Role of Helix 8 of the Thyrotropin-Releasing Hormone Receptor in Phosphorylation by G Protein-Coupled Receptor Kinase

Abstract

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 lysine and arginine residues between the NPXXY and residue 340 to glutamine (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 phenylalanine in the common GPCR motif NPXXY(X) 5-6 F(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 coexpression of a receptor with an intact helix 8 but without phosphorylation sites. Phosphorylation was inhibited but not completely prevented by alanine substitution for cysteine palmitoylation sites. Positively charged amino acids in the proximal tail of the ␤2-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 type 1 TRH receptor is a GPCR expressed in the anterior pituitary gland. In response to TRH, the receptor activates G q/11 , which in turn stimulates phospholipase C. The resulting increases in inositol trisphosphate and diacylglycerol cause rapid release of intracellular calcium and activation of protein kinase C. Like many GPCRs, the TRH receptor contains a canonical NPXXY at the end of transmembrane 7 in a NPXXY(X) 5-6 F(R/K) motif (Mirzadegan et al., 2003;Okuno et al., 2005;Swift et al., 2006) and is predicted to form an amphipathic eighth helix with a positively charged face ending at a downstream pair of cysteine residues, where palmitoylation occurs (Du et al., 2005).When TRH binds, the receptor undergoes rapid and quantitative phosphorylation at multiple sites in the cytoplasmic tail Hinkle, 2005, 2008;Jones et al., 2007). Agonist-stimulated phosphorylation of the receptor drives arrestin binding and arrestin-dependent receptor desensitization and internalization. The receptor tail is not detectably phosphorylated before activation.Agonist-dependent phosphorylation of GPCRs is usually carried out by either GRKs that recognize the activated state of the receptor or downstream kinases that are activated by receptor signaling. Th...

Helix 8 Plays a Crucial Role in Bradykinin B2 Receptor Trafficking and Signaling

“…Helix 8 and B 2 R-GRK Interaction-Whereas the publications so far have focused almost exclusively on a role of helix 8 in receptor expression or in G protein activation, it was just recently reported that helix 8 might also play a role in receptor phosphorylation and, therefore, in the interaction with GRKs (18). Mutation of basic residues in the helix 8 of the thyrotropin releasing hormone receptor resulted in reduced receptor internalization and phosphorylation that could be rescued partly by overexpression of GRK2 or -3 and fully by overexpression of GRK5 or -6.…”

“…Studies have not only shown the involvement of helix 8 in G protein activation (11-15) but also identified hydrophobic residues in helix 8 as essential for the surface expression of the GPCRs investigated (11,16,17). In addition, it has been published only recently that mutation of positively charged residues in helix 8 of the thyrotropin releasing hormone receptor prevented GRK-mediated receptor phosphorylation (18).As demonstrated by our research group, mutation of Tyr7.53 (Ballesteros/Weinstein nomenclature (19)) to an alanine in the NPXXY sequence resulted in constitutive receptor phosphorylation and internalization (20). Homology modeling indicated that Tyr7.53 in the B 2 R exerts an aromatic stacking interaction with the highly conserved phenylalanine Phe7.60 in helix 8.…”

“…Studies have not only shown the involvement of helix 8 in G protein activation (11-15) but also identified hydrophobic residues in helix 8 as essential for the surface expression of the GPCRs investigated (11,16,17). In addition, it has been published only recently that mutation of positively charged residues in helix 8 of the thyrotropin releasing hormone receptor prevented GRK-mediated receptor phosphorylation (18).…”

β-Subunit of the Ostα-Ostβ Organic Solute Transporter Is Required Not Only for Heterodimerization and Trafficking but Also for Function

“…HEK293 cells were transfected with 900 ng of V5-Ost␣ and 100 ng of each 3ϫHA-Ost␤ species DNA in 24-well plates. At 48 h after transfection, cell surface ELISA was conducted as described previously (17). Briefly, plates were incubated with monoclonal anti-V5 (Invitrogen) and anti-HA (Covance) at 1:5,000 followed by anti-mouse IgG-HRP (BioRad) at 1:5,000.…”

Deficiency or inhibition of lysophosphatidic acid receptor 1 protects against hyperoxia-induced lung injury in neonatal rats

“…Surface expression of N-terminally HA-tagged receptors was quantified as described (Gehret et al 2010). Briefly, the N-terminally tagged receptors were transiently expressed in COS-7 cells and 24 hours after transfection the cells were harvested, seeded in 12 well plates and serum-starved overnight.…”