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Mol Pharmacol. 2015 Aug;88(2):347-56. doi: 10.1124/mol.115.098293. Epub 2015 May 26.

Role of G Protein-Coupled Receptor Kinases 2 and 3 in μ-Opioid Receptor Desensitization and Internalization.

Author information

1
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.).
2
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.

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.

PMID:
26013542
PMCID:
PMC4518089
DOI:
10.1124/mol.115.098293
[Indexed for MEDLINE]
Free PMC Article

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