doi: 10.1161/01.RES.0000266971.15127.e8
© 2007 American Heart Association, Inc.
Editorials |
G Proteins
More Than Transducers of Receptor-Generated Signals?
From the Rudolf Virchow Center, DFG Research Center for Experimental Biomedicine, University of Wuerzburg, Germany.
Correspondence to Stefan Engelhardt, MD PhD, Rudolf Virchow Center, DFG Research Center for Experimental Biomedicine, University of Wuerzburg, Versbacher Strasse 9, 97078 Wuerzburg, Germany. E-mail stefan.engelhardt{at}virchow.uni-wuerzburg.de
See related article, pages 1191–1199
Key Words: nucleoside diphosphate kinases • G protein signaling • cAMP • ß-adrenergic receptor
Signaling through the activation of G proteins represents the most widely used signaling pathway in mammalian biology.1,2 Classically, a transmembrane receptor comprising seven transmembrane domains (G protein-coupled receptor, GPCR) is activated by an extracellular stimulus and transduces this information to heterotrimeric G proteins through a conformational change of the receptor protein. Throughout evolution, a large variety of GPCRs has evolved to detect a wide spectrum of signals ranging from photons and odorants to endogenous neurotransmitters and hormones such as the catecholamines. Consequently, the majority of currently used drugs act on GPCRs. On receptor-mediated activation of the G protein 
-subunit, the bound GDP is exchanged against GTP and both the GTP-bound -subunit as well as the ß
-subunits may activate downstream targets (Figure). As a GTPase, the -subunit then rapidly initiates its own inactivation through GTP-hydrolysis. This GTPase cycle of G protein activation and deactivation is subject to regulation by the RGS family of proteins (regulators of G protein signaling) that activate the GTPase function and thereby negatively regulate signaling of GPCRs.3,4
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Although the paradigm of G protein activation through activated GPCRs is generally accepted, there have been several reports, that G proteins can be activated independently from GPCR-activation. Lanier and coworkers have described a class of G protein interacting proteins, which they termed activators of G protein signaling (AGS-proteins5). AGS-proteins directly interact with G protein -subunits and ß dimers and are thereby capable to initiate G protein dependent signaling independent of GPCR-activation.
In addition, several reports have implied nucleoside-diphosphate kinase (NDPK) as a direct activator of G protein signaling. Originally believed to act solely as a source for GTP formed from GDP and ATP, NDPK increasingly appears to act as a highly integrated signaling molecule.6,7 Early reports described G protein activation through NDPK through a yet undefined mechanism and described a complex of NDPK and the stimulatory G protein (Gs).8 Subsequently it was shown, that the B isoform of NDPK acts as a histidine kinase that phosphorylates Gß at His266.9 This high energetic phosphate permits phosphate transfer onto GDP to form GTP in a local compartment in close vicinity to G subunits and thus mediates local activation of G proteins.
In this issue of Circulation Research, Hippe and colleagues now demonstrate, that this unique mechanism of G protein activation occurs in the mammalian heart and that it significantly contributes to the basal formation of cAMP.10 On expression of a mutant ß-complex specifically lacking His266 (and thus NDPK-mediated G activation), basal cAMP formation fell to less than 50%. A similar decrease of basal cAMP formation was observed on knockdown of NDPK B. Interestingly, ß-adrenergic receptor mediated cAMP formation and contractility were unaffected by disruption of NDPK-mediated Gß-phosphorylation. Thus NDPK-mediated G activation must be regarded as a separate pathway of G protein activation (Figure).
NDPKs are ubiquitious and constitutively expressed enzymes that provide the formation of nucleoside triphosphates. Despite their early discovery and biochemical characterization more than 40 years ago, NDPKs have long been thought to act solely through the transfer of a -phosphate group onto nucleoside-5-diphosphates by a mechanism involving the formation of a high energy phosphate intermediate on a histidine residue. However, evidence is accumulating, that NDPKs may act in addition as protein kinases. This has been recently demonstrated for a calcium regulated potassium channel KCa1.3.11 NDPK-mediated histidine phosphorylation was shown to be required for KCa1.3-activation, which is expressed in T cells and blood vessels.12 Another target of NDPK with likely cardiovascular relevance is AMP-kinase.13 NDPK mediated Gß-phosphorylation involves aspects of both the protein kinase and the nucleotide kinase mode of NDPK activity. On the one hand, Gß may serve as a simple intermediate storing station for high energetic phosphate transferred on to GDP. On the other hand, histidine phosphorylation of Gß may alter Gß-function in a yet to be determined way. Direct evidence for such a mode of action is still missing, but the close vicinity of the His266-residue to the interaction plane on Gß that interacts both with the G-subunit as well as with established downstream effectors of ß-subunits14 renders this possibility not unlikely. ß-Subunits regulate effector enzymes like PI3K, ACII, PLCß and G protein coupled receptor kinases 2 and 3 (GRKs). Increasing evidence points toward ß-dependent signals as critical regulators of cardiac pathology, namely myocardial remodelling. Both cardiomyocyte-specific inhibition of ß-dependent signaling with a C-terminal GRK2-fragment15 as well as GRK-independent scavenging of ß-subunits16 inhibited myocardial remodeling and the development of cardiac failure in experimental animal models. It will be crucial to assess how ß-dependent signaling pathways react on manipulation of NDPK-activity.
Given the central role of NDPKs in mammalian biology, many fundamental characteristics of the enzyme family are still unknown to date. Whether and how NDPK activity is regulated is largely unclear at the moment. Some recent publications indicate that intracellular translocation and complex formations may play an important role.13,17 Taking into account that the intermediately phosphorylated histidine residues on Gß but not those on NDPK B are substrates for a recently identified phosphohistidine phosphatase,18 it will be interesting to see, whether the nucleotide kinase and the histidine kinase functions are subject to individual regulation. Similarly, the downstream effectors of NDPKs, that have been identified likely represent only a small fraction of the entire picture (Figure).
Although the receptor-induced regulation of cAMP synthesis has been intensively studied, comparably little is know about the regulation of basal cAMP formation. Hippe and colleagues provide the first evidence that NDPK B-dependent activation of Gs contributes to the formation of basal cAMP in primary cardiac myocytes. In contrast, the ß-adrenergic receptor induced cAMP formation is not altered on disruption of NDPK mediated Gß phosphorylation. Does this proof independence of GPCR induced G protein activation? These experiments were performed in the presence of propranolol, effectively ruling out signaling through spontaneous activity of ß-adrenergic receptors. However, other Gs protein coupled GPCRs may still contribute to basal cAMP formation in cardiac myocytes. This would be plausible also with regard to the fact, that spontaneous activation and G protein coupling of GPCRs have been suggested to involve different modes of interaction than agonist-induced G protein activation.19 Studying the effect of NDPK mediated Gß phosphorylation in a cellular system with pronounced spontaneous activity of a Gs coupled GPCR may yield further insight in this respect.
In addition to spontaneously active GPCRs, basal cAMP may be derived from the spontaneous activity of G proteins as well as that of adenylyl cyclases. The work of Hippe et al indicates, that a major contribution to basal cAMP formation in cardiac myocytes originates upstream from adenylyl cyclase, likely with a significant contribution of Gs. In this respect, a direct comparison of the extent of cAMP-reduction through NDPK-inhibition to that achieved by inverse agonists at the ß-adrenergic receptors will be of interest.
The work of Hippe et al gains additional importance through the upregulation of NDPK, that has been observed in the membrane fraction of failing myocardium.17 Although total NDPK-levels appear to be unchanged, there is a relative enrichment of NDPK in the membrane fraction of failing myocardium. The mechanism underlying this translocation of NDPK is still unknown. However, the elevation of the membrane-associated NDPK was partially prevented in patients with congestive heart failure which have been treated with ß-adrenergic receptor antagonists. In agreement with these data, the progression of cardiac hypertrophy induced by chronic ß-adrenergic receptor stimulation was paralleled by the increase in membrane-associated NDPK.17 Consistent with recent reports showing NDPK translocation by stimulation of GPCRs,20,21 these data suggest that sustained stimulation of ß-adrenergic receptors increases association of NDPK with the plasma membrane. It is tempting to speculate, that this leads to enhanced formation of basal cAMP and thereby contributes to progression of the disease. This exciting question ultimately needs to be addressed in a relevant in vivo model, possibly through the use of Gß mutants or suppression of NDPK expression.
Taken together, the findings of Hippe et al add another facet to the complex function of G protein ß-subunits in cardiomyocytes. NDPK-mediated His266-phosphorylation of Gß appears to localize GTP-supply to G-subunits and thereby contributes to G protein activity independent from GPCR-activation.
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Acknowledgments |
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Sources of Funding
S.E. is supported by the Rudolf Virchow Center/DFG Research Center for Experimental Biomedicine supported by the Deutsche Forschungsgemeinschaft, Trigen, Sanofi-Aventis and the Bavarian Ministry of Economics. We would like to thank the Bundesministerium für Bildung und Forschung-Heart Failure Network (TP7) and the Interdisciplinary Center for Clinical Research, Wuerzburg for generous support. F.R. was supported by a fellowship of the Bayerische Forschungsstiftung.
Disclosures
None.
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Footnotes |
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The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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Related Article:
Dimer Complexes
Circ. Res. 2007 100: 1191-1199.
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