The absence of a direct correlation between the loss of [D-Ala(2),MePhe(4),Gly(5)-ol]enkephalin inhibition of adenylyl cyclase activity and agonist-induced mu-opioid receptor phosphorylation

Chronic activation of the mu-opioid receptor (MOR1TAG) results in the loss of agonist response that has been attributed to desensitization and down-re gulation of the receptor. It has been suggested that opioid receptor phosph orylation is the mechanism by which this desensitization and down-regulatio n occurs. When MOR1TAG; was stably expressed in both neuroblastoma neuro2A and human embryonic kidney HEK293 cells, the opioid agonist [D-Ala(2),MePhe (4),Gly(5)-ol]enkephalin (DAMGO) induced a time- and concentration-dependen t phosphorylation of the receptor, in both cell lines, that could be revers ed by the antagonist naloxone, Protein kinase C can phosphorylate the recep tor, but is not involved in DAMGO-induced MOR1TAG phosphorylation. The rapi d rate of receptor phosphorylation, occurring within minutes, did not corre late with the rate of the loss of agonist-mediated inhibition of adenylyl c yclase, which occurs in hours. This lack of correlation between receptor ph osphorylation and the loss of response was further demonstrated when recept or phosphorylation was increased by either calyculin A or overexpression of the G-protein receptor kinases. Calyculin A increased the magnitude of MOR 1TAG phosphorylation without altering the DAMGO-induced loss of the adenyly l cyclase response. Similarly, when mu- and delta-opioid (DOR1TAG) receptor s were expressed in the same system, overexpression of beta-adrenergic rece ptor kinase 2 elevated agonist-induced phosphorylation for both receptors, However, in the same cell lines under the same conditions, overexpression o f beta-adrenergic receptor kinase 2 and beta-arrestin 2 accelerated the rat e of DPDPE- but not DAMGO-induced receptor desensitization. Thus, these dat a show that phosphorylation of MOR1TAG is not an obligatory event for the D AMGO-induced loss in the adenylyl cyclase regulation by the receptor.