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J. Biol. Chem., Vol. 278, Issue 13, 11359-11368, March 28, 2003
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-Cells*
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
-cell line. These
-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
-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
-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
-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
-cells increases the likelihood that these proteins can interact with one another. Co-immunoprecipitation of IRS-1 and SERCA in CHO-T
cells and
-cells confirms that these proteins do indeed interact
directly. Pharmacological inhibition of SERCA in
-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.
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.
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