THE BETA-ADRENERGIC-RECEPTOR KINASE (GRK2) IS REGULATED BY PHOSPHOLIPIDS

The beta-adrenergic receptor kinase (beta ARK) is a member of growing family of G protein coupled receptor kinases (GRKs). beta ARK and othe r members of the GRK family play a role in the mechanism of agonist-sp ecific desensitization by virtue of their ability to phosphorylate G p rotein-coupled receptors in an agonist-dependent manner. beta ARK acti vation is known to occur following the interaction of the kinase with the agonist-occupied form of the receptor substrate and heterotrimeric G protein beta gamma subunits. Recently, lipid regulation of GRK2, GR K3, and GRK5 have also been described. Using a mixed micelle assay, GR K2 (beta ARK1) was found to require phospholipid in order to phosphory late the beta(2)-adrenergic receptor. As determined with a nonreceptor peptide substrate of beta ARK, catalytic activity of the kinase incre ased in the presence of phospholipid without a change in the K-m for t he peptide. Data obtained with the heterobifunctional cross-linking ag ent N-3-[I-125] azidophenylpropionamido-S-(2-thiopyridyl)-cysteine ([I -125]ACTP) suggests that the activation by phospholipid was associated with a conformational change in the kinase. [I-125]ACTP incorporation increased 2-fold in the presence of crude phosphatidylcholine, and th is increase in [I-125]ACTP labeling is completely blocked by the addit ion of MgATP. Furthermore, proteolytic mapping was consistent with the modification of a distinct site when GRK2 was labeled in the presence of phospholipid. While an acidic phospholipid specificity was demonst rated using the mixed micelle phosphorylation assay, a notable excepti on was observed with PIP2. In the presence of PIP2, kinase activity as well as [(125)]ACTP labeling was inhibited. These data demonstrate th e direct regulation of GRK2 activity by phospholipids and supports the hypothesis that this effect is the result of a conformational change within the kinase.