title: Chemical genetic engineering of G protein-coupled receptor kinase 2.
abstract: G protein-coupled receptor kinases (GRKs) play a pivotal role in receptor regulation. Efforts to study the acute effects of GRKs in intact cells have been limited by a lack of specific inhibitors. In the present study we have developed an engineered version of GRK2 that is specifically and reversibly inhibited by the substituted nucleotide analog 1-naphthyl-PP1 (1Na-PP1), and we explored GRK2 function in regulated internalization of the mu-opioid receptor (muOR). A previously described method that conferred analog sensitivity on various kinases, by introducing a space-creating mutation in the conserved active site, failed when applied to GRK2 because the corresponding mutation (L271G) rendered the mutant kinase (GRK2-as1) catalytically inactive. A sequence homology-based approach was used to design second-site suppressor mutations. A C221V second-site mutation produced a mutant kinase (GRK2-as5) with full functional activity and analog sensitivity as compared with wild-type GRK2 in vitro and in intact cells. The role of GRK2-as5 activity in the membrane trafficking of the muOR was also characterized. Morphine-induced internalization was completely blocked when GRK2-as5 activity was inhibited before morphine application. However, inhibition of GRK2-as5 during recycling and reinternalization of the muOR did not attenuate these processes. These results suggest there is a difference in the GRK requirement for initial ligand-induced internalization of a G protein-coupled receptor compared with subsequent rounds of reinternalization.
authors.affiliation: Chemistry and Chemical Biology Graduate Program, Department of Psychiatry, University of California, San Francisco, California 94143, USA.
authors.forename: Denise M
authors.initials: DM
authors.lastname: Kenski
authors.forename: Chao
authors.initials: C
authors.lastname: Zhang
authors.forename: Mark
authors.initials: M
authors.lastname: von Zastrow
authors.forename: Kevan M
authors.initials: KM
authors.lastname: Shokat
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D000939:Epitopes / Q000737:chemistry; D005434:Flow Cytometry; D054769:G-Protein-Coupled Receptor Kinase 2; D005818:Genetic Engineering; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009020:Morphine / Q000494:pharmacology; D016296:Mutagenesis; D009154:Mutation; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D017450:Receptors, Opioid, mu / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D013997:Time Factors; D014162:Transfection; D014404:Tubulin / Q000737:chemistry; D051552:beta-Adrenergic Receptor Kinases / Q000235:genetics*
chemical_list: D000939:Epitopes; D008024:Ligands; D017450:Receptors, Opioid, mu; D011994:Recombinant Proteins; D014404:Tubulin; D009020:Morphine; D000255:Adenosine Triphosphate; C496567:GRK2 protein, human; D051552:beta-Adrenergic Receptor Kinases; D054769:G-Protein-Coupled Receptor Kinase 2