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PMID: 7935368 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Characterization of an interaction between insulin receptor substrate 1 and the insulin receptor by using the two-hybrid system.

Molecular and cellular biology ·Vol. 14 ·No. 10 ·1994-10-00 ·Pages 6433-42

O'Neill TJ, Craparo A, Gustafson TA

Abstract

Insulin receptor substrate 1 (IRS-1) is a major substrate of the insulin receptor and has been implicated in insulin signaling. Although IRS-1 is thought to interact with the insulin receptor, the nature of the interaction has not been defined. In this study, we used the two-hybrid assay of protein-protein interaction in the yeast Saccharomyces cerevisiae to study the interaction between human IRS-1 and the insulin receptor. We demonstrate that IRS-1 forms a specific complex with the cytoplasmic domain of the insulin receptor when both are expressed as hybrid proteins in yeast cells. We show that the interaction is strictly dependent upon receptor tyrosine kinase activity, since IRS-1 shows no interaction with a kinase-inactive receptor hybrid containing a mutated ATP-binding site. Furthermore, mutation of receptor tyrosine 960 to phenylalanine eliminates IRS-1 interaction in the two-hybrid assay. These data suggest that the interaction between IRS-1 and the receptor is direct and provide evidence that the juxtamembrane domain of the receptor is involved. Furthermore, we show that a 356-amino-acid region encompassed by amino acids 160 through 516 of IRS-1 is sufficient for interaction with the receptor in the two-hybrid assay. Lastly, in agreement with our findings for yeast cells, we show that the insulin receptor is unable to phosphorylate an IRS-1 protein containing a deletion of amino acids 45 to 516 when expressed in COS cells. The two-hybrid assay should provide a facile means by which to pursue a detailed understanding of this interaction.

MeSH Terms
Amino Acid Sequence Animals Cells, Cultured DNA Mutational Analysis Humans Insulin Receptor Substrate Proteins Models, Biological Molecular Sequence Data Phosphoproteins/genetics,metabolism Phosphorylation Protein Binding Receptor Protein-Tyrosine Kinases/genetics,metabolism Receptor, Insulin/genetics,metabolism Recombinant Fusion Proteins/metabolism Saccharomyces cerevisiae/genetics Sequence Deletion Signal Transduction Structure-Activity Relationship Transformation, Genetic Tyrosine/metabolism
Chemicals
IRS1 protein, human Insulin Receptor Substrate Proteins Phosphoproteins Recombinant Fusion Proteins Tyrosine Receptor Protein-Tyrosine Kinases Receptor, Insulin
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
O'Neill T J
Department of Physiology, University of Maryland School of Medicine, Baltimore 21201.
Craparo A
Gustafson T A
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Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/mcb.14.10.6433-6442.1994
Published
1994-10-00
Pages
6433-42
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC359173
Subset
IM
Grants
NIDDK NIH HHS · DK44093 · United States
NIGMS NIH HHS · GM08181 · United States
External Links
PubMed source DOI: 10.1128/mcb.14.10.6433-6442.1994 PMC Full Text View journal details
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