Expression and characterization of two beta-adrenergic receptor kinase isoforms using the baculovirus expression system.
The beta-adrenergic receptor kinases, beta ARK1 and beta ARK2, are two recently cloned members of the G protein-coupled receptor kinase family. To further characterize these kinases, bovine beta ARK1 and beta ARK2 have been overexpressed in Sf9 insect cells using the baculovirus expression system. High yields (5-7 mg/L cells) of purified kinase preparations were obtained by sequential chromatography of infected Sf9 cell supernatant fractions on S-Sepharose and Heparin-Sepharose. The expressed kinases were fully active as evidenced by their ability to specifically phosphorylate the agonist-occupied beta 2-adrenergic receptor (beta 2AR) and light-activated rhodopsin. Similar initial rates and maximal stoichiometries of beta 2AR phosphorylation were observed for both beta ARK1 and beta ARK2. Moreover, G protein beta gamma subunits enhanced the initial rates of both beta ARK1 and beta ARK2 mediated beta 2AR phosphorylation by approximately tenfold. In the presence of beta gamma subunits the maximal stoichiometry of beta 2AR phosphorylation was increased from approximately 4 mol phosphate/mol receptor to approximately 10 mol/mol. Detailed kinetic analysis of rhodopsin phosphorylation suggests that both kinases follow a sequential mechanistic pathway and have similar Kms for rhodopsin (approximately 14 microM) and MgATP (60-90 microM). Peptide phosphorylation studies demonstrate that both kinases prefer acidic amino acids amino terminal to a serine. Heparin was found to be the most potent inhibitor for both kinases with IC50s of 1.4 and 1.1 microM for beta ARK1 and beta ARK2, respectively. These studies demonstrate that beta ARK1 and beta ARK2 share very similar kinetic properties and suggest that they may have a similar substrate specificity in vivo.
Kim
C M
CM
Department of Pharmacology, Jefferson Cancer Institute, Thomas Jefferson University, Philadelphia, PA 19107.
Dion
S B
SB
Onorato
J J
JJ
Benovic
J L
JL
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002413:Cations, Divalent / Q000378:metabolism; D002417:Cattle; D003001:Cloning, Molecular; D017868:Cyclic AMP-Dependent Protein Kinases; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D007546:Isoquinolines / Q000494:pharmacology; D008969:Molecular Sequence Data; D009036:Moths / Q000166:cytology; D009842:Oligopeptides / Q000138:chemical synthesis / Q000378:metabolism; D010766:Phosphorylation; D010879:Piperazines / Q000494:pharmacology; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D011943:Receptors, Adrenergic, beta / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D012243:Rhodopsin / Q000378:metabolism; D051552:beta-Adrenergic Receptor Kinases
D002413:Cations, Divalent; D007527:Isoenzymes; D007546:Isoquinolines; D009842:Oligopeptides; D010879:Piperazines; D047428:Protein Kinase Inhibitors; D011943:Receptors, Adrenergic, beta; D011994:Recombinant Proteins; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D012243:Rhodopsin; D011494:Protein Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D051552:beta-Adrenergic Receptor Kinases