EXPRESSION AND CHARACTERIZATION OF 2 BETA-ADRENERGIC-RECEPTOR KINASE ISOFORMS USING THE BACULOVIRUS EXPRESSION SYSTEM

The beta-adrenergic receptor kinases, betaARK1 and betaARK2, are two r ecently cloned members of the G protein-coupled receptor kinase family . To further characterize there kinases, bovine betaARK1 and betaARK2 have been overexpressed in Sf9 insect cells using the baculovirus expr ession system. High yields (5-7 mg/L cells) of purified kinase prepara tions were obtained by sequential chromatography of infected Sf9 cell supernatant fractions on S-Sepharose and Heparin-Sepharose. The expres sed kinases were fully active as evidenced by their ability to specifi cally phosphorylate the agonist-occupied beta2-adrenergic receptor (be ta2AR) and light-activated rhodopsin. Similar initial rates and maxima l stoichiometries of beta2AR phosphorylation were observed for both be taARK1 and beteARK2. Moreover, G protein betagamma subunits enhanced t he initial rates of both betaARK1 and betaARK2 mediated beta2AR phosph orylation by approximately tenfold. In the presence of betagamma subun its the maximal stoichiometry of beta2AR phosphorylation was increased from approximately 4 mol phosphate/mol receptor to approximately 10 m ol/mol. Detailed kinetic analysis of rhodopsin phosphorylation suggest s that both kinases follow a sequential mechanistic pathway and have s imilar K(m)s for rhodopsin (approximately 14 muM) and MgATP (60-90 muM ). Peptide phosphorylation studies demonstrate that both kinases prefe r 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 muM for betaARK1 and betaARK2, respectively. These studies demonstra te that betaARK1 and betaARK2 share very similar kinetic properties an d suggest that they may have a similar substrate specificity in vivo.