Structural Determinants of G-protein α Subunit Selectivity by Regulator of G-protein Signaling 2 (RGS2)

Abstract

"Regulator of G-protein signaling" (RGS) proteins facilitate the termination of G protein-coupled receptor (GPCR) signaling via their ability to increase the intrinsic GTP hydrolysis rate of G␣ subunits (known as GTPase-accelerating protein or "GAP" activity). RGS2 is unique in its in vitro potency and selectivity as a GAP for G␣ q subunits. As many vasoconstrictive hormones signal via G q heterotrimer-coupled receptors, it is perhaps not surprising that RGS2-deficient mice exhibit constitutive hypertension. However, to date the particular structural features within RGS2 determining its selectivity for G␣ q over G␣ i/o substrates have not been completely characterized. Here, we examine a trio of point mutations to RGS2 that elicits G␣ i -directed binding and GAP activities without perturbing its association with G␣ q . Using x-ray crystallography, we determined a model of the triple mutant RGS2 in complex with a transition state mimetic form of G␣ i at 2.8-Å resolution. Structural comparison with unliganded, wild type RGS2 and of other RGS domain/G␣ complexes highlighted the roles of these residues in wild type RGS2 that weaken G␣ i subunit association. Moreover, these three amino acids are seen to be evolutionarily conserved among organisms with modern cardiovascular systems, suggesting that RGS2 arose from the R4-subfamily of RGS proteins to have specialized activity as a potent and selective G␣ q GAP that modulates cardiovascular function. G protein-coupled receptors (GPCRs)4 form an interface between extracellular and intracellular physiology, as they convert hormonal signals into changes in intracellular metabolism and ultimately cell phenotype and function (1-3). GPCRs are coupled to their underlying second messenger systems by heterotrimeric guanine nucleotide-binding protein ("G-proteins") composed of three subunits: G␣, G␤, and G␥. Four general classes of G␣ subunits have been defined based on functional couplings (in the GTP-bound state) to various effector proteins. G s subfamily G␣ subunits are stimulatory to membrane-bound adenylyl cyclases that generate the second messenger 3Ј,5Ј-cyclic adenosine monophosphate (cAMP); conversely, G i subfamily G␣ subunits are generally inhibitory to adenylyl cyclases (4). G 12/13 subfamily G␣ subunits activate the small G-protein RhoA through stimulation of the GEF subfamily of RGS proteins, namely p115-RhoGEF, LARG, and PDZ-RhoGEF (5). G q subfamily G␣ subunits are potent activators of phospholipase-C␤ enzymes that generate the second messengers diacylglycerol and inositol triphosphate (6); more recently, two additional G␣ q effector proteins have been described: the receptor kinase GRK2 and the RhoA nucleotide exchange factor p63RhoGEF (7,8).The duration of GPCR signaling is controlled by the time G␣ remains bound to GTP before its hydrolysis to GDP. RGS proteins are key modulators of GPCR signaling by virtue of their ability to accelerate the intrinsic GTP hydrolysis activity of G␣ subunits (reviewed in Refs. 9 and 10). RGS2/G0S8, one of the first mammalian RGS ...

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