RGS3 Inhibits G Protein-Mediated Signaling via Translocation to the Membrane and Binding to Gα₁₁

Abstract

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 Gα₁₁ and to a lesser extent to Gα_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 Gα₁₁ (Gα₁₁-QL) resulted in the binding of RGS3, but not of its N-terminal fragment, to the membrane fraction and in its interaction with Gα₁₁-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.

ACKNOWLEDGMENTS

We thank Jean-Daniel Sraer for providing the human glomerular mesangial cell line, Abraham Rothman for providing the rat pulmonary arterial smooth muscle cell line, Hiroshi Itoh for providing α₁₁-WT and α₁₁-QL cDNA, and David J. Lacey for expert assistance in calcium measurements.

This work was supported by NIH grants HL 22563 and DK 41684 to M.J.D., a grant from the Milheim Foundation for Cancer Research to A.S., a grant-in-aid (96013570) from the National American Heart Association, and a grant from the Children’s Hospital of Wisconsin Foundation to S.J.E.