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 Galpha11 and to a lesser extent to Galphai3 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 Galpha11 (Galpha11-QL) resulted in the binding of RGS3, but not of its N-terminal fragment, to the membrane fraction and in its interaction with Galpha11-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.

Cytoplasmic, Nuclear, and Golgi Localization of RGS Proteins

“…The mechanism of this recruitment does not involve interaction of RGS4 with mutant G i ␣ because similar recruitment was observed with an RGS4 mutant that does not interact with G i ␣, and previous studies have shown that RGS4 does not complex with GTPase-deficient G i ␣ 1 (7). Dulin et al (31) reported the presence of RGS3 immunoreactivity in endothelin-or A23187-induced membrane ruffles in HMG/RGS3 cells, although most RGS3 remained in the cytoplasm. Whether RGS3 localization in membrane ruffles is unique to these mesangial cell transfectants and/or important in the regulatory effects of RGS3 in these cells is unknown.…”

R9AP, a membrane anchor for the photoreceptor GTPase accelerating protein, RGS9-1

“…Translocation and membrane targeting of RGS proteins have been proposed as regulatory mechanisms for modulating the intensity and duration of responses to extracellular signals. RGS3 was reported to undergo translocation to plasma membranes after agonist induction in both G ␣ -dependent (binding with G ␣11 ) or independent (not direct binding with G ␣11 ) pathways (21). RGS4 is recruited to cell membranes after G ␣ activation by unknown factors (29).…”

“…Closely related proteins like RGS7 and the RNA processing variant RGS9-2, are found in the cytoplasm or nucleus (16)(17)(18), as well as on plasma membranes. Other RGS proteins in general show highly varied distribution patterns (19)(20)(21)(22)(23)(24)(25)(26). Variable subcellular targeting of RGS proteins may be a mechanism for regulation of their functions (18,(27)(28)(29).…”

Activation of NF-κB by Bradykinin through a Gαq- and Gβγ-dependent Pathway That Involves Phosphoinositide 3-Kinase and Akt

“…The G␤ 1 and G␥ 2 constructs were gifts from Dr. Tatyana Voyno-Yasenetskaya (University of Illinois, Chicago). Expression plasmids for RGS3 and RGS3T were kindly provided by Dr. Nickolai Dulin (University of Illinois, Chicago) and were described previously (27). The expression vectors of G␤␥ scavengers, bovine transducin, and T8␤ARK-myc with a ␤ARK carboxyl-terminal fragment were kindly provided by Dr. Heidi Hamm (Northwestern University, Chicago) and Dr. Sivio Gutkind (National Institutes of Health, Bethesda, MD), respectively.…”