Non-SH2 Domains within Insulin Receptor Substrate-1 and SHC Mediate Their Phosphotyrosine-dependent Interaction with the NPEY Motif of the Insulin-like Growth Factor I Receptor (*)
- From the (1)Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201
- § To whom correspondence and reprint requests should be addressed: Dept. of Physiology, 510 Howard Hall, University of Maryland School of Medicine, 660 W. Redwood St., Baltimore, MD 21201. Tel.: 410-706-4253; Fax: 410-706-8341; E-mail: tgustafs{at}umabnet.ab.umd.edu.
Abstract
Insulin receptor substrate-1 (IRS-1) and SHC become rapidly phosphorylated upon tyrosines after insulin-like growth factor I receptor (IGFIR) activation. In this study we demonstrate that IRS-1, SHC, and the p85 subunit of phosphatidylinositol 3-kinase interact directly and specifically with the IGFIR. The interaction of all three proteins is dependent upon IGFIR kinase activity and, furthermore, substitution of Tyr-950 with Phe within the NPEY motif of the IGFIR eliminated interaction with both SHC and IRS-1 but had no effect upon p85 interaction. We show that residues 160-516 of IRS-1 and 1-238 of SHC are sufficient and necessary for receptor interaction in the yeast two-hybrid assay. We also demonstrate a direct in vitro interaction between the IGFIR and a fusion protein containing SHC amino acids 1-238. No interaction was observed with a SHC protein containing only the SH2 domain. We conclude that SHC and IRS-1 interact with the tyrosine-phosphorylated NPEY motif of the IGFIR, and that both proteins interact via related motifs located in their amino termini. We conclude that the interactions of SHC and IRS-1 with the IGFIR are similar to those which we have previously defined with the insulin receptor.
Footnotes
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↵* This work was supported by National Institutes of Health Grant DK44093, research grants from the American Diabetes Association and the Special Research Initiative Support from the University of Maryland (to T. A. G.), and by a training grant stipend from National Institutes of Health Grant GM08181 (to A. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵1 The abbreviations used are:
- IGFIR
-
insulin-like growth factor I receptor
- IR
-
insulin receptor
- IRS-1
-
insulin receptor substrate 1
- SHC
-
Src homology and collagen
- PI 3-kinase
-
phosphatidylinositol 3-kinase
- SH2
-
Src homology 2
- MBP
-
maltose-binding protein
- GST
-
glutathione S-transferase
- IL
-
interleukin
- X-Gal
-
5-bromo-4-chloro-3-indoyl β-D-galactoside.
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↵2 T. J. O'Neill and T. A. Gustafson, unpublished data.
