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Receptor Selectivity of the Cloned Opossum G Protein-Coupled Receptor Kinase 2 (GRK2) in Intact Opossum Kidney Cells: Role in Desensitization of Endogenous _2C-Adrenergic but Not Serotonin 1B Receptors

Department of Pharmacology and Therapeutics (P.M.C.L., M.H.G.) McGill University Montreal, Quebec, Canada H3G 1Y6
Departments of Medicine and Cellular and Molecular Medicine (P.R.A.) Neuroscience Research Institute University of Ottawa Ottawa, Ontario, Canada K1H 8M5

To characterize the specificity of endogenously expressed G protein-coupled receptor kinases (GRKs) for endogenous Gi-coupled _2C-adrenergic and serotonin 1B (5-HT1B) receptors in the opossum kidney (OK) cell line, we have isolated a 3.073-kb OK-GRK2 clone encoding a 689-amino acid protein that shares 94.2% amino acid identity with rat GRK2. Northern blot analysis revealed the presence of GRK2 mRNA transcripts of 5.0 and 3.0 kb in OK cells. In intact OK cells, preincubation (45 min) with agonist (5-HT or UK 14304, 1 µM) reduced the maximal inhibition of forskolin-induced cAMP accumulation mediated by endogenous 5-HT1B and _2C-adrenergic receptors by 12 ± 2% or 17 ± 4%, respectively. In transfected OK cells overexpressing OK-GRK2, agonist-induced desensitization of the _2C-adrenergic receptor, but not the 5-HT1B receptor, was enhanced by 2- to 4-fold. Conversely, in cells overexpressing the kinase-inactive mutant OK-GRK2-K220R, _2C-adrenergic receptor desensitization was selectively abolished, whereas desensitization of the 5-HT1B receptor was slightly enhanced. Similarly, depletion of GRK-2 protein by stable transfection of full-length antisense OK-GRK2 cDNA blocked the desensitization of _2C-adrenergic receptors but not of 5-HT1B receptors. These results represent the first evidence of the coexistence of GRK2-dependent (for _2C receptors) and GRK2-independent (for 5-HT1B receptors) mechanisms of desensitization in intact cells and demonstrate the selectivity of GRK2 for distinct Gi-coupled receptors.

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