Receptor density and recycling affect the rate of agonist-induced desensitization of mu-opioid receptor

Previously, we reported that the time course for the rapid phosphorylation rate of mu-opioid receptor expressed in human embryonic kidney (HEK)293 cel ls did not correlate with the slow receptor desensitization rate induced by [D-Ala(2),N-MePhe(4),Gly-ol(5)]-enkephalin (DAMGO). However, others have s uggested that receptor phosphorylation is the trigger for mu-opioid recepto r desensitization. In this study, we demonstrated the relatively slow rate of receptor desensitization could be attributed partially to the recycling of internalized receptor as determined by fluorescence-activated cell-sorti ng analysis. However, the blockade of the endocytic and Golgi transport eve nts in HEK293 cells with monensin and brefeldin A did not increase the init ial rate of receptor desensitization. But the desensitization rate was incr eased by reduction of the mu-opioid receptor level with beta-furnaltrexamin e (beta FNA). The reduction of the receptor level with 1 mu M beta FNA sign ificantly increased the rate of etorphine-induced receptor desensitization. By blocking the ability of receptor to internalize with 0.4 M sucrose, a s ignificant degree of receptor being rapidly desensitized was observed in HE K293 cells pretreated with beta FNA. Hence, mu-opioid receptor is being res ensitized during chronic agonist treatment. The significance of resensitiza tion of the internalized receptor in affecting receptor desensitization was demonstrated further with human neuroblastoma SHSY5Y cells that expressed a low level of mu-opioid receptor. Although DAMGO could not induce a rapid desensitization in these cells, in the presence of monensin and brefeldin A , DAMGO desensitized the mu-opioid receptor's ability to regulate adenylyl cyclase with a t(1/2) = 9.9 +/- 2.1 min and a maximal desensitized level at 70 +/- 4.7%. Furthermore, blockade of receptor internalization with 0.4 M sucrose enhanced the DAMGO-induced receptor desensitization, and the inclus ion of monensin prevented the resensitization of the mu-opioid receptor aft er chronic agonist treatment in SHSY5Y cells. Thus, the ability of the mu-o pioid receptor to resensitize and to recycle, and the relative efficiency o f the receptor to regulate adenylyl cyclase activity, contributed to the ob served slow rate of mu-opioid receptor desensitization in HEK293 cells.