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Molecular and Cellular Biology, January 1999, p. 714-723, Vol. 19, No. 1
Department of Medicine and Cardiovascular
Research Center, Medical College of Wisconsin, Milwaukee, Wisconsin
53226,1 and
Laboratory of
Immunoregulation, National Institute of Allergy and Infectious
Diseases, National Institutes of Health, Bethesda, Maryland
20892-18762
Received 12 March 1998/Returned for modification 18 August
1998/Accepted 9 October 1998
In the present study, we investigated the function and the
mechanism of action of RGS3, a member of a family of proteins called regulators of G protein signaling (RGS). Polyclonal antibodies against
RGS3 were produced and characterized. An 80-kDa protein was identified
as RGS3 by immunoprecipitation and immunoblotting with anti-RGS3
antibodies in a human mesangial cell line (HMC) stably transfected with
RGS3 cDNA. Coimmunoprecipitation experiments in RGS3-overexpressing
cell lysates revealed that RGS3 bound to aluminum fluoride-activated
G
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Copyright © 1999, American Society for Microbiology. All rights reserved.
RGS3 Inhibits G Protein-Mediated Signaling via
Translocation to the Membrane and Binding to
G
11
11 and to a lesser extent to G
i3 and
that this binding was mediated by the RGS domain of RGS3. A role of
RGS3 in postreceptor signaling was demonstrated by decreased calcium
responses and mitogen-activated protein (MAP) kinase activity induced
by endothelin-1 in HMC stably overexpressing RGS3. Moreover, depletion
of endogenous RGS3 by transfection of antisense RGS3 cDNA in NIH 3T3
cells resulted in enhanced MAP kinase activation induced by
endothelin-1. The study of intracellular distribution of RGS3 indicated
its unique cytosolic localization. Activation of G proteins by
AlF4
, NaF, or endothelin-1 resulted in
redistribution of RGS3 from cytosol to the plasma membrane as
determined by Western blotting of the cytosolic and particulate
fractions with RGS3 antiserum as well as by immunofluorescence
microscopy. Agonist-induced translocation of RGS3 occurred by a dual
mechanism involving both C-terminal (RGS domain) and N-terminal
regions of RGS3. Thus, coexpression of RGS3 with a constitutively
active mutant of G
11 (G
11-QL) resulted in
the binding of RGS3, but not of its N-terminal fragment, to the
membrane fraction and in its interaction with G
11-QL in vitro without any stimuli. However, both full-length RGS3 and its
N-terminal domain translocated to the plasma membrane upon stimulation
of intact cells with endothelin-1 as assayed by immunofluorescence microscopy. The effect of endothelin-1 was also mimicked by calcium ionophore A23187, suggesting the importance of Ca2+ in the
mechanism of redistribution of RGS3. These data indicate that RGS3
inhibits G protein-coupled receptor signaling by a complex mechanism
involving its translocation to the membrane in addition to its
established function as a GTPase-activating protein.
*
Corresponding author. Mailing address: Department of
Medicine & Cardiovascular Research Center, Medical College of
Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226. Phone: (414)
456-8213. Fax: (414) 456-6560. E-mail: mdunn{at}mcw.edu.
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