title: Role of G Protein-Coupled Receptor Kinases 2 and 3 in μ-Opioid Receptor Desensitization and Internalization.
abstract: There is ongoing debate about the role of G protein-coupled receptor kinases (GRKs) in agonist-induced desensitization of the μ-opioid receptor (MOPr) in brain neurons. In the present paper, we have used a novel membrane-permeable, small-molecule inhibitor of GRK2 and GRK3, Takeda compound 101 (Cmpd101; 3-[[[4-methyl-5-(4-pyridyl)-4H-1,2,4-triazole-3-yl] methyl] amino]-N-[2-(trifuoromethyl) benzyl] benzamidehydrochloride), to study the involvement of GRK2/3 in acute agonist-induced MOPr desensitization. We observed that Cmpd101 inhibits the desensitization of the G protein-activated inwardly-rectifying potassium current evoked by receptor-saturating concentrations of methionine-enkephalin (Met-Enk), [d-Ala(2), N-MePhe(4), Gly-ol(5)]-enkephalin (DAMGO), endomorphin-2, and morphine in rat and mouse locus coeruleus (LC) neurons. In LC neurons from GRK3 knockout mice, Met-Enk-induced desensitization was unaffected, implying a role for GRK2 in MOPr desensitization. Quantitative analysis of the loss of functional MOPrs following acute agonist exposure revealed that Cmpd101 only partially reversed MOPr desensitization. Inhibition of extracellular signal-regulated kinase 1/2, protein kinase C, c-Jun N-terminal kinase, or GRK5 did not inhibit the Cmpd101-insensitive component of desensitization. In HEK 293 cells, Cmpd101 produced almost complete inhibition of DAMGO-induced MOPr phosphorylation at Ser(375), arrestin translocation, and MOPr internalization. Our data demonstrate a role for GRK2 (and potentially also GRK3) in agonist-induced MOPr desensitization in the LC, but leave open the possibility that another, as yet unidentified, mechanism of desensitization also exists.
authors.affiliation: School of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom (J.D.L., H.S.S., A.E.C., E.T., S.L.W., E.K., G.H.); Department of Pharmacology, University of Washington School of Medicine, Seattle, Washington (C.C.); and Department of Pharmacy and Pharmacology, University of Bath, Bath, United Kingdom (M.O., S.M.H., C.P.B.).
authors.forename: Janet D
authors.initials: JD
authors.lastname: Lowe
authors.affiliation: School of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom (J.D.L., H.S.S., A.E.C., E.T., S.L.W., E.K., G.H.); Department of Pharmacology, University of Washington School of Medicine, Seattle, Washington (C.C.); and Department of Pharmacy and Pharmacology, University of Bath, Bath, United Kingdom (M.O., S.M.H., C.P.B.).
authors.forename: Helen S
authors.initials: HS
authors.lastname: Sanderson
authors.affiliation: School of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom (J.D.L., H.S.S., A.E.C., E.T., S.L.W., E.K., G.H.); Department of Pharmacology, University of Washington School of Medicine, Seattle, Washington (C.C.); and Department of Pharmacy and Pharmacology, University of Bath, Bath, United Kingdom (M.O., S.M.H., C.P.B.).
authors.forename: Alexandra E
authors.initials: AE
authors.lastname: Cooke
authors.affiliation: School of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom (J.D.L., H.S.S., A.E.C., E.T., S.L.W., E.K., G.H.); Department of Pharmacology, University of Washington School of Medicine, Seattle, Washington (C.C.); and Department of Pharmacy and Pharmacology, University of Bath, Bath, United Kingdom (M.O., S.M.H., C.P.B.).
authors.forename: Mehrnoosh
authors.initials: M
authors.lastname: Ostovar
authors.affiliation: School of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom (J.D.L., H.S.S., A.E.C., E.T., S.L.W., E.K., G.H.); Department of Pharmacology, University of Washington School of Medicine, Seattle, Washington (C.C.); and Department of Pharmacy and Pharmacology, University of Bath, Bath, United Kingdom (M.O., S.M.H., C.P.B.).
authors.forename: Elena
authors.initials: E
authors.lastname: Tsisanova
authors.affiliation: School of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom (J.D.L., H.S.S., A.E.C., E.T., S.L.W., E.K., G.H.); Department of Pharmacology, University of Washington School of Medicine, Seattle, Washington (C.C.); and Department of Pharmacy and Pharmacology, University of Bath, Bath, United Kingdom (M.O., S.M.H., C.P.B.).
authors.forename: Sarah L
authors.initials: SL
authors.lastname: Withey
authors.affiliation: School of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom (J.D.L., H.S.S., A.E.C., E.T., S.L.W., E.K., G.H.); Department of Pharmacology, University of Washington School of Medicine, Seattle, Washington (C.C.); and Department of Pharmacy and Pharmacology, University of Bath, Bath, United Kingdom (M.O., S.M.H., C.P.B.).
authors.forename: Charles
authors.initials: C
authors.lastname: Chavkin
authors.affiliation: School of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom (J.D.L., H.S.S., A.E.C., E.T., S.L.W., E.K., G.H.); Department of Pharmacology, University of Washington School of Medicine, Seattle, Washington (C.C.); and Department of Pharmacy and Pharmacology, University of Bath, Bath, United Kingdom (M.O., S.M.H., C.P.B.).
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Husbands
authors.affiliation: School of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom (J.D.L., H.S.S., A.E.C., E.T., S.L.W., E.K., G.H.); Department of Pharmacology, University of Washington School of Medicine, Seattle, Washington (C.C.); and Department of Pharmacy and Pharmacology, University of Bath, Bath, United Kingdom (M.O., S.M.H., C.P.B.).
authors.forename: Eamonn
authors.initials: E
authors.lastname: Kelly
authors.affiliation: School of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom (J.D.L., H.S.S., A.E.C., E.T., S.L.W., E.K., G.H.); Department of Pharmacology, University of Washington School of Medicine, Seattle, Washington (C.C.); and Department of Pharmacy and Pharmacology, University of Bath, Bath, United Kingdom (M.O., S.M.H., C.P.B.).
authors.forename: Graeme
authors.initials: G
authors.lastname: Henderson
authors.affiliation: School of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom (J.D.L., H.S.S., A.E.C., E.T., S.L.W., E.K., G.H.); Department of Pharmacology, University of Washington School of Medicine, Seattle, Washington (C.C.); and Department of Pharmacy and Pharmacology, University of Bath, Bath, United Kingdom (M.O., S.M.H., C.P.B.) c.p.bailey@bath.ac.uk.
authors.forename: Chris P
authors.initials: CP
authors.lastname: Bailey
mesh_terms: D000818:Animals; D001549:Benzamides / Q000494:pharmacology*; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)- / Q000494:pharmacology*; D054769:G-Protein-Coupled Receptor Kinase 2 / Q000378:metabolism*; D054770:G-Protein-Coupled Receptor Kinase 3 / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008125:Locus Coeruleus / Q000187:drug effects*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D051381:Rats; D017208:Rats, Wistar; D017450:Receptors, Opioid, mu / Q000378:metabolism*
chemical_list: D001549:Benzamides; D017450:Receptors, Opioid, mu; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D054770:G-Protein-Coupled Receptor Kinase 3; D054769:G-Protein-Coupled Receptor Kinase 2