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Originally published In Press as doi:10.1074/jbc.M209521200 on January 10, 2003

J. Biol. Chem., Vol. 278, Issue 13, 11359-11368, March 28, 2003
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Insulin Receptor Substrate 1 Regulation of Sarco-endoplasmic Reticulum Calcium ATPase 3 in Insulin-secreting beta -Cells*

Prabhakar D. Borge Jr. and Bryan A. WolfDagger

From the Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia and University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

We have previously characterized an insulin receptor substrate 1 (IRS-1)-overexpressing beta -cell line. These beta -cells demonstrated elevated fractional insulin secretion and elevated cytosolic Ca2+ levels compared with wild-type and vector controls. This effect of IRS-1 may be mediated via an interaction with the sarco-endoplasmic reticulum calcium ATPase (SERCA). Here we demonstrate that IRS-1 and IRS-2 localize to an endoplasmic reticulum (ER)-enriched fraction in beta -cells using subcellular fractionation. We also observe co-localization of both IRS-1 and IRS-2 with ER marker proteins using immunofluorescent confocal microscopy. Furthermore, immuno-electron microscopy studies confirm that IRS-1 and SERCA3b localize to vesicles derived from the ER. In Chinese hamster ovary-T (CHO-T) cells transiently transfected with SERCA3b alone or together with IRS-1, SERCA3b co-immunoprecipitates with IRS-1. This interaction is enhanced with insulin treatment. SERCA3b also co-immunoprecipitates with IRS-1 in wild-type and IRS-1-overexpressing beta -cell lines. Ca2+ uptake in ER-enriched fractions prepared from wild-type and IRS-1-overexpressing cell lines shows no significant difference, indicating that the previously observed decrease in Ca2+ uptake by IRS-1-overexpressing cells is not the result of a defect in SERCA. Treatment of wild-type beta -cells with thapsigargin, an inhibitor of SERCA, resulted in an increase in glucose-stimulated fractional insulin secretion similar to that observed in IRS-1-overexpressing cells. The colocalization of IRS proteins and SERCA in the ER of beta -cells increases the likelihood that these proteins can interact with one another. Co-immunoprecipitation of IRS-1 and SERCA in CHO-T cells and beta -cells confirms that these proteins do indeed interact directly. Pharmacological inhibition of SERCA in beta -cells results in enhanced secretion of insulin. Taken together, our data suggest that interaction between IRS proteins and SERCA is an important regulatory step in insulin secretion.

* This work was supported by grants from the American Diabetes Association (to P. D. B. and B. A. W.) and by National Institutes of Health (NIH) Grant DK49814 (to B. A. W.). The Radioimmunoassay Core and Biomedical Imaging Core of the Penn Diabetes Center are supported by NIH Grant DK19525.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia, 5135 Main, 34th St. and Civic Center Blvd., Philadelphia, PA 19104-4399. Tel.: 215-590-2869; Fax: 215-590-1021; E-mail: wolfb@mail.med.upenn.edu.

Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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