The β-adrenergic receptor kinase (GRK2) is regulated by phospholipids

Abstract

The β-adrenergic receptor kinase (βARK) is a member of growing family of G protein coupled receptor kinases (GRKs). βARK and other members of the GRK family play a role in the mechanism of agonist-specific desensitization by virtue of their ability to phosphorylate G protein-coupled receptors in an agonist-dependent manner. βARK activation is known to occur following the interaction of the kinase with the agonist-occupied form of the receptor substrate and heterotrimeric G protein βγ subunits. Recently, lipid regulation of GRK2, GRK3, and GRK5 have also been described. Using a mixed micelle assay, GRK2 (βARK1) was found to require phospholipid in order to phosphorylate the β2-adrenergic receptor. As determined with a nonreceptor peptide substrate of βARK, catalytic activity of the kinase increased in the presence of phospholipid without a change in the K(m) for the peptide. Data obtained with the heterobifunctional cross-linking agent N-3-[125I]iodo- 4-azidophenylpropionamido-S-(2-thiopyridyl)-cysteine ([125I]ACTP) suggests that the activation by phospholipid was associated with a conformational change in the kinase. [125I]ACTP incorporation increased 2-fold in the presence of crude phosphatidylcholine, and this increase in [125I]ACTP labeling is completely blocked by the addition 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 demonstrated using the mixed micelle phosphorylation assay, a notable exception was observed with PIP2. In the presence of PIP2, kinase activity as well as [125I]ACTP labeling was inhibited. These data demonstrate the direct regulation of GRK2 activity by phospholipids and supports the hypothesis that this effect is the result of a conformational change within the kinase.