Morphine Activates Opioid Receptors without Causing Their Rapid Internalization

Abstract

We have examined the endocytic trafficking of epitopetagged ␦ and opioid receptors expressed in human embryonic kidney (HEK) 293 cells. These receptors are activated by peptide agonists (enkephalins) as well as by the alkaloid agonist drugs etorphine and morphine. Enkephalins and etorphine cause opioid receptors to internalize rapidly (t1 ⁄2 ϳ 6 min) by a mechanism similar to that utilized by a number of other classes of receptor, as indicated by localization of internalized opioid receptors in transferrin-containing endosomes and inhibition of opioid receptor internalization by hypertonic media. Remarkably, morphine does not stimulate the rapid internalization of either ␦ or opioid receptors, even at high concentrations that strongly inhibit adenylyl cyclase. These data indicate that agonist ligands, which have similar effects on receptor-mediated signaling, can have dramatically different effects on the intracellular trafficking of a G protein-coupled receptor.Opioid receptors constitute a class of G protein-coupled receptors that mediate the effects of endogenously produced opioid peptides in the central and peripheral nervous systems. An interesting feature of these receptors is that they are activated both by native peptides and by structurally distinct non-peptide alkaloid ligands (1). Following activation, opioid receptors are regulated by multiple mechanisms, which modulate the functional plasticity of the endogenous opioid system and contribute to the development of opiate tolerance and dependence (2-5).Previous studies have described two distinguishable processes of opioid receptor regulation, termed desensitization and down-regulation (3, 4, 6 -9). Both desensitization and downregulation of opioid receptors are stimulated by peptide and alkaloid agonists (4, 6). However, individual agonists may have substantially different effects on the rapid regulation of opioid receptors (10 -12). We have observed that opioid receptors are regulated by a process of rapid internalization that exhibits a remarkable degree of agonist specificity not observed previously in studies of the intracellular trafficking of other receptors. EXPERIMENTAL PROCEDURES Construction and Expression of Epitope-tagged ␦ and OpioidReceptors-cDNAs encoding murine ␦ (13) and (14) opioid receptors were epitope-tagged in the amino-terminal extracellular domain utilizing a shuttle vector containing a signal-FLAG cassette (kindly provided by Drs. Jeff Reagan and Brian Kobilka) (15) and subcloned into pcDNA3 (Invitrogen) for transfection. The structure of each mutant cDNA was confirmed by dideoxy sequencing (Sequenase, U. S. Biochemical Corp.).Human embryonic kidney (HEK) 1 293 cells (ATCC) were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (University of California San Francisco Cell Culture Facility) and transfected using calcium phosphate coprecipitation (16). Stably transfected cells were isolated following neomycin selection (Geneticin, Life Technologies, Inc.) as described previously (17), and satur...

Regulatory mechanisms that modulate signalling by G-protein-coupled receptors

“…However, mutants of receptors that couple to G s and G q have been identified that either signal normally yet exhibit impaired internalization, or are deficient in G-protein coupling and yet are internalized normally [191,192]. Further evidence against this mechanism of endocytosis has been obtained from recent observations of the µ-opioid receptor in transfected cells and enteric neurons of the intact animal [193,194]. In these systems enkephalin and the agonist etorphine stimulate receptor endocytosis in the expected manner.…”

Internalization and trafficking of opioid receptor ligands in rat cortical neurons

“…In particular, it has been linked to receptor resensitization allowing for functional recovery (Grady et al, 1995;Ferguson et al, 1998), as well as to regulation of gene transcription (Belcheva et al, 1996;Ventura et al, 1998;Ignatova et al, 1999;Souazé et al, 1997;Sarret et al, 1999). Most of our knowledge concerning the mechanisms of OR internalization emanates from studies in vitro, in immortal cells expressing either native or recombinant receptors (von Zastrow et al, 1993;Arden et al, 1995;Keith et al, 1996;Trapaidze et al, 1996;Gaudriault et al, 1997). Thus, studies from our laboratory have demonstrated that OR and ␦OR, when coexpressed in the same cells, internalized through partly distinct endocytic pathways and were sorted via different endocytic vesicles (Gaudriault et al, 1997).…”

Dimerization of the δ Opioid Receptor:

“…Morphine, unlike most of the opioid agonists, does not induce rapid internalization of the opioid receptors (5,8). An exact mechanism of the opioid receptor internalization is not known, although it has been suggested that the rapid endocytosis of the receptors is mediated through the classic endocytic pathway (5,7). Possible events that would precede the receptor internalization, such as the interaction of the receptor with an adapter protein or another receptor, have not yet been determined.…”

“…Different opioid ligands induce different effects on rapid internalization of the opioid receptors. Morphine, unlike most of the opioid agonists, does not induce rapid internalization of the opioid receptors (5,8). An exact mechanism of the opioid receptor internalization is not known, although it has been suggested that the rapid endocytosis of the receptors is mediated through the classic endocytic pathway (5,7).…”

“…Short-term treatment with opioid ligands causes rapid loss of receptors from the surface of the cell as a result of the receptor endocytosis (5)(6)(7). Both of these effects require the intact Cterminal tail of the receptor (4,7).…”