title: Lipid-mediated regulation of G protein-coupled receptor kinases 2 and 3.
abstract: G protein-coupled receptor-mediated signaling is attenuated by a process referred to as desensitization, wherein agonist-dependent phosphorylation of receptors by G protein-coupled receptor kinases (GRKs) is proposed to be a key initial event. However, mechanisms that activate GRKs are not fully understood. In one scenario, beta gamma-subunits of G proteins (G beta gamma) activate certain GRKs (beta-adrenergic receptor kinases 1 and 2, or GRK2 and GRK3), via a pleckstrin homology domain in the COOH terminus. This interaction has been proposed to translocate cytosolic beta-adrenergic receptor kinases (beta ARKs) to the plasma membrane and facilitate interaction with receptor substrates. Here, we report a novel finding that membrane lipids modulate beta ARK activity in vitro in a manner that is analogous and competitive with G beta gamma. Several lipids, including phosphatidylserine (PS), stimulated, whereas phosphatidylinositol 4,5-bisphosphate inhibited, the ability of these GRKs to phosphorylate agonist-occupied m2 muscarinic acetylcholine receptors. Furthermore, both PS and phosphatidylinositol 4,5-bisphosphate specifically bound to beta ARK1, whereas phosphatidylcholine, a lipid that did not modulate beta ARK activity, did not bind to beta ARK1. The lipid regulation of beta ARKs did not occur via a modulation of its autophosphorylation state. PS- and G beta gamma-mediated stimulation of beta ARK1 was compared and found strikingly similar; moreover, their effects together were not additive (except at initial stages of reaction), which suggests that PS and G beta gamma employed a common interaction and activation mechanism with the kinase. The effects of these lipids were prevented by two well known G beta gamma-binding proteins, phosducin and GST-beta ARK-(466-689) fusion protein, suggesting that the G beta gamma-binding domain (possibly the pleckstrin homology domain) of the GRKs is also a site for lipid:protein interaction. We submit the intriguing possibility that both lipids and G proteins co-regulate the function of GRKs.
authors.affiliation: Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, Chicago, Illinois 60611.
authors.forename: S K
authors.initials: SK
authors.lastname: DebBurman
authors.forename: J
authors.initials: J
authors.lastname: Ptasienski
authors.forename: E
authors.initials: E
authors.lastname: Boetticher
authors.forename: J W
authors.initials: JW
authors.lastname: Lomasney
authors.forename: J L
authors.initials: JL
authors.lastname: Benovic
authors.forename: M M
authors.initials: MM
authors.lastname: Hosey
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000096:biosynthesis / Q000378:metabolism*; D004789:Enzyme Activation; D054770:G-Protein-Coupled Receptor Kinase 3; D019204:GTP-Binding Proteins / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D010743:Phospholipids / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D011976:Receptors, Muscarinic / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera; D014162:Transfection; D051552:beta-Adrenergic Receptor Kinases
chemical_list: D046911:Macromolecular Substances; D010743:Phospholipids; D011976:Receptors, Muscarinic; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D020794:Receptor Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D054770:G-Protein-Coupled Receptor Kinase 3; C517848:GRK3 protein, human; D051552:beta-Adrenergic Receptor Kinases; D019204:GTP-Binding Proteins