Identification of G protein-coupled receptor kinase 2 phosphorylation sites responsible for agonist-stimulated delta-opioid receptor phosphorylation

Agonist-induced receptor phosphorylation is an initial step in opioid recep tor desensitization, a molecular mechanism of opioid tolerance and dependen ce. Our previous research suggested that agonist-induced delta-opioid recep tor (DOR) phosphorylation occurs at the receptor carboxyl terminal domain. The current study was carried out to identify the site of DOR phosphorylati on during agonist stimulation and the kinases catalyzing this reaction. Tru ncation (Delta 15) or substitutions (T358A, T361A, and S363G single or trip le mutants) at the DOR cytoplasmic tail caused 80 to 100% loss of opioid-st imulated receptor phosphorylation, indicating that T358, T361, and S363 all contribute and are cooperatively involved in agonist-stimulated DOR phosph orylation. Coexpression of GRK2 strongly enhanced agonist-stimulated phosph orylation of the wild-type DOR (WT), but Delta 15 or mutant DOR (T358A/T361 A/S363G) failed to show any detectable phosphorylation under these conditio ns. These results demonstrate that T358, T361, and S363 are required for ag onist-induced and GRK-mediated receptor phosphorylation. Agonist-induced re ceptor phosphorylation was severely impaired by substitution of either T358 or S363 with aspartic acid residue, but phosphorylation of the T361D mutan t was comparable with that of WT. In the presence of exogenously expressed GRK2, phosphorylation levels of T358D and S363D mutants were approximately half of that of WT, whereas significant phosphorylation of the T358/S363 do uble-point mutant was not detected. These results indicate that both T358 a nd S363 residues at the DOR carboxyl terminus are capable of serving cooper atively as phosphate acceptor sites of GRK2 in vivo. Taken together, we hav e demonstrated that agonist-induced opioid receptor phosphorylation occurs exclusively at two phosphate acceptor sites (T358 and S363) of GRK2 at the DOR carboxyl terminus. These results represent the identification of the GR K phosphorylation site on an opioid receptor for the first time and demonst rate that GRK is the prominent kinase responsible for agonist-induced opioi d receptor phosphorylation in vivo.