Interaction of a GRB-IR splice variant (a human GRB10 homolog) with the insulin and insulin-like growth factor I receptors. Evidence for a role in mitogenic signaling.
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 approximately 135-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.
O'Neill
T J
TJ
Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
Rose
D W
DW
Pillay
T S
TS
Hotta
K
K
Olefsky
J M
JM
Gustafson
T A
TA
D000273:Adipose Tissue / Q000737:chemistry; D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002455:Cell Division / Q000187:drug effects; D005347:Fibroblasts / Q000187:drug effects; D051383:GRB10 Adaptor Protein; D000882:Haplorhini; D006367:HeLa Cells; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D051379:Mice; D008845:Microinjections; D008969:Molecular Sequence Data; D019000:Phosphotyrosine / Q000378:metabolism; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017526:Receptor, IGF Type 1 / Q000378:metabolism*; D011972:Receptor, Insulin / Q000378:metabolism*; D015398:Signal Transduction; D015003:Yeasts
C096358:GRB10 protein, human; C495959:Grb10 protein, mouse; D007328:Insulin; D011506:Proteins; D012333:RNA, Messenger; D051383:GRB10 Adaptor Protein; D019000:Phosphotyrosine; D017526:Receptor, IGF Type 1; D011972:Receptor, Insulin