Mechanism of beta-adrenergic receptor kinase activation by G proteins.
The beta-adrenergic receptor kinase (beta-ARK) specifically phosphorylates the activated form of various G protein-coupled receptors such as the beta 2-adrenergic receptor (beta 2-AR). Recently, G protein beta gamma subunits have been demonstrated to activate beta-ARK-mediated receptor phosphorylation. To further elucidate beta-ARK/G protein interactions, we have developed a direct binding assay. The direct binding of [35S]methionine-labeled beta-ARK to either brain Gi/Go or beta gamma subunits was rapid and saturable with similar Kd values of approximately 58 and approximately 32 nM, respectively. Both heterotrimeric G proteins and beta gamma subunits enhanced the initial rate of beta 2-AR and rhodopsin phosphorylation approximately 10-fold. Kinetic studies demonstrate that beta gamma enhances beta-ARK-mediated beta 2-AR phosphorylation both by decreasing the Km for the beta 2-AR approximately 4-fold and increasing the stoichiometry of phosphorylation from approximately 4 to approximately 11 mol/mol. An agonist- and ATP-dependent binding of beta-ARK to the reconstituted beta 2-AR was also demonstrated. In addition, beta-ARK binding was enhanced in the presence of both the activated beta 2-AR and beta gamma subunits suggesting the formation of a transient ternary complex consisting of beta-ARK, beta gamma, and beta 2-AR. Overall, these studies suggest that the specific association of beta-ARK with heterotrimeric G proteins may play an important role in promoting receptor/kinase interaction and subsequent receptor phosphorylation.
Department of Pharmacology, Jefferson Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.
C M
CM
Kim
S B
SB
Dion
J L
JL
Benovic
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000502:physiology*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009036:Moths; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D011943:Receptors, Adrenergic, beta / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D051552:beta-Adrenergic Receptor Kinases
D011943:Receptors, Adrenergic, beta; D011994:Recombinant Proteins; D011494:Protein Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D051552:beta-Adrenergic Receptor Kinases; D019204:GTP-Binding Proteins