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Effects of General Receptor for Phosphoinositides 1 on Insulin and Insulin-Like Growth Factor I-Induced Cytoskeletal Rearrangement, Glucose Transporter-4 Translocation, and Deoxyribonucleic Acid Synthesis¹

Division of Endocrinology and Metabolism, Department of Medicine, Veterans Administration Medical Center, University of California-San Diego (M.C., P.V., N.N., S.M., J.M.O.), La Jolla, California 92093; and the Program in Molecular Medicine and Department of Biochemistry and Molecular Biology, University of Massachusetts Medical Center (J.K., M.P.C.), Worcester, Massachusetts 01605

Address all correspondence and requests for reprints to: Dr. Jerrold M. Olefsky, Department of Medicine, University of California-San Diego, 9500 Gilman Drive, La Jolla, California 92093-0673.

We investigated the effects of general receptor for phosphoinositides-1 (GRP1), a recently cloned protein that binds 3,4,5-phosphatidylinositol [PtdIns(3,4,5)P3] with high affinity, but not PtdIns(3,4)P2 nor PtdIns(3)P, on insulin and insulin-like growth factor I (IGF-I)-induced cytoskeletal rearrangement, glucose transporter-4 (GLUT4) translocation, and DNA synthesis. GRP1 consists of an NH₂-terminally located coiled coil domain followed by a Sec7 domain and a COOH-terminal pleckstrin homology (PH) domain that is required for PtdIns binding. We used microinjection of glutathione-S-transferase fusion proteins containing residues 239–399 (PH domain), residues 52–260 (Sec7 domain), residues 5–71 (N-terminal domain), full-length GRP1, and an antibody (AB) raised against full-length GRP1 coupled with immunofluorescent detection of actin filament rearrangement, GLUT4 translocation, and 3'-bromo-5'-deoxyuridine incorporation. Microinjection of these constructs and the AB had no effect on insulin-induced GLUT4 translocation or DNA synthesis. However, microinjection of the GRP1-PH and the GRP1-Sec7 domain as well as the -GRP1-AB significantly inhibited insulin- and IGF-I-stimulated actin rearrangement in an insulin receptor-overexpressing cell line (HIRcB) compared with that in control experiments. Coinjection of GRP1-Sec7 along with constitutively active Rac (Q67L) did not inhibit Rac-induced actin rearrangement. Furthermore, GRP1 is not able to bind and act as a nucleotide exchange factor for the small GTP-binding proteins of the Rho family. As GRP1 acts as a guanine nucleotide exchange factor for ARF6 proteins, we propose a signaling pathway distinct from the small GTP-binding protein Rac, connecting PtdIns(3,4,5)P3 via GRP1 to ARF6, leading to insulin- and IGF-I-induced actin rearrangement.

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