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PMID: 8039498 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

A complex of GRB2-dynamin binds to tyrosine-phosphorylated insulin receptor substrate-1 after insulin treatment.

The EMBO journal ·Vol. 13 ·No. 13 ·1994-07-01 ·Pages 3033-8

Ando A, Yonezawa K, Gout I, Nakata T, Ueda H, Hara K, Kitamura Y, Noda Y, Takenawa T, Hirokawa N

Abstract

Insulin drives the formation of a complex between tyrosine-phosphorylated IRS-1 and SH2-containing proteins. The SH2-containing protein Grb2 also possesses adjacent SH3 domains, which bind the Ras guanine nucleotide exchange factor Sos. In this report, we examined the involvement of another SH3 binding protein, dynamin, in insulin signal transduction. SH3 domains of Grb2 as GST fusion proteins bound dynamin from lysates of CHO cells expressing wild-type insulin receptor (IR) (CHO-IR cells) in a cell-free system (in vitro). Immunoprecipitation studies using specific antibodies against Grb2 revealed that Grb2 was co-immunoprecipitated with dynamin from unstimulated CHO-IR cells. After insulin treatment of CHO-IR cells, anti-dynamin antibodies co-immunoprecipitated the IR beta-subunit and IRS-1, as tyrosine-phosphorylated proteins and PI 3-kinase activity. However, purified rat brain dynamin did not bind directly to either the IR, IRS-1 or the p85 subunit of PI 3-kinase in vitro. Together, these results suggest that in CHO-IR cells, insulin stimulates the binding of dynamin to tyrosine-phosphorylated IRS-1 via Grb2 and that IRS-1 also associates with PI 3-kinase in response to insulin. This complex formation was reconstituted in vitro using recombinant baculovirus-expressed IRS-1, GST-Grb2 fusion proteins and dynamin peptides containing proline-rich sequences. Furthermore, dynamin GTPase activity was found to be stimulated when an IRS-1-derived phosphopeptide, containing the Grb2 binding site, was added to the dynamin-Grb2 complex in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

MeSH Terms
Adaptor Proteins, Signal Transducing Amino Acid Sequence Animals Brain/metabolism CHO Cells Cricetinae Dynamins GRB2 Adaptor Protein GTP Phosphohydrolases/metabolism Insulin/pharmacology Insulin Receptor Substrate Proteins Membrane Proteins/metabolism Molecular Sequence Data Phosphatidylinositol 3-Kinases Phosphoproteins/metabolism Phosphorylation/drug effects Phosphotransferases (Alcohol Group Acceptor)/genetics,metabolism Protein Binding/drug effects Proteins/genetics,metabolism Rats Receptor, Insulin/genetics,metabolism Recombinant Fusion Proteins/genetics,metabolism Son of Sevenless Proteins Tyrosine/metabolism
Chemicals
Adaptor Proteins, Signal Transducing GRB2 Adaptor Protein Grb2 protein, rat Insulin Insulin Receptor Substrate Proteins Irs1 protein, rat Membrane Proteins Phosphoproteins Proteins Recombinant Fusion Proteins Son of Sevenless Proteins Tyrosine Phosphatidylinositol 3-Kinases Phosphotransferases (Alcohol Group Acceptor) Receptor, Insulin GTP Phosphohydrolases Dynamins
Authors & Affiliations
10 authors, click to expand affiliations / ORCID
Ando A
Second Department of Internal Medicine, Kobe University School of Medicine, Japan.
Yonezawa K
Gout I
Nakata T
Ueda H
Hara K
Kitamura Y
Noda Y
Takenawa T
Hirokawa N
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36 references, click to expand
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Article Info
Journal
The EMBO journal
Abbr.
EMBO J
ISSN
0261-4189
Published
1994-07-01
Pages
3033-8
Language
English
Region
England
NLM ID
8208664
PMCID
PMC395193
Subset
IM
External Links
PubMed source PMC Full Text View journal details
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