In a previous work, we described a differential desensitization of the human
delta-opioid receptor (hDOP-R) by
etorphine (a non-selective and
alkaloid agonist) and delta-selective and peptidic agonists (
DPDPE ([D-Pen(2,5)]
enkephalin) and
deltorphin I (Tyr-D-
Ala-Phe-Asp-
Val-Val-Gly-NH(2))) in the
neuroblastoma cell line SK-N-BE (Allouche et al., Eur. J. Pharmacol., 371, 235, 1999). In the present study, we explored the putative role of different
kinases in this differential regulation. First, selective chemical inhibitors of PKA, PKC and
tyrosine kinases were used and we showed a significant reduction of
etorphine-induced
opioid receptor desensitization by the
bisindolylmaleimide I (PKC inhibitor) while
genistein (
tyrosine kinase inhibitor) was potent to impair desensitization induced by the different agonists. When the PKA was inhibited by
H89 pretreatment, no modification of
opioid receptor desensitization was observed whatever the agonist used. Second, we further studied the role of
G protein-coupled receptor kinases (GRKs) and by using western-blot experiments we observed that only the GRK2
isoform was expressed in the SK-N-BE cells. Next, the
neuroblastoma cells were transfected with the wild type GRK2 or its dominant negative mutant GRK2-K220R and the inhibition on cAMP level was determined in naïve and agonist-pretreated cells. We showed that over-expression of GRK2-K220R totally abolished
etorphine-induced receptor desensitization while no effect was observed with peptidic agonists and over-expression of GRK2 selectively impaired cAMP inhibition promoted by
etorphine suggesting that this
kinase was involved in the regulation of hDOP-R activated only by
etorphine. Third, correlation between functional experiments and phosphorylation of the hDOP-R after agonist activation was assessed by western-blot using the specific anti-phospho-DOP-R Ser(363) antibody. While all agonists were potent to increase phosphorylation of
opioid receptor, we showed no impairment of receptor phosphorylation level after PKC inhibitor pretreatment. Upon agonist activation, no enhancement of receptor phosphorylation was observed when the GRK2 was over-expressed while the GRK2-K220R partially reduced the hDOP-R Ser(363) phosphorylation only after peptidic agonists pretreatment. In conclusion, hDOP-R desensitization upon
etorphine exposure relies on the GRK2, PKC and
tyrosine kinases while
DPDPE and
deltorphin I mediate desensitization at least via
tyrosine kinases. Although the Ser(363) was described as the primary phosphorylation site of the mouse DOP-R, we observed no correlation between desensitization and phosphorylation of this
amino acid.