IDENTIFICATION OF PHOSPHORYLATION SITES IN THE G-PROTEIN-COUPLED RECEPTOR FOR PARATHYROID-HORMONE - RECEPTOR PHOSPHORYLATION IS NOT REQUIRED FOR AGONIST-INDUCED INTERNALIZATION

In some G protein-coupled receptors (GPCRs), agonist-dependent phospho rylation by specific GPCR kinases (GRKs) is an important mediator of r eceptor desensitization and endocytosis. Phosphorylation and the subse quent events that it triggers, such as arrestin binding, have been sug gested to be regulatory mechanisms for a wide variety of GPCRs. In the present study, we investigated whether agonist-induced phosphorylatio n of the PTH receptor, a class II GPCR, also regulates receptor intern alization. Upon agonist stimulation, the PTH receptor was exclusively phosphorylated on serine residues. Phosphoamino acid analysis of a num ber of receptor mutants in which individual serine residues had been r eplaced by threonine identified serine residues in positions 485, 486, and 489 of the cytoplasmic tail as sites of phosphorylation after ago nist treatment. When serine residues at positions 483, 485, 486, 489, 495, and 498 were simultaneously replaced by alanine residues, the PTH receptor was no longer phosphorylated either basally or in response t o PTH. The substitution of these serine residues by alanine affected n either the number of receptors expressed on the cell surface nor the a bility of the receptor to signal via Gs. Overexpression of GRK2, but n ot GRK3, enhanced PTH-stimulated receptor phosphorylation, and this ph osphorylation was abolished by alanine mutagenesis of residues 483, 48 5, 486, 489, 495, and 498. Thus, phosphorylation of the PTH receptor b y the endogenous kinase in HEK-293 cells occurs on the same residues t argeted by overexpressed GRK2. Strikingly, the rate and extent of PTH- stimulated internalization of mutated PTH receptors lacking phosphoryl ation sites were identical to that observed for the wild-type PTH rece ptor. Moreover, overexpressed GRK2, while enhancing the phosphorylatio n of the wild-type PTH receptor, had no affect on the rate or extent o f receptor internalization in response to PTH. Thus, the agonist-occup ied PTH receptor is phosphorylated by a kinase similar or identical to GRK2 in HEK-293 cells, but this phosphorylation is not requisite for efficient receptor endocytosis.