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 synthesis.
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 alpha-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.
Clodi
M
M
Department of Medicine, Veterans Administration Medical Center, University of California-San Diego, La Jolla 92093, USA.
Vollenweider
P
P
Klarlund
J
J
Nakashima
N
N
Martin
S
S
Czech
M P
MP
Olefsky
J M
JM
D016475:3T3 Cells; D017667:Adipocytes / Q000378:metabolism; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002460:Cell Line; D003599:Cytoskeleton / Q000187:drug effects* / Q000648:ultrastructure; D004247:DNA / Q000096:biosynthesis*; D005347:Fibroblasts / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000502:physiology; D051275:Glucose Transporter Type 4; D007328:Insulin / Q000494:pharmacology*; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D051379:Mice; D008845:Microinjections; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D009124:Muscle Proteins; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000502:physiology*
D051275:Glucose Transporter Type 4; D007328:Insulin; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D018160:Receptors, Cytoplasmic and Nuclear; C495693:Slc2a4 protein, mouse; C495694:Slc2a4 protein, rat; C040985:phosphatidylinositol receptors; D007334:Insulin-Like Growth Factor I; D004247:DNA; D019204:GTP-Binding Proteins