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Vol. 58, Issue 5, 1050-1056, November 2000
-Opioid
Receptor Phosphorylation
National Laboratory of Medical Neurobiology, Fudan University
Medical Center, Shanghai, People's Republic of China (J.G., J.Z.,
L.M.); Shanghai Institute of Cell Biology, Chinese Academy of Sciences,
Shanghai, People's Republic of China (Y.-L.W., W.-B.Z., G.P.); and
Department of Pharmacology, New York University School of Medicine, New
York, New York (L.A.D.)
Agonist-induced receptor phosphorylation is an initial step in opioid
receptor desensitization, a molecular mechanism of opioid tolerance and
dependence. Our previous research suggested that agonist-induced
-opioid receptor (DOR) phosphorylation occurs at the receptor
carboxyl terminal domain. The current study was carried
out to identify the site of DOR phosphorylation during agonist
stimulation and the kinases catalyzing this reaction. Truncation
(
15) or substitutions (T358A, T361A, and S363G single or triple
mutants) at the DOR cytoplasmic tail caused 80 to 100% loss of
opioid-stimulated receptor phosphorylation, indicating that T358, T361,
and S363 all contribute and are cooperatively involved in
agonist-stimulated DOR phosphorylation. Coexpression of GRK2 strongly
enhanced agonist-stimulated phosphorylation of the wild-type DOR (WT),
but 15 or mutant DOR (T358A/T361A/S363G) failed to show any
detectable phosphorylation under these conditions. These results
demonstrate that T358, T361, and S363 are required for agonist-induced
and GRK-mediated receptor phosphorylation. Agonist-induced receptor
phosphorylation was severely impaired by substitution of either T358 or
S363 with aspartic acid residue, but phosphorylation of the T361D
mutant 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 double-point mutant was not detected. These results
indicate that both T358 and S363 residues at the DOR carboxyl terminus
are capable of serving cooperatively as phosphate acceptor sites of
GRK2 in vivo. Taken together, we have 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 GRK
phosphorylation site on an opioid receptor for the first time and
demonstrate that GRK is the prominent kinase responsible for
agonist-induced opioid receptor phosphorylation in vivo.
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