1. G Protein-coupled Receptor Kinase GRK2 Is a Phospholipid-dependent Enzyme That Can Be Conditionally Activated by G Protein βγ Subunits
- Author
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M. Marlene Hosey, Shubhik K. DebBurman, Jeffrey L. Benovic, and Judy Ptasienski
- Subjects
Phosphatidylinositol 4,5-Diphosphate ,Rhodopsin ,G-Protein-Coupled Receptor Kinase 3 ,G protein ,Chick Embryo ,Inositol 1,4,5-Trisphosphate ,Protein Serine-Threonine Kinases ,Biology ,Biochemistry ,Structure-Activity Relationship ,chemistry.chemical_compound ,Adenosine Triphosphate ,Cricetulus ,Phosphatidylinositol Phosphates ,GTP-Binding Proteins ,Cricetinae ,Animals ,Humans ,Phosphatidylinositol ,Phosphorylation ,Protein kinase A ,Molecular Biology ,Cells, Cultured ,G protein-coupled receptor ,Receptor, Muscarinic M2 ,G protein-coupled receptor kinase ,Kinase ,Myocardium ,Receptor Protein-Tyrosine Kinases ,Cell Biology ,Cyclic AMP-Dependent Protein Kinases ,Receptors, Muscarinic ,Cell biology ,Enzyme Activation ,Pleckstrin homology domain ,chemistry ,beta-Adrenergic Receptor Kinases ,lipids (amino acids, peptides, and proteins) - Abstract
G protein-coupled receptor kinases (GRKs) mediate agonist-dependent phosphorylation of G protein-coupled receptors (GPRs) and initiate homologous receptor desensitization. Previously, we reported that charged phospholipids directly interacted with the two GRK isoforms, GRK2 and GKR3, via a pleckstrin homology (PH) domain to regulate GRK activity (DebBurman, S. K., Ptasienski, J., Boetticher, E., Lomasney, J. W., Benovic, J. L., and Hosey, M. M. (1995) J. Biol. Chem. 270: 5742-5747). Here, evidence is provided to support the hypothesis that charged phospholipids are required for agonist-dependent phosphorylation of receptors by GRK2. In the absence of charged phospholipids, the purified human m2 muscarinic acetylcholine receptor (hm2mAChR) reconstituted in pure phosphatidylcholine vesicles or in a noninhibitory detergent was not a substrate for GRK2. However, these receptor preparations were stoichiometrically phosphorylated in an agonist-dependent manner upon addition of charged phospholipids. The known ability of G protein betagamma subunits to stimulate mAChR phosphorylation also was found to be absolutely dependent on the presence of charged phospholipids, including phosphatidylinositol 4,5-bisphosphate (PIP2). Phospholipids also regulated GRK-mediated phosphorylation of casein, a nonreceptor-soluble substrate. Among lipids tested, lipid inositol phosphates, PIP2 and phosphatidylinositol 4-monophosphate, were found to be the most potent activators of GRK2 and were the only lipids that regulated GRK2 in a complex biphasic manner. At low micro concentrations, PIP2 activated GRK2 via an interaction with the GRK pleckstrin homology domain; however, at high micro concentrations, PIP2 inhibited GRK2, apparently via another mechanism. PIP2-mediated inhibition could be partly relieved by increasing ATP. The results support the hypothesis that GRK2 is a lipid-dependent protein kinase that requires charged phospholipids for enzyme activation, for regulation by Gbetagamma subunits, and potentially for membrane association.
- Published
- 1996