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

Martin Clodi, Peter Vollenweider, Jes Klarlund, Naoki Nakashima, Stuart Martin, Michael P. Czech, Jerrold M. Olefsky

Research output: Contribution to journalArticle

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Abstract

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 α-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.

Original languageEnglish
Pages (from-to)4984-4990
Number of pages7
JournalEndocrinology
Volume139
Issue number12
DOIs
Publication statusPublished - Jan 1 1998
Externally publishedYes

Fingerprint

Facilitative Glucose Transport Proteins
Insulin-Like Growth Factor I
Insulin
DNA
Microinjections
Phosphatidylinositols
Actins
Antibodies
phosphatidylinositol receptors
rac GTP-Binding Proteins
Guanine Nucleotide Exchange Factors
rho GTP-Binding Proteins
Deoxyuridine
Proteins
Insulin Receptor
Glutathione Transferase
Actin Cytoskeleton
Nucleotides

All Science Journal Classification (ASJC) codes

  • Endocrinology

Cite this

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. / Clodi, Martin; Vollenweider, Peter; Klarlund, Jes; Nakashima, Naoki; Martin, Stuart; Czech, Michael P.; Olefsky, Jerrold M.

In: Endocrinology, Vol. 139, No. 12, 01.01.1998, p. 4984-4990.

Research output: Contribution to journalArticle

Clodi, Martin ; Vollenweider, Peter ; Klarlund, Jes ; Nakashima, Naoki ; Martin, Stuart ; Czech, Michael P. ; Olefsky, Jerrold M. / 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. In: Endocrinology. 1998 ; Vol. 139, No. 12. pp. 4984-4990.
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abstract = "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 α-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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