang, J. S., Seeburg, D. H., Gray, E., Ullrich, A., Scrace, G., Stroobant, P. & Waterfield, M. D. (1984) EMBO J. 3, 921-928. 4. Josephs, S. F., Guo, C., Ratner, L. & Wong-Staal, F. (1984) Science 223, 487-490. 5. Betsholtz, C., Johnsson, A., Heldin, C.-H., Westermark, B., Lind, P., Urdea, M. S., Eddy, R., Shows, T. B., Philpott, K., Mellor, A. L., Knott, T. J. & Scott, J. (1986) Nature (London) 320, 695699. 6. Hammacher, A., Heilman, U., Johnsson, A., Ostman, A., Gunnarsson, K., Westermark, B., Wasteson, A. & Heldin, C.-H. (1988) J. Biol. Chem. 263, 16493-16498. 7. Heldin, C.-H., Johnsson, A., Wennergren, S., Wernstedt, C., Betsholtz, C. & Westermark, B. (1986) Nature (London) 319, 511-514. 8. Stroobant, P. & Waterfield, M. D. (1984) EMBO J. 3, 2963-2967. 9. Heldin, C.-H., Bickstr6m, G., Ostman, A., Hammacher, A., Rdnnstrand, L., Rubin, K., Nistdr, M. & Westermark, B. (1988) EMBO J. 7, 1387-1394. 10. Hart, C. E., Forstrom, J. W., Kelly, J. D., Seifert, R. A., Smith, R. A., Ross, R., Murray, M. & Bowen-Pope, D. F. (1988) Science 240, 1529-1531. 11. Yarden, Y., Escobedo, J. A., Kuang, W.-J., Yang-Feng, T. L., Daniel, T. O., Tremble, P. M., Chen, E. Y., Ando, M. E., Harkins, R. N., Francke, U., Friend, V. A., Ullrich, A. & Williams, L. T. (1986) Nature (London) 323, 226-232. 12. Gronvald, R. G. K., Grant, F. J., Haldeman, B. A., Hart, C. E., O'Hara, P. J., Hagen, F. S., Ross, R., Bowen-Pope, D. F. & Murray, M. (1988) Proc. Natl. Acad. Sci. USA 85, 3435-3439. 13. Claesson-Welsh, L., Eriksson, A., Mordn, A., Severinsson, L., Ek, B., Ostman, A., Betsholtz, C. & Heldin, C.-H. (1988) Mol. Cell. Biol. 8, 3476-3486. 14. Ek, B. & Heldin, C.-H. (1984) J. Biol. Chem. 259, 11145-11152. 15. Escobedo, J. A. & Williams, L. T. (1988) Nature (London) 335, 85-87. 16. Ostman, A., Backstr6m, G., Fong, N., Betsholtz, C., Wernstedt, C., Hellman, U., Westermark, B., Valenzuela, P. & Heldin, C.-H. (1989) Growth Factors, in press. 17. Claesson-Welsh, L., Hammacher, A., Westermark, B., Heldin, C.-H. & Nistdr, M. (1989) J. Biol. Chem. 264, 1742-1747. 18. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J. & Rutter, W. J. (1979) Biochemistry 18, 5294-5299. 19. Nistdr, M., Wedell, B., Betsholtz, C., Bywater, M., Pettersson, M., Westermark, B. & Mark, J. (1987) Cancer Res. 47, 4953-4969. 20. Huynh, T. V., Young, R. A. & Davis, R. W. (1986) in DNA Cloning, ed. Glover, D. M. (IRL, Oxford), Vol. 1, pp. 49-78. 21. Denhardt, D. T. (1966) Biochem. Biophys. Res. Commun. 23, 641-646. 22. Truett, M. A., Blacher, R., Burke, R. L., Caput, D., Chu, C., Dina, D., Hartog, K., Kuo, C. H., Masiarz, F. R., Merryweather, J. P., Najarian, R., Pachl, C., Potter, S. J., Puma, J., Quiroga, M., Rall, L. B., Randolph, A., Urdea, M. S., Valenzuela, P., Dahl, H. H., Favalaro, J., Hansen, J., Nordfang, 0. & Ezban, M. (1985) DNA 4, 333-349. 23. Wigler, M., Sweet, R., Sim, G. K., Wold, B., Pellicier, A., Lacy, E., Maniatis, T., Silverstein, S. & Axel, R. (1979) Cell 16, 777-785. 24. Ronnstrand, L., Beckmann, M. P., Faulders, B., Ostman, A., Ek, B. & Heldin, C.-H. (1987) J. Biol. Chem. 262, 2929-2932. 25. Heldin, C.-H., Johnsson, A., Ek, B., Wennergren, S., Ronnstrand, L., Hammacher, A., Faulders, B., Wasteson, A.& Westermark, B. (1987) Methods Enzymol. 147, 3-17. 26. Bolton, A. E. & Hunter, W. M. (1973) Biochem. J. 133, 529-539. 27. Smart, J. E., Opperman, H., Czernilofsky, A. P., Purchio, A. F., Erikson, R. L. & Bishop, J. M. (1981) Proc. Natl. Acad. Sci. USA 78, 6013-6017. 28. Meijlink, P., Curran, T., Miller, A. D. & Verma, I. M. (1985) Proc. Nat!. Acad. Sci. USA 82, 4987-4991. 29. Shaw, G. & Kamen, R. (1986) Cell 46, 659-667. 30. Kozak, M. (1986) Cell 44, 283-292. 31. von Heijne, G. (1986) Nucleic Acids Res. 14, 4683-4690. 32. Coussens, L., Van Beveren, D., Smith, D., Chen, E., Mitchell, R. L., Isacke, C. M., Verma, I. M. & Ullrich, A. (1986) Nature (London) 320, 277-280. 33. Yarden, Y., Kuang, W.-J., Yang-Feng, T., Coussens, L., Munemitsu, S., Dull, T. J., Chen, E., Schlessinger, J., Francke, U. & Ullrich, A. (1987) EMBO J. 6, 3341-3351. 34. White, M. F., Livingstone, J. N., Backer, J. M., Lauris, V., Dull, T. J., Ullrich, A. & Kahn, C. R. (1988) Cell 54, 641-649. 35. Matsui, T., Heidaran, M., Miki, T., Popescu, N., La Rochelle, W., Kraus, M., Pierce, J. & Aaronson, S. (1989) Science 243, 800-804.