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Molecular and Cellular Biology, January 1999, p. 714-723, Vol. 19, No. 1
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

RGS3 Inhibits G Protein-Mediated Signaling via Translocation to the Membrane and Binding to Galpha 11

Nickolai O. Dulin,1 Andrey Sorokin,1 Eleanor Reed,1 Stephen Elliott,1 John H. Kehrl,2 and Michael J. Dunn1,*

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 Galpha 11 and to a lesser extent to Galpha 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 Galpha 11 (Galpha 11-QL) resulted in the binding of RGS3, but not of its N-terminal fragment, to the membrane fraction and in its interaction with Galpha 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.

Molecular and Cellular Biology, January 1999, p. 714-723, Vol. 19, No. 1
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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