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Circulation Research. 2006;98:585-586
doi: 10.1161/01.RES.0000215568.88246.b8
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(Circulation Research. 2006;98:585.)
© 2006 American Heart Association, Inc.

Editorials

Specificity and Diversity in Gi/o-Mediated Signaling

How the Heart Operates the RGS Brake Pedal

Thomas Wieland, Stefan Herzig

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 stefan.herzig{at}uni-koeln.de



See related article, pages 659–666


Key Words: RGS • G-protein • GPCR • heart rate

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{alpha}ß{gamma}), thereby promoting the dissociation of the heterotrimer into a free GTP-liganded G{alpha}-subunit and a Gß{gamma} dimer. Both G{alpha} and Gß{gamma}-dimer then regulate the activity of effectors, eg, second-messenger producing enzymes and ion channels. The duration of G protein activation is primarily controlled by the intrinsic GTPase activity of G{alpha}. On GTP hydrolysis, G{alpha} returns to the GDP-bound conformation and reassembles with the Gß{gamma} 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{alpha}, but they also serve as protein scaffolds.1,2 RGS proteins contribute to the complexity in Gi/o-mediated signaling. All RGS proteins share a 120-aa RGS homology domain, which contains the GTPase accelerating activity for the {alpha} 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 Gß{gamma}-regulated potassium channel (GIRK) in atrial myocytes via Ca2+/calmodulin and phosphatidylinositol 3,4,5-trisphosphate (PIP3).5,6 At rest, PIP3 inhibits RGS GAP activity, but during depolarisation Ca2+ entering through Ca2+-channels forms a Ca2+/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 (ß2, A1, and M2, respectively), PTX-sensitive Gi/o proteins (G{alpha}i2, G{alpha}i3, and different splice variants of G{alpha}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 (ß1-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 Gß{gamma} 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 ß- and {alpha}2{delta}-subunits) depend on inhibition of cAMP production (with direct or PKA-mediated effects, respectively) through G{alpha}i2/3 and G{alpha}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{alpha}o or G{alpha}i2. Using a knock-in of such an RGS-insensitive G{alpha}i2 mutant, they demonstrate a major sensitization of rate regulation by muscarinic agonist on elimination of RGS protein control of G{alpha}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{alpha}i2–Gß{gamma}–GIRK. Similarly, the authors show involvement of RGS proteins in preferential A1–G{alpha}o coupling, and ß2–G{alpha}i2 (>G{alpha}o) coupling. Because pharmacological blockers remain imperfect tools,16 the ion channel species encountered have not been directly identified in this study. However, the strength of the current approach lies in the ingenious method, which preserves the natural arrangement and stoichiometry of signaling proteins, and can be scrutinized from molecular to in vivo levels. The concentration dependence of agonist effects confirms that under physiological conditions, there is no exclusive coupling through specific Gi/o proteins. However, in the classical case of muscarinic control of inward rectifier potassium channels, the role of RGS proteins deserves as much attention as the discoveries of nondissociation of activated G{alpha}ß{gamma},17 or coregulation of GIRK by G{alpha}.18,19

In a next step, thorough understanding of RGS protein function may be achieved by double-mutant approaches, specifically rescuing one pair of RGS protein and G{alpha} subunit species. Indeed, such an approach has been reported for the interactions of RGS16 and RGS4 with G{alpha}i1 and G{alpha}q, respectively.20 A highly conserved Glu residue was replaced by Lys on the RGS protein. The complementary Lys residue on the G{alpha} subunit which forms an interacting salt bridge at the RGS–G{alpha} interaction surface was changed to a Glu, producing RGS and G{alpha} mutants which exhibit significantly reduced interactions with their "natural" counterparts. Both mutants, however, form a fully functional RGS protein–G{alpha} subunit pair as proven by in vitro GAP activity and functional inhibition at the cellular level. By applying such mutant pairs to the system described by Fu et al,9 divergent functions of apparently redundant G{alpha} isoforms21,22 and RGS proteins23,24 could be addressed at a new level of scientific rigor and physiological significance. Embryonic stem cell–derived cardiocytes hold promise as an excellent system for use of the molecular-genetic toolbox in modern cell biology and electrophysiology, provided that their developmental biology, eg, of signal transduction,25,26 is taken into account. Ultimately, the creation of transgenic animals, as done by Fu et al,9 and the possibility of crossing of such transgenic animals will provide fertile ground for novel and unsuspected insights into signaling specificity, with possible therapeutic relevance beyond cardiovascular diseases.


*    Acknowledgments
 
The Authors’ work is supported by the Deutsche Forschungsgemeinschaft (He 1578 13-1 to S.H., Wi 1373 9-1 and SFB-TR 23 TP B6 to T.W.), the Faculty of Clinical Medicine Mannheim, and the Center for Molecular Medicine Cologne (CMMC A5 to S.H.).


*    Footnotes
 
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.


*    References
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Related Article:

Endogenous RGS Proteins and G{alpha} Subtypes Differentially Control Muscarinic and Adenosine-Mediated Chronotropic Effects
Ying Fu, Xinyan Huang, Huailing Zhong, Richard M. Mortensen, Louis G. D’Alecy, and Richard R. Neubig
Circ. Res. 2006 98: 659-666. [Abstract] [Full Text] [PDF]




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