ROLE OF CARBOXYL-TERMINUS OF MU-OPIOID AND DELTA-OPIOID RECEPTOR IN AGONIST-INDUCED DOWN-REGULATION

Chronic exposure of mu- and delta-opioid receptors to their agonists l eads to different rates in receptor down-regulation. In order to analy ze the role of the carboxyl terminus of mu- and delta-opioid receptors in the difference in the rate of down-regulation, two chimeras of the se receptors were generated by swapping the carboxyl termini; MORTAGDT and DORTAGMT. These chimeras were tagged at the N-terminus with hemag glutinin (HA) epitope (YPYDVPDYA), which can be recognized by the mono clonal antibody 12CA5, and then stably expressed in Neuro 2A (N2A) cel ls. The swapping of the carboxyl termini did not alter the ligand sele ctivity of these receptor chimeras. However, they did exhibit a reduct ion in agonist potency to inhibit forskolin-stimulated adenylyl cyclas e activity for all agonists tested except etorphine which had a potenc y comparable to that of wild type receptors. Treatment of the N2A cell s expressing MORTAGDT with 50 nM etorphine produced a faster rate of r eceptor down-regulation when compared to the wild type mu-opioid recep tor. Immunofluorescence microscopy of the MORTAGDT chimera using a mon oclonal antibody against HA confirmed internalization of the receptors after treatment with etorphine for 1 and 6 h. There was a reduction i n the HA-immunoreactivity at the cell surface of the MORTAGDT chimera concurrent with more noticeable HA-immunoreactivity inside the cell co mpared to the wild type receptor. On the other hand, the rate of down- regulation of DORTAGMT receptors was seen to be the same as the wild t ype delta-opioid receptor after etorphine treatment. Immunofluorescenc e studies showed more reduction in cell surface staining of the DORTAG MT chimera compared to the wild type receptor. These data suggest the involvement of the carboxyl terminus in agonist-induced down-regulatio n and internalization of the mu-opioid receptor. However, different me chanisms that are unrelated to the carboxyl terminus may operate in th e down-regulation of delta-opioid receptor. (C) 1998 Elsevier Science B.V.