Grb10, a positive, stimulatory signaling adapter in platelet-derived growth factor BB-, insulin-like growth factor I-, and insulin-mediated mitogenesis

Grb10 has been described as a cellular partner of several receptor tyrosine kinases, including the insulin receptor (IR) and the insulin-like growth f actor I (IGF-I) receptor (IGF-IR), Its cellular role is still unclear and a positive as well as an inhibitory role in mitogenesis depending on the cel l context has been implicated. We have tested other mitogenic receptor tyro sine kinases as putative Grb10 partners and have identified the activated f orms of platelet derived growth factor (PDGF) receptor beta (PDGFR beta), h epatocyte growth factor receptor (Met), and fibroblast growth factor recept or as candidates. We have mapped Y771 as a PDPGR beta site that is involved in the association with Grb10 via its SH2 domain. We have further investig ated the putative role of Grb10 in mitogenesis with four independent experi mental strategies and found that all consistently suggested a role as a pos itive, stimulatory signaling adaptor in normal fibroblasts. (i) Complete Gr b10 expression from cDNA with an ecdysone-regulated transient expression sy stem stimulated PDGF-BB-, IGF-I, and insulin- but not epidermal growth fact or (EGF)-induced DNA synthesis in an ecdysone dose-responsive fashion. (ii) Microinjection of the (dominant-negative) Grb10 SH2 domain interfered with PDGF-BB- and insulin-induced DNA synthesis. (iii) Alternative experiments were based on cell-permeable fusion peptides with the Drosophila antennaped ia homeodomain which effectively traverse the plasma membrane of cultured c ells. A cell-permeable Grb10 SH2 domain similarly interfered with PDGP-BB-, IGF-I-, and insulin-induced DNA synthesis. In contrast, a cell-permeable G rb10 Pro-rich putative SH3 domain binding region interfered with IGF-I- and insulin- but not with PDGF-BB- or EGF-induced DNA synthesis. (iv) Transien t overexpression of complete Grb10 increased whereas cell-permeable Grb10 S H2 domain fusion peptides substantially decreased the cell proliferation ra te (as measured by cell numbers) in normal fibroblasts. These experimental strategies independently suggest that Grb10 functions as a positive, stimul atory, mitogenic signaling adapter in PDGF-BB, IGF-I, and insulin action. T his function appears to involve the Grb10 SH2 domain, a novel sequence term ed BPS, and the Pro-rich putative SH3 domain binding region in IGF-I- and i nsulin-mediated mitogenesis, In contrast, PDGF-BB-mediated mitogenesis appe ars to depend on the SH2 but not on the Pro-rich region and may involve oth er, unidentified Grb10 domains. Distinct protein domains may help to define specific Grb10 functions in different signaling pathways.