TY - JOUR T1 - ROLE OF HELIX 8 OF THE THYROTROPIN-RELEASING HORMONE RECEPTOR IN PHOSPHORYLATION BY G PROTEIN-COUPLED RECEPTOR KINASE JF - Molecular Pharmacology JO - Mol Pharmacol DO - 10.1124/mol.109.059733 SP - mol.109.059733 AU - Austin U. Gehret AU - Brian W. Jones AU - Phuong N. Tran AU - Laurie B. Cook AU - Emileigh K. Greuber AU - Patricia M. Hinkle Y1 - 2009/11/11 UR - http://molpharm.aspetjournals.org/content/early/2009/11/11/mol.109.059733.abstract N2 - The thyrotropin-releasing hormone (TRH) receptor undergoes rapid and extensive agonist-dependent phosphorylation attributable to G protein-coupled receptor (GPCR) kinases (GRKs), particularly GRK2. Like many GPCRs, the TRH receptor is predicted to form an amphipathic helix, helix 8, between the NPXXY motif at the cytoplasmic end of the seventh transmembrane domain and palmitoylation sites at Cys335 and Cys337. Mutation of all six Lys and Arg residues between the NPXXY and residue 340 to Gln (6Q-receptor) did not prevent the receptor from stimulating inositol phosphate turnover but almost completely prevented receptor phosphorylation in response to TRH. Phosphorylation at all sites in the cytoplasmic tail was inhibited. The phosphorylation defect was not reversed by long incubation times or high TRH concentrations. As expected for a phosphorylation-defective receptor, the 6Q-TRH receptor did not recruit arrestin, undergo the typical arrestin-dependent increase in agonist affinity, or internalize well. Lys326, directly before Phe in the common GPCR motif NPXXY(X)5-6F(R/K), was critical for phosphorylation. The 6Q-TRH receptor was not phosphorylated effectively in cells overexpressing GRK2, or in in vitro kinase assays containing purified GRK2. Phosphorylation of the 6Q-receptor was partially restored by co-expression of a receptor with an intact helix 8 but without phosphorylation sites. Phosphorylation was inhibited but not completely prevented by Ala-substitution for Cys palmitoylation sites. Positively charged amino acids in the proximal tail of the β2-adrenergic receptor were also important for GRK-dependent phosphorylation. The results indicate that positive residues in helix 8 of GPCRs are important for GRK-dependent phosphorylation.The American Society for Pharmacology and Experimental Therapeutics ER -