Interaction of a GRB-IR Splice Variant (a Human GRB10 Homolog) with the Insulin and Insulin-like Growth Factor I Receptors

Abstract

We have utilized the yeast two-hybrid system to identify proteins that interact with the cytoplasmic domain of the insulin receptor. We identified a human cDNA that is a splice variant of the human GRB10 homolog GRB-IR, which we term GRB10/IR-SV1 (for GRB10/ GRB-IR splice variant 1). The protein encoded by the GRB10/IR-SV1 cDNA contains an SH2 domain and a pleckstrin homology domain. Cloning of a full-length human cDNA revealed a predicted coding sequence that was similar to the mouse GRB10 protein, although GRB10/IR-SV1 contained an 80-amino acid deletion. The GRB10/IR-SV1 cDNA is a splice variant of the GRB-IR cDNA such that GRB10/IR-SV1 contains an intact pleckstrin homology domain and a distinct amino terminus. The interaction of GRB10/IR-SV1 with the insulin receptor and the insulin-like growth factor I (IGF-I) receptor is mediated by the SH2 domain, and we show that glutathione S-transferase-SH2 domain fusion proteins interact specifically in vitro with the insulin receptor derived from mammalian cells. The GRB10/IR-SV1 SH2 domain also interacted with an ϳ135-kDa phosphoprotein from unstimulated cell lysates, an interaction that decreased after insulin stimulation. We present evidence that the GRB10/IR-SV1 protein plays a functional role in insulin and IGF-I signaling by showing that microinjection of an SH2 domain fusion protein inhibited insulin-and IGF-I-stimulated mitogenesis in fibroblasts, yet had no effect on mitogenesis induced by epidermal growth factor. Our findings suggest that GRB10/IR-SV1 may serve to positively link the insulin and IGF-I receptors to an uncharacterized mitogenic signaling pathway.

The SH2-containing adapter protein GRB10 interacts with BCR-ABL

“…None of these mutants lost the ability to bind to the Grb10-SH2 domain in vitro (Figure 3b and data not shown). The inability to exactly determine a single Bcr-Abl autophosphorylation site responsible for Grb10 binding was also reported by several other investigators (Frantz et al, 1997;O'Neill et al, 1996). Therefore, the binding site of the Grb10-SH2 domain seems to be more complex and might involve several autophosphorylation sites or additional sequences in the Brc region aa243-446.…”

“…Among the clones identi®ed was a new SH2-containing adapter molecule, Grb10, which was independently isolated six times from a mouse embryo library. In addition, a human splice variant of Grb10 (hGrb10svIR) (Liu et al, 1995;O'Neill et al, 1996) could be isolated in the yeast two-hybrid screen with a library obtained from a CML cell line (K562). Grb10 belongs to an emerging family of SH2-containing adapter molecules including Grb7 and the recently identi®ed Grb14 (Ooi et al, 1995).…”

“…Hereby, Grb10 is perhaps able to deliver a pro-mitogenic signal. Experiments with a putative dominant-negative mutant of Grb10 (the isolated SH2 domain) expressed in ®broblasts show inhibition of DNA synthesis (O'Neill et al, 1996). Furthermore, overexpression of Grb7 in certain types of breast cancer and Grb14 in estrogen receptor-positive breast cancer cells and prostate cancer suggest a role for this adapter family in oncogenesis (Stein et al, 1994;Daly et al, 1996).…”

“…Despite its cloning in conjunction with the EGF receptor, Grb10 could not be shown to bind to the EGF receptor in vivo (Ooi et al, 1995). In contrast, recently an in vivo association of Grb10 with the Ret receptor, the Insulin receptor and the ELK receptor was demonstrated suggesting that Grb10 may play a role in the biology of these receptors (Hansen et al, 1996;Pandey et al, 1995;Stein et al, 1996;Frantz et al, 1997;O'Neill et al, 1996).…”

“…Grb10 was recently cloned by screening of an expression library with the tyrosinephosphorylated carboxy-terminus of the epidermal growth factor receptor (EGFR) (Ooi et al, 1995). Three distinct splice variants of Grb10 have been described (mGrb10, hGrb-IR, hGrbsvIR) (Liu and Roth, 1995;O'Neill et al, 1996;Frantz et al, 1997). Grb10 belongs to an emerging family of SH2-containing signaling molecules including Grb7 and Grb14.…”