Regulation Of Cardiac Camp Synthesis And Contractility By Nucleoside Diphosphate Kinase B/G Protein Beta Gamma Dimer Complexes

Heterotrimeric G proteins are pivotal regulators of myocardial contractility. In addition to the receptor-induced GDP/GTP exchange, G protein alpha subunits can be activated by a phosphate transfer via a plasma membrane-associated complex of nucleoside diphosphate kinase B (NDPK B) and G protein beta gamma-dimers (G beta gamma). To investigate the physiological role of this phosphate transfer in cardiomyocytes, we generated a G beta(1)gamma(2)-dimer carrying a single amino acid exchange at the intermediately phosphorylated His-266 in the beta(1) subunit (G beta(1)H266L gamma(2)). Recombinantly expressed G beta(1)H266L gamma(2) were integrated into heterotrimeric G proteins in rat cardiomyocytes but were deficient in intermediate G beta phosphorylation. Compared with wild-type G beta(1)gamma(2) (G beta 1WT gamma(2)), overexpression of G beta(1)H266L gamma(2) suppressed basal cAMP formation up to 55%. A similar decrease in basal cAMP production occurred when the formation of NDPK B/G beta gamma complexes was attenuated by siRNA-mediated NDPK B knockdown. In adult rat cardiomyocytes expressing G beta(1)H266L gamma(2), the basal contractility was suppressed by approximate to 50% which correlated to similarly reduced basal cAMP levels and reduced Ser16-phosphorylation of phospholamban. In the presence of the beta-adrenoceptor agonist isoproterenol, the total cAMP formation and contractility were significantly lower in G beta(1)H266L gamma(2) than in G beta 1WT gamma(2) expressing cardiomyocytes. However, the relative isoproterenol-induced increased was not affected by G beta(1)H266L gamma(2). We conclude that the receptor-independent activation of G proteins via NDPK B/G beta gamma complexes requires the intermediate phosphorylation of G protein beta subunits at His-266. Our results highlight the histidine kinase activity of NDPK B for G beta and demonstrate its contribution to the receptor-independent regulation of cAMP synthesis and contractility in intact cardiomyocytes.