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 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. beta ARK activation 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, GRK3, and GRK5 have also been described. Using a mixed micelle assay, GRK2 (beta ARK1) was found to require phospholipid in order to phosphorylate the beta 2-adrenergic receptor. As determined with a nonreceptor peptide substrate of beta ARK, catalytic activity of the kinase increased in the presence of phospholipid without a change in the Km for the peptide. Data obtained with the heterobifunctional cross-linking agent N-3-[125I]iodo-4-azidophenylpropionamido-S-(2-thiopyridyl)-c ysteine ([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.
Onorato
J J
JJ
Department of Medicine, University of Wisconsin, Madison 53706, USA.
Gillis
M E
ME
Liu
Y
Y
Benovic
J L
JL
Ruoho
A E
AE
D000595:Amino Acid Sequence; D000818:Animals; D001386:Azides; D016367:Baculoviridae / Q000235:genetics; D002384:Catalysis; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D003001:Cloning, Molecular; D006224:Cricetinae; D003432:Cross-Linking Reagents; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D007457:Iodine Radioisotopes; D008969:Molecular Sequence Data; D010743:Phospholipids / Q000378:metabolism*; D011487:Protein Conformation; D018411:Spodoptera; D013379:Substrate Specificity; D051552:beta-Adrenergic Receptor Kinases
D001386:Azides; D003432:Cross-Linking Reagents; D007457:Iodine Radioisotopes; D010743:Phospholipids; C057771:N-(3-iodo-4-azidophenylpropionamido)-S-(2-thiopyridyl)cysteine; D017868:Cyclic AMP-Dependent Protein Kinases; D051552:beta-Adrenergic Receptor Kinases