Characterization of Drosophila Insulin Receptor Substrate

Rachel M Kulansky Poltilove,Aviva R Jacobs,Carol Renfrew Haft,Pin Xu,Simeon I Taylor

doi:10.1074/jbc.m003579200

Rachel M Kulansky Poltilove, Aviva R Jacobs + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m003579200

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Abstract

Insulin receptor substrate (IRS) proteins are phosphorylated by multiple tyrosine kinases, including the insulin receptor. Phosphorylated IRS proteins bind to SH2 domain-containing proteins, thereby triggering downstream signaling pathways. The Drosophila insulin receptor (dIR) C-terminal extension contains potential binding sites for signaling molecules, suggesting that dIR might not require an IRS protein to accomplish its signaling functions. However, we obtained a cDNA encoding Drosophila IRS (dIRS), and we demonstrated expression of dIRS in a Drosophila cell line. Like mammalian IRS proteins, the N-terminal portion of dIRS contains a pleckstrin homology domain and a phosphotyrosine binding domain that binds to phosphotyrosine residues in both human and Drosophila insulin receptors. When coexpressed with dIRS in COS-7 cells, a chimeric receptor (the extracellular domain of human IR fused to the cytoplasmic domain of dIR) mediated insulin-stimulated tyrosine phosphorylation of dIRS. Mutating the juxtamembrane NPXY motif markedly reduced the ability of the receptor to phosphorylate dIRS. In contrast, the NPXY motifs in the C-terminal extension of dIR were required for stable association with dIRS. Coimmunoprecipitation experiments demonstrated insulin-dependent binding of dIRS to phosphatidylinositol 3-kinase and SHP2. However, we did not detect interactions with Grb2, SHC, or phospholipase C-gamma. Taken together with published genetic studies, these biochemical data support the hypothesis that dIRS functions directly downstream from the insulin receptor in Drosophila.

Highlights

From the §Diabetes Branch, NIDDKD, National Institutes of Health, Bethesda, Maryland 20892 and the ‡Graduate Genetics Program, The George Washington University, District of Columbia 20052
Sequence Analysis—We identified a sequence in the EST data base (AA536319) corresponding to a cDNA encoding Drosophila Insulin receptor substrate (IRS)
We identified at least six potential sites of tyrosine phosphorylation that are present in the context of known consensus binding sequences for SH2 domain-containing proteins (Table I)

Summary

Introduction

From the §Diabetes Branch, NIDDKD, National Institutes of Health, Bethesda, Maryland 20892 and the ‡Graduate Genetics Program, The George Washington University, District of Columbia 20052. The Drosophila insulin receptor (dIR) C-terminal extension contains potential binding sites for signaling molecules, suggesting that dIR might not require an IRS protein to accomplish its signaling functions. Like mammalian IRS proteins, the N-terminal portion of dIRS contains a pleckstrin homology domain and a phosphotyrosine binding domain that binds to phosphotyrosine residues in both human and Drosophila insulin receptors. Phosphotyrosine residues in IRS molecules interact with various SH2 domain-containing proteins, such as Grb and PI 3-kinase, thereby activating several signaling pathways [1, 2]. One isoform closely resembles mammalian receptors, and the other contains a 368-amino acid C-terminal extension Because this extension contains many potential sites for tyrosine phosphorylation, including consensus binding sites for the p85 subunit of PI 3-kinase, it was suggested that the Drosophila insulin receptor might mediate signaling activities normally performed by IRS molecules. This raised the question as to whether Drosophila might have an endogenous IRS molecule

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