Structural and functional aspects of the receptors for platelet-derived growth factor

Progress in Growth Farm Research, Vol. I, pp. 253-266. Printed in Great Britain. All rights reserved.

1989

095>2235/89 $0.00 + 50 T 1990 Pergamon Press plc

STRUCTURAL AND FUNCTIONAL ASPECTS OF THE RECEPTORS FOR PLATELETDERIVED GROWTH FACTOR Bengt Westermark*, Lena Claesson-Welsh? and Carl-Henrik Heldint * Department of Pathology, University Hospital S-751 85 Uppsala, Sweden i Ludwig Institute for Cancer Research, Box 595, Biomedical Center S-751 23 Uppsala, Sweden

Platelet-derivedgrowth factor (PDGF) is a 30 kDa dimer of disulfide-bonded A and B chains. Three isoforms of PDGFhave been isolated (PDGF-AA, PDGF-AB and PDGFBB). These bind with different affinities and spect@cities to two structurally related cell surface receptors, viz. the u-receptor and the preceptor. The receptors are transmembrane proteins with an intracellular, ligand-stimulatable protein tyrosine kinase domain. Activation of the receptors is intimately associated with receptor dimerization, and available data suggest that PDGF is a divalent ligand such that one molecule of PDGFbinds anddimerizes two receptor molecules. Stimulation of PDGFreceptors leads to a cascade of cellular events, which have been shown to require an intact receptor tyrosine kinase activity. However, ligand-induced internalization and degradation of the preceptor occur essentially independent of the receptor kinase activity. Receptor activation leads to the phosphorylation on tyrosine residues of three enzymes, probably by direct phosphorylation: phospholipase C-y, phosphatidylinositol3’ kinase and Raf-I. In certain cells, PDGF preceptor expression is inducible such that cells in normal tissue in vivo do not express receptors; only in inflammatory lesions or when cells are explantedin vitro. are receptors being expressed. Transformation by the v-sis oncogene is mediated by an autocrine PDGF-like growth factor. Although both the u- and preceptors are structurally related to the v-fms and v-kit oncogenes, it is not known tf the PDGF receptors have a transforming potential. In conclusion. the finding of three isoforms of PDGF that interact with two structurally related receptors implies a jinely tuned regulatory network, the role of which in cell growth and transformation remains to be clarified.

INTRODUCTION Platelet-derived growth factor (PDGF) was discovered as a growth promoting constituent of platelet alpha-granules. Subsequent studies have shown that PDGF is synthesized and released by a number of different normal and transformed cell types (see Refs l--4 for reviews) and it is generally assumed that PDGF has a pivotal role in the regulation of normal cell proliferation. It has been suggested that PDGF is also a mediator of pathological cell growth, e.g. in tumor development and in the generation 253

254

B. Westermark et ai.

of atherosclerosis and other nonneoplastic proliferative disorders (see Ref. 4 for a review). Initial structural analysis of PDGF isolated from human platelets showed that the factor is a 30 kDa dimeric protein consisting of disulfide-bonded polypeptide chains (denoted A and B), of similar sizes [5]. The A and B chains are encoded by separate genes and are about 60% similar in amino acid sequence [6]. Purification and microsequencing of various isolates of PDGF have shown that all three isoforms of PDGF exist: the heterodimer (PDGF-AB) has been identified in human platelet PDGF [7, 81 and in the conditioned medium of human glioma cells [9], PDGF-AA has been found in the conditioned media of various human tumor cell lines [9-l I], and PDGFBB is a constituent of human [7,8] and porcine [ 121platelet PDGF. Moreover, the v-si.s oncogene of simian sarcoma virus (SSV) encodes a transforming protein that is structurally similar to PDFG-BB [ 13, 151. Receptors for PDGF were initially discovered on mesenchyme- and gha-derived cells in culture [ 161 and found to possess a ligand-stimulatable protein tyrosine kinase activity [ 17, 181. Molecular cloning of mouse [19] and human [20, 211 PDGF receptor cDNA revealed a structural similarity to other protein tyrosine kinases. the most closely related ones being the c-fms and c-kit proto-oncogene products [ 191. The recent availability of all three isoforms of PDGF has led to the identification of two different, but related types of PDGF receptors, which differ in ligand specificity. The PDGF cxreceptor binds all three isoforms of PDGF with high affinities whereas the Preceptor binds PDGF-BB with high affinity, PDGF-AB with lower affinity, and does not bind PDGF-AA with any appreciable affinity [22, 231. The preceptor is identical to the receptor species that was initially discovered and structurally and functionally defined as the “PDGF receptor”. cDNA for the a-receptor has recently been cloned [24. 251. STRUCTURAL

ASPECTS

OF PDGF RECEPTORS

The nucleotide sequences of human CZ-[24, 251 and p [ 19-211 PDGF receptor cDNAs predict polypeptides of 1066 and 1074 amino acid residues, respectively, not including the signal peptides. The two molecules are structurally related with an overall Human SS

% IDENTITY

PDGF

receptors

ED1

ED2

ED3

30

ED4

ED5

TMJM

4383

TKI

Kl

8:

35

74

27

FIGURE 1. Domain structure of the PDGF receptors. Tbe extracellular portion of each receptor is mnde up by five immunoglobulin-like domains. Each receptor has a single transmem brane segment. The cytoplasmic parts contain tbe protein tyrosioe kinase domains which are split by iaaert domaim that bave ao sequeoce similarity to kinase domains. Figures represent sequence simiJarities between tbe two receptor molecules. Ss, signal sequence; ED1 to ED5, extracellular domainq TM, transmembrane segment JM, juxtamembrane segment; TKI, TK2, tyrosine kinase domains; KI, kinase insert, CT, carboxy-termiad tail. Open circles represent conserved cysteine residues. Vertical bars represent putative sites for N-linked glycosylation.

PDGF Receptors

255

amino acid sequence similarity of 44%. A schematic representation of the receptors is provided in Fig. 1. Each molecule is divided into an extracellular and an intracellular portion by a single putative membrane spanning stretch of about 25 hydrophobic amino acid residues. The extracellular portion of each molecule contains 10 similarly spaced cysteine residues, the positions of which are compatible with the presence of five immunoglobulin-like domains. The extracellular portions of the 01-and Preceptors contain 8 and 11 putative sites for N-linked glycosylation, respectively. Only a couple of these appear to be conserved with regard to their positions in the two receptor molecules. The cytoplasmic portion of each receptor contains the protein tyrosine kinase domain which in both cases is split by an inserted sequence of some 100 amino acid residues, without any homology to protein kinases. The kinase domains start 49 amino acids from the plasma membrane. It is notable that the juxtamembrane portions are 83% similar in amino acid sequence, whereas the homology of the kinase inserts is only 35%. The C-terminal tails of the receptors are also more divergent in sequence (27% identity). The PDGF a- and Breceptors are structurally related to the protein products of the c-fnu and c-kit proto-oncogenes with regard to overall amino acid sequence, spacing of the 10 cysteines in the extracellular domain, and presence of an insert in the tyrosine kinase domain (cf. Fig. 2). c-fms encodes the receptor for the macrophage growth factor CSF- 1 [26] and has been transduced as the V-&Y oncogene in the McDonough strain of feline sarcoma virus. v-kit is the oncogene of HZCfeline sarcoma virus [27]. The function of the c-kit product is unknown; its structure strongly implies that it too is a receptor for a growth factor [28,29], the identity of which remains to be established. BIOSYNTHESIS,

PROCESSING, INTERNALIZATION OF PDGF RECEPTORS

AND DEGRADATION

The PDGF a- and Preceptors are glycoproteins that are synthesized as precursors carrying immature N-linked carbohydrate complexes. Metabolic labeling and immunoprecipitation of human fibroblasts [30-321 have demonstrated that the receptors are synthesized as precursors of M, 140,000 (a-receptor) and M, 160,000 (p receptor). These are processed to the M, 170,000 (m-receptor) and M, 180,000 (/% receptor) mature forms that are present at the cell surface. The turnover of the cell surface receptors is fairly rapid; it is markedly increased by the addition of saturating concentrations of ligand, which leads to an almost complete internalization and down regulation of receptors [33]. Studies at the ultrastructural level have shown that receptor-bound PDGF is collected in coated pits, is rapidly internalized, accumulates in receptosome-like structures and is then translocated to the lysosomal compartment [34], where it is degraded [33]. The efficiency of ligand-induced down-regulation, in conjunction with the finding that reappearance of cell surface receptors following down-regulation requires de nova protein synthesis [33], indicate that the receptor is not recycled after internalization. The mechanism of ligand-induced PDGF receptor internalization is poorly understood. We have recently addressed this question by transfecting PDGF p receptors in porcine endothelial cells that lack endogenous PDGF receptor expression (A. Sorkin et al., in preparation). Two constructs were used, one with the full length human Preceptor cDNA and one in which the lysine residue in the assumed

FIGURE 2. Comparison of the amino acid sequences of the extracelhdar and transmembrane (A) and intracellular(B) parts of the human PDGF c+ and preceptor, tbe human CSF-1 receptor 1721 and the human c-kitproduct [Zs]. ‘llte comparison includes the signal sequences and extends to the C-terminal tails of the respective proteins. Identical residues are boxed. The protein tyrosine kinase domains are indicated by brackets. Conserved cysteine residues are marked with asterisks.

Fig. 2 continued.

258

B. Westermark

et al.

ATP-binding site was mutated to alanine (K634A mutant). We found that both wild type and mutant receptor underwent ligand-induced internalization, degradation and down-regulation at 37°C. However, these processes were somewhat slower in the cells expressing the K634A mutant receptor. Escobedo et al. [35] reported that inactivation of the protein tyrosine kinase activity of the preceptor, yields a receptor that is internalized, down-regulated and degraded essentially similarly to the wild type receptor, when expressed in CHO cells. Similar studies on the EGF receptor have shown that inactivation of the protein tyrosine kinase activity leads to loss of internalization [36] or rapid recycling after internalization [37]. LIGAND-INDUCED

RECEPTOR ACTIVATION: DIMERIZATION

ROLE OF RECEPTOR

As the ligand-binding extracellular domains of the PDGF receptors are linked to the intracellular catalytic domain by a single stretch of amino acids, it is not easy to conceive how the signal is transmitted across the plasma membrane. Schlessinger and coworkers have in a series of reports suggested that, in the case of the EGF receptor, ligand binding stabilizes the receptor in a dimeric or oligomeric configuration, allowing a direct interaction of the intracellular portions (reviewed in Ref. 38). Analogously, PDGF-BB has been shown to induce dimerization of purified porcine PDGF Preceptor in a dose-dependent fashion [39]. Dimerization was found to be closely associated with activation of the receptor tyrosine protein kinase. Additional evidence for ligand-induced dimerization of PDGF receptors has been published [40,41]. Since PDGF is a dimer and potentially a bivalent ligand, it is possible that each molecule of PDGF binds to two receptor molecules. In this respect it is interesting that dimerization of purified porcine PDGF preceptor decreases at higher concentrations of PDGF-BB [39]; at very high ligand concentrations, ligands bind receptors in a monovalent fashion, making dimerization, and hence activation of the kinase impossible. If one considers the binding pattern of the two homodimeric isoforms of PDGF, to the two PDGF receptor types, it is reasonable to believe that PDGF-AA dimerizes two a-receptors only (PDGFR-u), PDGF-AB can induce dimerization of either two CTreceptors or one OL-and one @receptor (PDGFR-acr, -@I) whereas PDGF-BB, owing to its promiscuous character, can dimerize receptors in all three configurations (PDGFR-aa; -c$, -/$I) (Fig. 3). As briefly outlined below, we have obtained circumstantial evidence in favor of this model. To explore the possibility that PDGF receptor dimerization occurs in the intact cell, and is intimately associated with the generation of the biological response, we have utilized the finding that PDGF-AB and PDGF-BB, but not PDGF-AA, induce actin reorganization and membrane ruffling in human foreskin fibroblasts [42]. The isoform-dependency of this reaction indicates that the response is only mediated by the activated preceptor. Our studies showed (i) that PDGF-AA is a competitive inhibitor of ruffling induced by PDGF-AB, but not PDGFBB, (ii) that down-regulation of the a-receptor abolished the effect elicited by PDGFAB, but not by PDGF-BB and (iii) that PDGF-AB competes for the effect of PDGFBB in cells with down-regulated a-receptors. In summary, these experiments are compatible with a model by which the Preceptor is functionally active with regard to the initiation of membrane ruffling only in a dimeric configuration (either homo- or

259

PDGF Receptors

PDGF-AA

aci

CUR

aa

FIGURE 3. Schemrtic representationof the interaction of the thee isoforms of PDGF with two PDGF receptor types (0~ and /heceptor). ‘he model is based on the notion that PDGF is a bivalent Rgand, and that the A chain of PDGF ooly interacts with the a-receptor whereas tbe B&aia heracts with both receptor typea. The made1 infers a hierarchy mwag the PDGF isoforms, as described in the text. PDGF-BB is the uaiversal PDGF in the sense that it interacts with all three wceptor dimers.

heterodimeric); hence, a biological response to PDGF-AB requires the presence of both a- and preceptors. This view is substantiated by the finding that an efficient downregulation as well as autophosphorylation of Preceptors by PDGF-AB requires the presence of a-receptors [42]. Evidence for heterodimerization has also been obtained from experiments showing that human-specific preceptor antibodies, bring down mouse a-receptors from a mixture of human and mouse fibroblast membranes incubated in the presence of PDGF-AB [40]. The model presented above does not preclude the possibility that PDGF receptor dimerization also involves a ligand-induced conformational change of the ligandbinding domain of the receptor. Thus, it is conceivable that binding of the bivalent ligand constitutes the initial event in receptor dimerization, followed by a conformation-dependent stabilization of the dimer, which in the intact cell may be followed by receptor oligomerization and clustering. That PDGF indeed induces a conformational change of the receptor is evidenced by the finding that binding to cells is essentially irreversible at neutral pH [33]. SIGNAL

TRANSDUCTION

EVENTS

The binding of PDGF to its receptor elicits a cascade of early cellular events, including protein phosphorylation, inositol lipid breakdown, ion fluxes and gene

260

B. Westermark et al.

expression. Attempts to link the receptor protein tyrosine kinase activity to these responses remained futile for a long time. Recent studies have shown that an intact kinase activity is essential for signal transduction of the preceptor; this is likely to hold true also for the a-receptor, although this has not yet been experimentally proven. Thus, a mutated PDGF preceptor, devoid of kinase activity is unable to mediate inositol lipid breakdown, calcium release, alteration of intracellular pH, gene expression, DNA synthesis [35], actin reorganization, or chemotaxis [43]. A PDGF-induced activation of phospholipase C was suggested by the finding that the addition of PDGF to responsive cells leads to the degradation of phosphatidylinositol-4,Sbisphosphate (PI-4,5-P,) [44, 451 and the generation of two second messengers, i.e. inositol- 1,4,5-trisphosphate (Ins- 1,4,5-P,), which induces liberation of intracellularly stored calcium [46], and diacylglycerol, that activates protein kinase C [47]. Recent studies have shown that phospholipase C-y is a substrate for the PDGF receptor protein-tyrosine kinase, both in vivo and in vitro [48, 491. PDGF-induced phosphorylation of PLC-y occurs at high stoichiometry, correlates well with PLC activity, and is probably caused by a direct interaction between the PDGF receptor and PLC-y, i.e. no intermediate kinase is required for the reaction to occur. The latter assumption is also supported by the finding that the PDGF receptor and PLC-ycan be co-immunoprecipitated. Moreover, the same tyrosine residues are phosphorylated in vivo as in vitro using purified receptor [48]. Inherent difficulties in the assay system have so far made it impossible to determine whether phosphorylation of PLC-y has any consequences for its catalytic activity. In addition to the effect on PI-4,5-P, hydrolysis, PDGF has been shown to regulate PI metabolism in yet another fashion. Thus, PDGF activates a unique PI-3’ kinase which phosphorylates the inositol ring on the D-3 position [50,51]. This novel pathway gives rise to three primary products, PI-3-P, PI-3,4-P, and PIP,. The two latter products are absent in unstimulated cells but present in low amounts after stimulation with PDGF-BB. It is interesting that the PI-3’ kinase is also activated by the pp6WYrr‘/ polyoma middle T complex [52, 531; its activity may thus be related both to growth stimulation and transformation, although a functional role of the products has not been found. The finding that the PI-3’ kinase is precipitated both by ligand-activated PDGF Preceptors [5 1] and phosphotyrosine antibodies [50] suggests that the enzyme is a substrate for the receptor kinase. A third potentially interesting candidate for the PDGF /?-receptor kinase is the serine/threonine protein kinase Raf-1. Exposure of cells to PDGF leads to multiple phosphorylation and a change in the electrophoretic mobility of Raf-1 [54. 551. Although most of the increase of phosphorylation was found to occur on serine and threonine residues, a minor proportion of tyrosine phosphorylation was also detected. Studies with isolated PDGF Preceptor and Raf-1 indicated that Raf-1 is a direct substrate for the PDGF Preceptor protein tyrosine kinase. Moreover, an examination of the tyrosine-phosphorylated Raf-1 protein showed that its serine/threonine kinase activity was increased several-fold, compared to the untreated control. Co-precipitation studies have revealed a physical association between the PDGF receptor and Raf-1. This association was found to occur efficiently only with the phosphorylated receptor, implying two roles of the receptor protein tyrosine kinase in this situation: autophosphorylation joins the Raf-1 protein with the receptor and transphosphorylation increases the catalytic activity of Raf-1. These studies provide circumstantial evidence that Raf-1 may have an important function in the PDGF-dependent signal

%I

PDGF Receptors

transmission pathway. This notion is reinforced by previous studies on the transforming potential of Raf-1 . Thus, oncogenic activation of Raf- 1 by amino-terminal truncation is associated with an increase in the constitutive kinase activity of the product; microinjection of this modified product leads to mitogenesis. CONSTITUTIVE

AND INDUCIBLE

PDGF RECEPTOR EXPRESSION

Receptors for PDGF have been found on a variety of mesenchyme-derived connective tissue cells in culture. The most commonly used target cells include dermal and lung fibroblasts, vascular smooth muscle cells, 3T3 cells, Rat 1 cells and NRK cells. All these cell types contain both cc-and preceptors; the number of Preceptors most commonly exceeds that of a-receptors. There are up to now only two cases in which normal cells have been shown to express only one of the two receptor types. Thus, the O2A glial progenitor cell of the rat optic nerve shows binding characteristics which are compatible with the expression of a-receptors only [%I. In addition, capillary endothelial cells of the brain express /Sreceptors only, as demonstrated by ligandbinding studies [57]. PDGF receptors are also expressed in a variety of human mesenchyme- and gliaderived tumor cell lines. Northern blot analysis of mRNA levels have shown a great variation in receptor expression with no apparent consistent pattern [24] (M. Nister et al., unpublished; P. LevCen et al., unpublished). It appears that the two receptors are independently expressed, even in tumors of the same histogenetic origin. Whether this reflects the expression pattern of the normal progenitor cells, from which the tumor has evolved, or is related to transformation and tumorigenesis, is not known. Immunohistochemistry staining of PDGF Preceptors in intact tissues has provided important information on the in vivo expression of this receptor; no such information is as yet available regarding the a-receptor. It appears that fibroblasts of normal connective tissue are devoid of receptors, or express receptors at a very low level. Receptors are, however, present on such cells in inflammatory tissue, such as in the synovia of patients with acute rheumatoid arthritis [58], and arginduced upo’n explantation of cells in culture [59]. However, also cells of certain noninflammatory tissues, such as endometrial cells of the uterus, constitutively express preceptors in vivo [59]. The studies cited above indicate that the responsiveness to PDGF may be regulated at the level of receptor expression. It is therefore of great importance to identify those factors, humoral and others, that are involved in the regulation of the synthesis of PDGF receptors. The pattern of c+ and Preceptor distribution in normal and malignant cells, indicates that the two PDGF receptor types may be regulated by different mechanisms. This view is substantiated by the finding that a growth inhibiting substance, viz. transforming growth factor-/3 (TGF-/I), down-regulates a-receptors in cultured cells without so much as affecting preceptor expression [60]. ROLE OF PDGF RECEPTORS IN TRANSFORMATION TUMORIGENESIS

AND

There is ample evidence that transformation by the v-sis oncogene is mediated by an autocrine PDGF-like growth factor, resembling a homodimer of B chains [61-64]. Several investigators have addressed the question whether the autocrine factor can

262

B. Westermark et al.

activate the receptor in an intracellular compartment or if the receptor has to beexpressed at the cell surface in order to be able to successfully interact with the ligand. This issue had been unexpectedly difficult to settle. Compelling evidence has been presented that the protein tyrosine kinase activity of the PDGF receptor is stimulated intracellularly, leading to the autophosphorylation of an immature receptor [65: 661. Furthermore, a recombinant v-sis protein equipped with a C-terminal KDEL sequence, which retains the product in the lumen of the endoplasmic reticulum, has the same transforming activity as the native v-sis product [67]. Other studies, however, indicate that the mitogenic signal is transduced at the cell surface. Thus, PDGF antibodies inhibit SSV-transformation of human fibroblasts [62] and reverses the phenotype of some other SSV-transformed cells [68]. Suramin, an agent that displaces receptor-bound PDGF [69] or v-sis product [63,70], reverses SSV-transformation [63]. Moreover, alteration of the intracellular transport system by monensin led to the autophosphorylation of an immature PDGF receptor but inhibited c-fos expression [71]. Most data obtained are compatible with the hypothesis that the v-k product and the receptor interact inside the v-sis transformed cells and that this leads to receptor autophosphorylation. However, in order to generate a mitogenic signal, the ligandreceptor complex has to reach the cell surface, perhaps in order to interact with the proper substrates for the activated receptor kinase. The fact that the PDGF a- and /&receptors are structurally related to the products of the v-fms and v-kit oncogenes raises the question whether the PDGF receptors too have transforming potentials. This question may now be addressed by a systematic search for structural rearrangements of the receptor genes in malignant tumors as well as by manipulative experiments, where the transforming activity of deliberately mutated receptors is analyzed. CONCLUSION Recent information on PDGF and its receptors has revealed a growth regulatory system of unexpected complexity; three isoforms of PDGF have been identified and these interact with different specificities with two homologous cell surface receptors. It thus appears that the PDGF family of ligands and receptors operate in a finely tuned growth regulatory system. We hope that future studies will lead to a better understanding of the PDGF-dependent signal pathways in cell growth and transformation. REFERENCES 1. Westermark B, Heldin C-H. Activation of proto-oncogenes coding for growth factors or growth factor receptors. In: Klein G, ed. Cellular oncogene activation. New York: Marcel Dekker; 1988: 149-180. 2. Heldin C-H, Westermark B. Growth factors as transforming proteins. Eur J Biochem. 1989; 184: 487496. 3. Hannink M, Donoghue DJ. Structure and function of platelet-derived growth factor (PDGF) and related proteins. Biochim Biophys Acta 1989; 989: l-10. 4. Ross R, Raines EW, Bowen-Pope DF. The biology of platelet-derived growth factor. Cell 1986; 46: 155-169. 5. Johnsson A, Heldin C-H, Westermark B, Wasteson A. Platelet-derived growth factor: Identification of constituent polypeptide chains. Biochem Biophys Res Commun. 1982; 104: 66-74. 6. Betsholtz C, Johnsson A, Heldin C-H, Westermark B, Lind P, Urdea MS, Eddy R, Shows TB, Philpott

PDGF Receptors

7. 8. 9. 10. 11.

12. 13. 14. 15. 16. 17. 18. 19.

20. 21. 22. 23. 24. 25. 26.

263

K. Mellor A, Knott TJ, Scott J. cDNA sequence and chromosomal localization of platelet-derived growth factor A-chain and its expression in tumour cell lines. Nature 1986; 320: 695-699. Hammacher A, Hellman U, Johnsson A, Gunnarsson K, dstman A, Westermark B, Wasteson A. Heldin C-H. A major part of PDGF purified from human platelets is a heterodimer of one A chain and one B chain. J Biol Chem. 1988; 263: 16493-16498. Bowen-Pope DF, Hart CE, Seifert RA. Sera and conditioned media contain different isoforms of platelet-derived growth factor (PDGF) which bind to different classesof PDGF receptor. J Biol Chem. 1989; 264: 2502-2508. Hammacher A, Nister M, Westermark B, Heldin C-H. A human glioma cell line secretes three structurally and functionally different dimeric forms of PDGF. Eur J B&hem. 1988; 176: 179-l 86. Heldin C-H, Johnsson A, Wennergren S, Wemstedt C, Betsholtz C, Wasteson A. A human osteosarcoma cell line secretes a growth factor structurally related to a homodimer of PDGF A chains. Nature 1986; 319: 51 I-515. Westermark B, Johnsson A, Paulsson Y, Betsholtz C, Heldin C-H, Herlyn M, Rodeck U, Koprowski H. Human melanoma cell lines of primary and metastatic origin express the genes encoding the chains of platelet-derived growth factor (PDGF) and produce a PDGF-like growth factor. Proc Nat1 Acad Sci USA. 1986; 83: 7197-7200. Stroobant P, Waterfield MD. Purification and properties of porcine platelet-derived growth factor. EMBO J. 1984; 3: 2963-2967. Devare SG, Reddy EP, Law JD, Robbins KC, Aaronson SA. Nucleotide sequence of the simian sarcoma virus genome: Demonstration that its acquired cellular sequences encode the transforming gene product ~28”“. Proc Nat1 Acad Sci USA. 1983; 80: 731-735. Waterfield MD, Scrace GT, Whittle N, Stroobant P, Johnsson A, Wasteson A, Westermark B, Heldin C-H, Huang JS, Deuel TF. Platelet-derived growth factor is structurally related to the putative transforming protein ~28”’ of simian sarcoma virus. Nature 1983; 304: 35-39. Doolittle RF, Hunkapiller MW, Hood LE, Devare SG, Robbins KC, Aaronson SA, Antoniades HN. Simian sarcoma virus oncogene, v-h, is derived from the gene (or genes) encoding a platelet-derived growth factor. Science 1983; 221: 275-277. Heldin C-H, Westermark B, Wasteson A. Specific receptors for platelet-derived growth factor on cells derived from connective tissue and glia. Proc Nat1 Acad Sci USA. 1981; 78: 3664-3558. Ek B, Westermark B, Wasteson A, Heldin C-H. Stimulation of tyrosine-specific phosphorylation by platelet-derived growth factor. Nature 1982; 295: 419-420. Ek B, Heldin C-H. Use of an antiserum against phosphotyrosine for the identification of phosphorylated components in human fibroblasts stimulated by platelet-derived growth factor. J Biol Chem. 1984; 259: 11145-l 1152. Yarden Y, Escobedo JA, Kuang W-J, Yang-Feng TL, Daniel TO, Tremble PM, Chen EY, Ando ME. Harkins RN, Francke U, Friend VA, Ullrich A, Williams LT. Structure of the receptor for plateletderived growth factor helpsdefine a family ofclosely related growth factor receptors. Nature 1986: 323: 226232. Gronwald RGK, Grant FJ, Haldeman BA, Hart CE, G’Hara PJ, Hagen FS, Ross R, Bowen-Pope DF. Murray M. Cloning and expression of a cDNA coding for the human platelet-derived growth factor receptor: Evidence for more than one receptor class. Proc Nat1 Acad Sci USA. 1988; 85: 3435-3439. Claesson-Welsh L, Eriksson A, Moren A. Severinsson L, Ek B, Gstman A, Betsholtz C, Heldin C-H. cDNA cloning and expression of a human platelet-derived growth factor (PDGF) receptor specific for B-chain-containing PDGF molecules. Mol Cell Biol. 1988; 8: 3476-3486. Heldin C-H, Backstrom G, &tman A, Hammacher A, Riinnstrand L. Rubin K, Nister M, Westermark B. Binding of different dimeric forms of PDGF to human fibroblasts: Evidence for two separate receptor types. EMBO J. 1988; 7: 1387-1394. Hart CE, Forstrom JW, Kelly JD, Seifert RA, Smith RA, Ross R, Murray MJ, Bowen-Pope DF. Two classes of PDGF receptors recognize different isoforms of PDGF. Science 1988; 240: 1529-l 53 1. Matsui T, Heidaran M, Miki T, Toru M, Popescu N, La Rochelle W, Kraus M, Pierce J, Aaronson SA. Isolation of a novel receptor cDNA establishes the existence of two PDGF receptor genes. Science 1989; 243: S&803. Claesson-Welsh L, Eriksson A, Westermark B, Heldin C-H. cDNA cloning and expression of the human A type PDGF receptor establishes structural similarity to the B type PDGF receptor. Proc Nat1 Acad Sci USA. 1989; 86: 49174921. Sherr CJ. Rettenmier CW, Sacca R, Roussel MF. Look AT, Stanley ER. The c:fms proto-oncogene

264

27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47.

B. Westermark et al. product is related to the receptor for the mononuclear phagocyte growth factor, CSF- I. Cell 1985: 41: 665-616. Besmer P, Murphy JE, George PC, Qui F, Bergold PJ, Lederman L, Snyder HW Jr, Brodeur D, Zuckerman EE, Hardy WD. A new acute transforming feline retrovirus and relationship of its oncogene v-kit with the protein kinase gene family. Nature 1986; 320: 415421. Yarden Y, Kuang W-J, Yang-Feng T, Coussens L, Munemitsu S, Dull TJ, Chen E, Schlessinger J, Francke U. Ullrich A. Human protooncogene c-kif: A new cell surface receptor tyrosine kinase for an unidentified ligand. EMBO J. 1987; 6: 3341-3351. Qiu F, Ray P, Brown K, Barker PE, Jhanwar S, Ruddle FH, Besmer P. Primary structure of c-kit: relationship with the CSF-l/PDGF receptor kinase family-oncogenic activation of v-kit involves deletion of extracellular domain and C terminus. EMBO J. 1988; 7: 1003-1011. Hart CE, Seifert RA, Ross R, Bowen-Pope DF. Synthesis, phosphorylation and degradation of multiple forms of the platelet-derived growth factor receptor studied using a monoclonal antibody. J Biol Chem. 1987; 262: 10780-10785. Keating MT, Williams LT. Processing of the platelet-derived growth factor receptor. Biosynthetic and degradation studies using anti-receptor antibodies. J Biol Chem. 1987; 262: 7932-7937. Claesson-Welsh L, Hammacher A, Westermark B, Heldin C-H, Nister M. Identification and structural analysis ofthe A type receptor for PDGF: Similarities with the B type receptor. J Biol Chem. 1989; 264: 1742-1747. Heldin C-H, Wasteson A, Westermark B. Interaction of platelet-derived growth factor with its fibroblast receptor. J Biol Chem. 1982; 257: 42164221. Nilsson J, Thyberg J, Heldin C-H, Westermark B, Wasteson A. Surface binding and internalization of platelet-derived growth factor in human fibroblasts. Proc Nat1 Acad Sci USA. 1983; 80: 5592-5596. Escobedo JA, Barr PJ, Williams LT. Role of tyrosine kinase and membrane-spanning domains in signal transduction by the platelet-derived growth factor receptor. Mol Cell Biol. 1988; 8: 51265131. Glenney JR Jr, Chen WS, Lazar CS, Walton GM, Zokas LM, Rosenfeld MG, Gill GN. Ligandinduced endocytosis of the EGF receptor is blocked by mutational inactivation and by microinjection of anti-phosphotyrosine antibodies. Cell 1988; 52: 675684. Honegger AM, Dull TJ, Felder S, Van Obberghen E, Bellot F, Szapary D, Schmidt A, Ullrich A, Schlessinger J. Point mutation at the ATP binding site of EGF receptor abolishes protein-tyrosine kinase activity and alters cellular routing. Cell 1987; 51: 199-209. Schlessinger J. The epidermal growth factor receptor as a multifunctional allosteric protein. Biochemistry 1988; 27: 3119-3123. Heldin C-H. Ernlund A, Rorsman C, Riinnstrand L. Dimerization of B type PDGF receptors occur after ligand binding and is closely associated with receptor kinase activation. J Biol Chem. 1989; 264: 8905-8912. Seifert RA, Hart CE, Phillips PE, Forstrom JW, Ross R, Murray M, Bowen-Pope DF. Two different subunits associate to create isoform-specific platelet-derived growth factor receptors. J Biol Chem. 1989; 264: 8771-8778. Bishayee S, Majumdar S, Khire J, Das M. Ligand-induced dimerization of the platelet-derived growth factor receptor. Monomerdimer interconversion occurs independent of receptor phosphorylation. J Biol Chem. 1989; 264: 11699-l 1705. Hammacher A, Mellstrijm K, Heldin C-H, Westermark B. Isoform-specific induction of actin reorganization by platelet-derived growth factor suggests that the functionally active receptor is a dimer. EMBO J. 1989; 8: 2489-2495. Westermark B, Siegbahn A, Heldin C-H, Claesson-Welsh L. The B type receptor for platelet-derived growth factors mediates a chemotactic response by means of ligand-induced activation of the receptor protein tyrosine kinase. Proc Nat1 Acad Sci USA., in press. Habenicht AJR, Glomset JA, King WC, Nist C, Mitchell CD, Ross R. Early changes in phosphatidylinositol and arachidonic acid metabolism in quiescent Swiss 3T3 cells stimulated to divide by platelet-derived growth factor. J Biol Chem. 1981; 256: 12329-12235. Berridge M, Heslop J, Irvine RF, Brown KD. Inositol trisphosphate formation and calcium mobilization in Swiss 3T3 cells in response to platelet-derived growth factor. Biochem J. 1984; 222: 195-201. Berridge MJ, Irvine RF. Inositol trisphosphate, a novel second messenger in cellular signal transduction. Nature 1984; 312: 315-321. Nishizuka Y. The role of protein kinase C in cell surface signal transduction and tumor promotion.

PDGF Receptors

265

Nature 1984; 308: 693-698. 48. Meisenhelder J, Suh P-G, Rhee SG, Hunter T. Phospholipase C-y is a substrate for the PDGF and EGF receptor protein tyrosine kinases in viva and in vitro. Cell 1989; 57: 1109-l 122. 49. Wahl MI, Olashaw NE, Nishibe S, Rhee SG, Pledger WJ, Carpenter G. Platelet-derived growth factor induces rapid and sustained tyrosine phosphorylation of phospholipase C-yin quiescent BALB/c 3T3 cells. Mol Cell Biol. 1989; 9: 2934-2943. 50. Auger KR, Serunian LA, Soltoff SP, Libby P, Cantley LC. PDGF-dependent tyrosine phosphorylation stimulates production of novel polyphosphoinositides in intact cells. Cell 1989: 57: 167-175. 51. Coughlin SR, Escobedo JA, Williams LT. Role of phosphatidylinositol kinase in PDGF receptor signal transduction. Science 1989; 243: 1191-l 194. 52. Kaplan DR, Whitman M, Schaflhausen B, Pallas DC, White M, Cantley L, Roberts TM. Common elements in growth factor stimulation and oncogenic transformation: 85 kDa phosphoprotein and phosphatidylinositol kinase activity. Cell 1987; 50: 1021-1029. 53. Courtneidge SA, Heber A. An 81 kd protein complexed with middle T antigen and ~~60”“‘: A possible phosphatidylinositol kinase. Cell 1987; 50: 1031-1037. 54. Morrison DK. Kaplan DR, Rapp U, Roberts TM. Signal transduction from membrane to cytoplasm: Growth factors and membrane-bound oncogene products increase Raf-1 phosphorylation and associated protein kinase activity. Proc Nat1 Acad Sci USA. 1988; 85: 8855-8859. 55. Morrison DK, Kaplan DR. Escobedo JA, Rapp UR, Roberts TM, Williams LT. Direct activation of the serine/threonine kinase activity of Raf-1 through tyrosine phosphorylation by the PDGF /% receptor. Cell 1989; 58: 649-657. 56. Hart IK, Richardson WD, Heldin C-H, Westermark B, Raff MC. PDGF receptors on cells of the oligodendrocyte-type-2 astrocyte (O-2A) cell lineage. Development 1989; 105: 595-3. 57. Smits A, Hermansson M, Nister M, Kamushina I, Heldin C-H, Westermark B. Funa K. Rat brain capillary endothelial cells express functional PDGF B-type receptors. Growth Factors, in press. 58. Rubin K, Tingstriim A, Hansson GK, Larsson E, Rijnnstrand L, Klareskog L, Claesson-Welsh L. Heldin C-H. Fellstrom B, Terracio L. Induction of B-type receptors for platelet-derived growth factor in vascular inflammation: possible implications for development of vascular proliferative lesions. Lancet 1988; i: 1353-1355. 59. Terracio L, Ronnstrand L, Tingstrijm A, Rubin K, Claesson-Welsh L, Funa K, Heldin C-H. Induction of platelet-derived growth factor receptor expression in smooth muscle cells and fibroblasts upon tissue culturing. J Cell Biol. 1988; 107: 1947-1958. 60. Gronwald RGK, Seifert RA, Bowen-Pope DF. Differential regulation of platelet-derived growth factor subunits by transforming growth factor-p. J Biol Chem. 1989; 264: 812&8125. 61. Robbins KC, Antoniades HN, Devare SG, Hunkapiller MW, Aaronson SA. Structural and immunological similarities between simian sarcoma virus gene product(s) and human platelet-derived growth factor. Nature 1983; 305: 605608. 62. Johnsson A, Betsholtz C, Heldin C-H, Westermark B. Antibodies against platelet-derived growth factor inhibit acute transformation by simian sarcoma virus. Nature 1985; 317: 438440. 63. Betsholtz C, Johnsson A, Heldin C-H, Westermark B. Efficient reversion of SSV-transformation and inhibition of growth factor-induced mitogenesis by suramin. Proc Nat1 Acad Sci USA. 1986: 83: 644& 6444. 64. Westermark B, Betsholtz C, Johnsson A, Heldin C-H. Acute transformation by simian sarcoma virus is mediated by an externalized PDGF-like growth factor. In: Kjelgaard NO, Forchhammmer J, eds. Viral carcinogens. Munksgaard: Copenhagen; 1978: 445-457. 65. Keating MT. Williams LT. Autocrine stimulation ofintracellular PDGF receptors in v-k transformed cells. Science 1988; 239: 914915. 66. Huang SS, Huang JS. Rapid turnover of the platelet-derived growth factor receptor in s&transformed cells and reversal by suramin. Implications for the mechanism of autocrine transformation, J Biol Chem. 1988; 263: 12608-12618. 67. Bejcek BE, Li DY, Deuel TF. Transformation by v-sis occurs by an internal autoactivation mechanism. Science 1989; 245: 1496-1499. 68. Huang JS, Huang SS, Deuel TF. Transforming protein of simian sarcoma virus stimulates autocrine growth of SSV-transformed cells through PDGF cell-surface receptors. Cell 1984; 39: 79-87. 69. Williams LT. Tremble PM, Lavin MF, Sunday ME. Platelet-derived growth factor receptors form a high affinity state in membrane preparations. J Biol Chem. 1984; 259: 5287-5294.

266

B. Westermark et al.

70. Garrett JS, Coughlin SR, Niman HL, Tremble PM, Giels GM, Williams LT. Blockade of autocrine stimulation in simian sarcoma virus-transformed cells reverses down-regulation of platelet-derived growth factor receptors. Proc Nat1 Acad Sci USA. 1984; 81: 74667470. 71. Hannink M, Donoghue DJ. Autocrine stimulation by the v-sis gene product requires a ligand-receptor interaction at the cell surface. J Cell Biol. 1988; 107: 287-298. 72. Coussens L, Van Beveren C, Smith D, Chen E, Mitchell RL, Isacke C, Verma IA, Ullrich A. Structural alteration of viral homologue of receptor protooncogenefmr at carboxyterminus. Nature 1986; 320: 277-280.