EFFECTS OF GENERAL RECEPTOR FOR PHOSPHOINOSITIDES-1 ON INSULIN AND INSULIN-LIKE-GROWTH-FACTOR I-INDUCED CYTOSKELETAL REARRANGEMENT, GLUCOSETRANSPORTER-4 TRANSLOCATION, AND DEOXYRIBONUCLEIC-ACID SYNTHESIS

We investigated the effects of general receptor for phosphoinositides- 1 (GRP1), a recently cloned protein that binds 3,4,5-phosphatidylinosi tol [PtdIns(3,4,5)P3] with high affinity, but not PtdIns(3,4)P2 nor Pt dIns(3)P, on insulin and insulin-like growth factor I (IGF-I)induced c ytoskeletal 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 micr oinjection of glutathione-S-transferase fusion proteins containing res idues 239-399 (PH domain), residues 52-260 (Sec7 domain), residues 5-7 1 (N-terminal domain), full-length GRP1, and an antibody (AB) raised a gainst full-length GRP1 coupled with immunofluorescent detection of ac tin filament rearrangement, GLUT4 translocation, and 3'-bromo-5'-deoxy uridine incorporation. Microinjection of these constructs and the AB h ad no effect on insulin-induced GLUT4 translocation or DNA synthesis. However, microinjection of the GRP1-PH and the GRP1-Sec7 domain as wel l as the alpha-GRP1-AB significantly inhibited insulin- and IGF-I-stim ulated 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 bin d and act as a nucleotide exchange factor for the small GTP-binding pr oteins of the Rho family. As GRP1 acts as a guanine nucleotide exchang e factor for ARF6 proteins, we propose a signaling pathway distinct fr om the small GTP-binding protein Rac, connecting PtdIns(3,4,5)P3 via G RP1 to ARF6, leading to insulin- and IGF-I-induced actin rearrangement .