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 betagamma-dimers (Gbetagamma). To investigate the physiological role of this phosphate transfer in cardiomyocytes, we generated a Gbeta1gamma2-dimer carrying a single amino acid exchange at the intermediately phosphorylated His-266 in the beta1 subunit (Gbeta1H266Lgamma2). Recombinantly expressed Gbeta1H266Lgamma2 were integrated into heterotrimeric G proteins in rat cardiomyocytes but were deficient in intermediate Gbeta phosphorylation. Compared with wild-type Gbeta1gamma2 (Gbeta1WTgamma2), overexpression of Gbeta1H266Lgamma2 suppressed basal cAMP formation up to 55%. A similar decrease in basal cAMP production occurred when the formation of NDPK B/Gbetagamma complexes was attenuated by siRNA-mediated NDPK B knockdown. In adult rat cardiomyocytes expressing Gbeta1H266Lgamma2, the basal contractility was suppressed by approximately 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 Gbeta1H266Lgamma2 than in Gbeta1WTgamma2 expressing cardiomyocytes. However, the relative isoproterenol-induced increased was not affected by Gbeta1H266Lgamma2. We conclude that the receptor-independent activation of G proteins via NDPK B/Gbetagamma complexes requires the intermediate phosphorylation of G protein beta subunits at His-266. Our results highlight the histidine kinase activity of NDPK B for Gbeta and demonstrate its contribution to the receptor-independent regulation of cAMP synthesis and contractility in intact cardiomyocytes.
Hippe
Hans-Joerg
HJ
Innere Medizin III-Kardiologie, Universität Heidelberg, Heidelberg, Germany. hans-joerg.hippe@med.uni-heidelberg.de
Luedde
Mark
M
Lutz
Susanne
S
Koehler
Henrike
H
Eschenhagen
Thomas
T
Frey
Norbert
N
Katus
Hugo A
HA
Wieland
Thomas
T
Niroomand
Feraydoon
F
D000818:Animals; D002461:Cell Line, Transformed; D000242:Cyclic AMP / Q000096:biosynthesis* / Q000235:genetics / Q000502:physiology; D019281:Dimerization; D020962:Heterotrimeric GTP-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D006801:Humans; D009200:Myocardial Contraction / Q000235:genetics / Q000502:physiology*; D032383:Myocytes, Cardiac / Q000378:metabolism*; D054778:NM23 Nucleoside Diphosphate Kinases; D009701:Nucleoside-Diphosphate Kinase / Q000096:biosynthesis* / Q000235:genetics / Q000502:physiology; D021122:Protein Subunits / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D051381:Rats
D054778:NM23 Nucleoside Diphosphate Kinases; D021122:Protein Subunits; D000242:Cyclic AMP; C517940:NME2 protein, human; D009701:Nucleoside-Diphosphate Kinase; D020962:Heterotrimeric GTP-Binding Proteins