Cell, Vol 52, 791-799. March 25, 1988. Copyright 0 1988 by Cell Press A Glioma-Derived PDGF A Chain Homodimer Has Different Functional Activities from a PDGF AB Heterodimer Purified from Human Platelets Monica Nist&,* Annet Hammacher,t Karin MellstrGm, Agneta Siegbahn,+ Lars RGnnstrand,t Bengt Westermark,’ and Carl-Henrik Heldin+ l Department of Pathology University Hospital S-751 85 Uppsala, Sweden t Ludwig Institute for Cancer Research Biomedical Center S-751 23 Uppsala, Sweden *Department of Clinical Chemistry University Hospital S-751 85 Uppsala, Sweden Summary Glioma-derived growth factor I (GDGF-I) is structurally similar to a platelet-derived growth factor (PDGF) A chain homodimer, whereas PDGF purified from human platelets is a heterodimer of one A and one B chain. Binding experiments revealed that GDGF-I and PDGF bound to a common receptor on human fibroblasts, but also suggested the presence of a second receptor type recognizing only PDGF. In contrast to PDGF, GDGF-I had only a limited mitogenic activity, a low ability to stimulate receptor autophosphorylation and actin reorganization, and no chemotactic activity. GDGF-I did, however, cause transmodulation of EGF receptors, suggesting that it, like PDGF, activates pro- tein kinase C in fibroblasts. These data indicate that different PDGF-like growth factors have different func- tional activities, which are possibly mediated via different receptors. Introduction Platelet-derived growth factor (PDGF) is a major mitogen in serum for connective tissue-derived cells and glial cells (for reviews, see Heldin et al., 1985; Ross et al., 1986). Structurally, it is a disulphide-bonded dimer of two differ- ent homologous polypeptide chains, denoted A and B (Johnsson et al., 1982). The A and B chains are encoded by separate genes, located on the human chromosomes 7 (Betsholtz et al., 1986) and 22 (Dalla Favera et al., 1982; Swan et al., 1982), respectively, which appear to have evolved through duplication of a common ancestor. The possible evolutionary advantage of the conservation of two closely related PDGF chains is not understood. Hu- man PDGF is assembled as a heterodimer (Hammacher et al., submitted), whereas porcine PDGF is a B chain homodimer (Stroobant and Waterfield, 1984). PDGF-like growth factors are produced by a variety of normal and transformed cell types, and it has been suggested that this may lead to autocrine stimulation of cell growth in cases where the producer cell carries the cognate recep- tor (reviewed in Heldin et al., 1986a). A large proportion of human tumor cell lines have been found to express PDGF A and/or B chain mRNA and to produce PDGF-like factors (NistBr et al., 1984; Nist& et al., 1987; Nister et al., submitted). Recently, different PDGF-like factors were identified and structurally characterized from medium conditioned by the human clonal glioma cell line U-343 MGa Cl 2:6 (Hammacher et al., submitted). One of the fac- tors, GDGF-I, was found to be a 31 kd protein structurally similar to a homodimer of PDGF A chains; an AB heterodi- mer and a B chain homodimer were also identified. The aim of the present investigation was to compare the func- tional characteristics of the PDGF A chain homodimer GDGF-I with those of an AB heterodimer purified from hu- man platelets. PDGF exerts its mitogenic effect via binding to a 170-185 kd cell surface receptor. The receptor is a trans- membrane protein with an external ligand binding do- main, and a protein tyrosine kinase activity associated with the cytoplasmic part (Ek et al., 1982; Yarden et al., 1986). Binding of PDGF to the external domain induces autophosphorylation of the receptor and phosphorylation on tyrosine residues of certain cytoplasmic substrates (reviewed in Heldin and RBnnstrand, 1988). Other early cellular effects of PDGF, which are believed to be of sig- nificance in the mitogenic pathway, include induction of an increased cytoplasmic Ca2+ concentration (Moolenaar et al., 1984), cytoplasmic alkalization (Burns and Rozen- gurt, 1983), activation of protein kinase C leading to trans- modulation of EGF receptors (Rozengurt et al., 1983), and induction of specific genes (Cochran et al., 1983). Binding of PDGF to its receptor induces an increased cell surface motility with actin reorganization and forma- tion of circular ruffles (Mellstrtim et al., 1983). The ruffles are of a specific morphology and are found on normal cells only after PDGF-stimulation and spontaneously oc- curring on a few tumor cell lines (MellstrBm et al., unpub- lished data). Of potential importance for its in vivo function is the effect of PDGF on chemotaxis of fibroblasts (Seppa et al., 1982), smooth muscle cells (Grotendorst et al., 1982), granulocytes and monocytes (Deuel et al., 1982). The biochemical mediators of the effects of PDGF on chemotaxis and ruffling are not known. In this work we report that, whereas PDGF and GDGF-I at least partially compete for binding to the same receptor. GDGF-I Induces a lower mitogenic response. GDGF-I has only limited effects on stimulation of autophosphorylation of the PDGF receptor, induction of ruffling, or induction of chemotaxis, but does induce transmodulation of EGF receptors. The data indicate that different PDGF-like fac- tors have different functional characteristics. Results Receptor Competing Activity of GDGF-I and PDGF The addition of 50,000 cpm of 1251-labeled GDGF-I or PDGF to confluent cultures of human foreskin fibroblasts led to a total binding of 7% and 8%, respectively, of added radioactivity. PDGF was as potent as GDGF-I in compet- ing with 1251-GDGF-I for binding to fibroblasts (Figure 1A).