Specificity and diversity in Gi/o-mediated signaling: how the heart operates the RGS brake pedal.

G-Protein–coupled receptors (GPCRs) are involved in the regulation of virtually every physiological process. These receptors operate by catalyzing the GDP/GTP exchange at a coupled heterotrimeric G protein (G ), thereby promoting the dissociation of the heterotrimer into a free GTP-liganded G -subunit and a G dimer. Both G and G -dimer then regulate the activity of effectors, eg, secondmessenger producing enzymes and ion channels. The duration of G protein activation is primarily controlled by the intrinsic GTPase activity of G . On GTP hydrolysis, G returns to the GDP-bound conformation and reassembles with the G dimer. More than 100 different GPCRs have been detected in cardiovascular cells, some of which are coupled to members of the pertussis-toxin (PTX)-sensitive Gi/o subfamily of heterotrimeric G proteins. An intense focus of investigation has been the mechanism(s) by which such a wide array of specific signals can be channeled through a very limited number of multifunctional G protein subunits, and yet retain specificity when reaching their ultimate molecular targets, such as enzymes or ion channels. In recent years, such “preferential coupling” has been attributed to spatially restricted signaling complexes, formed in lipid rafts and caveolae, and held together by anchoring or scaffolding proteins. Within this context, regulators of G protein signaling (RGS) proteins are of particular interest. RGS proteins were first identified as GTPase Activating Proteins (GAPs) which speed up GTP hydrolysis of G , but they also serve as protein scaffolds.1,2 RGS proteins contribute to the complexity in Gi/omediated signaling. All RGS proteins share a 120-aa RGS homology domain, which contains the GTPase accelerating activity for the subunits. The majority of RGS proteins terminate activation of Gi/o and Gq/11 proteins and are therefore often called “promiscuous.”1,2 This, together with the observed coexpression of several functionally equivalent RGS protein species within the same cell,3 suggests the possibility that RGS proteins may exhibit specificity in other contexts. RGS proteins do not interact exclusively with G proteins but can mediate their effects at other levels within signaling pathways: (1) Certain GPCRs, including cardiovascular GPCRs,1,2 are preferentially regulated by RGS proteins via protein–protein interactions, possibly requiring additional scaffold proteins.4 (2) RGS proteins regulate the activity of a G -regulated potassium channel (GIRK) in atrial myocytes via Ca /calmodulin and phosphatidylinositol 3,4,5-trisphosphate (PIP3). At rest, PIP3 inhibits RGS GAP activity, but during depolarisation Ca entering through Ca -channels forms a Ca /Calmodulin (CaM) complex which relieves the PIP3-mediated inhibition and allows the RGS protein to accelerate GTP hydrolysis. The reassociation of the G protein heterotrimer then terminates the activation of the GIRK channel. The interaction of RGS proteins with CaM is a spatial restricted phenomenon as it requires intact lipid rafts.7 (3) As shown for RGS2, RGS proteins can directly bind and thereby inhibit the activity of adenylyl cyclases type V, which is abundant in the heart.8 In summary, RGS proteins can affect preferential coupling in a number of different ways, interacting with GPCRs, G-proteins, accessory proteins, and effectors. In this context, the study of Fu et al9 focused on the negative chronotropic effects of some classical autacoids (adrenaline, adenosine, acetylcholine [ACh]). Autacoid effects are mediated through receptors ( 2, A1, and M2, respectively), PTX-sensitive Gi/o proteins (G i2, G i3, and different splice variants of G o), and a set of ion channels (IK,ACh, If, and ICa,L). Each of these channels can affect the spontaneous rate of depolarization, depending on the region of the heart, developmental or disease state, and level of sympathetic ( 1-adrenoceptor) tone. The ACh-activated inward rectifier potassium current IK,ACh is carried by G protein–regulated potassium channels (GIRK, a tetramer consisting of Kir3.1 and Kir3.4 subunits). For opening, it requires G dimers released from activated Gi/Go proteins.7 Inhibition of cardiac If (HCN1,2 and 4 tetramers, see reference 10), and of ICa,L (a heterotrimer of a Cav1.2 pore and accessory and 2 -subunits) depend on inhibition of cAMP production (with direct or PKA-mediated effects, respectively) through G i2/3 and G o, as revealed by their PTX-sensitivity and, more specifically, in knockout mice.11–13 Given this functional diversity of bradycardic signals and mechanisms, what is the role of RGS proteins? Instead of laboriously creating knockout mice of the many, and likely functionally redundant, RGS proteins (note that despite 10 years of RGS protein research only 2 knockout mice14,15 with specific phenotypes, RGS2 / and RGS9 / , have been published), Fu et al9 took advantage of a very simple molecular switch that specifically prevents any RGS GAP effect on specific mutants of G o or G i2. Using a knock-in of such an RGS-insensitive G i2 mutant, they demonstrate a major sensitization of rate regulation by muscarinic agonist on elimination of RGS protein control of G i2, both in stem cell–derived cardiocytes and living mice. Of note, this sensitization is entirely abolished by a blocker of GIRK, arguing for an RGS-sensitive preferential coupling chain of M2–G i2–G –GIRK. Similarly, the authors show involveThe opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association. From the Department of Experimental and Clinical Pharmacology and Toxicology (T.W.), University of Heidelberg, Mannheim, and the Department of Pharmacology (S.H.), University of Cologne, Germany. Correspondence to Stefan Herzig, Department of Pharmacology, University of Cologne, Gleueler Strasse 24, 50931 Koeln, Germany. E-mail [email protected] (Circ Res. 2006;98:585-586.) © 2006 American Heart Association, Inc.