Sequestration of the δ Opioid Receptor

Abstract

Exposure to opiates causes decreased sensitivity to the drug. This receptor desensitization is a process whereby continuous or repeated exposure to a high concentration of the opiates results in a reduced cellular response (1). The mechanism of desensitization of the opiate receptor is thought to be similar to the mechanism of desensitization of the well explored prototypic ␤ 2 -adrenergic receptor (␤ 2 AR) 1 (2). Rapid desensitization is thought to result from the alterations in the receptor conformation that interferes with its coupling to a G protein and by sequestration and internalization of the receptor into intracellular compartments. Longer desensitization is thought to be due to the receptor down-regulation with a net loss of binding sites within the cell (3). The cellular mechanisms involved in this desensitization process of the opioid receptors or the intracellular compartments involved in this process have not been well established. The molecular cloning of the cDNA encoding opioid receptors has made the studies to address the mechanism of receptor desensitization feasible. The primary structure of the opioid receptors has revealed that they are members of "G proteincoupled receptor (GPCR) family" (4, 5). Many of the structural features that are conserved in other GPCRs are found in the opioid receptors; these include consensus N-linked glycosylation sites near the N terminus, a palmitoylation site in the C-terminal tail, disulfide bonds in the extracellular loop between the third and fourth transmembrane domain, and sites for phosphorylation in the C-terminal tail and in the first and third intracellular domain (6). In the case of other GPCRs, C-terminal tail of the receptor is shown to be phosphorylated, and this is implicated in receptor desensitization.Mutational analysis of many members of the GPCR family provide evidence that the third cytoplasmic loop and the Cterminal tail are involved in the coupling of the membrane receptors with intracellular G proteins (2, 3). The same regions are shown to be involved in the control of receptor sequestration and internalization. A consensus sequence in the receptor for G protein coupling or for receptor internalization have not yet been clearly identified among the cloned GPCRs. It is thought that the membrane proximal regions of the third intracellular loop of these receptors are required for coupling, and multiple regions in the cytoplasmic loops and C-terminal tail are involved in the receptor internalization (7). The domain involved in the G protein coupling or the receptor sequestration/internalization of the opioid receptor is not known.In order to address the questions regarding agonist-induced internalization of the opioid receptor, we have used mutants of the ␦ opioid receptor cDNA stably expressed in Chinese hamster ovary (CHO) cells. We have generated two types of mutant receptors, (i) deletion mutants lacking various portions of Cterminal tail or (ii) point mutants of the various Ser or Thr in the C-terminal tail. We used antisera against the...

Dimerization of the δ Opioid Receptor:

“…Unique restriction enzyme sites were used to replace a corresponding region of the wild type receptor with the polymerase chain reaction amplification product. CHO cells stably expressing Flag epitope-tagged wild type or ⌬C15 deletion mutant receptors were generated, and their binding affinities, coupling to adenylyl cyclase, internalization, and down-regulation properties were characterized as described previously (4,7). For the experiments with immunoprecipitation and Western blotting, CHO or COS cells were transfected with 20 g of c-Myc and/or Flag-tagged DOR using the Ca 2ϩ phosphate method (18).…”

“…Receptor-The Flag epitope-tagged DOR was generated as described previously (4,7). c-Myc epitope-tagged ␦-opioid receptor (DOR) was generated by an overlapping extension polymerase chain reaction, using Flag epitope-tagged DOR cDNA as the template.…”

“…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.…”

“…Both of these effects require the intact Cterminal tail of the receptor (4,7). Although deletion of the C-terminal tail substantially reduces the extent of both downregulation and rapid internalization, point mutations within this region reduce the extent of internalization without affecting down-regulation, suggesting that these two responses are differentially regulated (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).…”