Dimerization of G-protein-coupled receptors has been increasingly noted in the regulation of their biological activity. However, its involvement in agonist-induced receptor internalization is not well understood. In this study, we examined the ability of mouse ␦-opioid receptors to dimerize and the role of receptor dimerization in agonist-induced internalization. Using differentially (Flag and c-Myc) epitope-tagged receptors we show that ␦-opioid receptors exist as dimers. The level of dimerization is agonist dependent. Increasing concentrations of agonists reduce the levels of dimer with a corresponding increase in the levels of monomer. Interestingly, morphine does not affect the levels of either form. It has been shown that morphine, unlike other opioid agonists, does not induce receptor internalization. This suggests a relationship between the ability of agonists to reduce the levels of dimer and to induce receptor internalization. The time course of the agonist-induced decrease of ␦-opioid receptor dimers is shorter than the time course of internalization, suggesting that monomerization precedes the agonist-induced internalization of the receptor. Furthermore, we found that a mutant ␦-opioid receptor, with a 15-residue C-terminal deletion, does not exhibit dimerization. This mutant receptor has been shown to lack the ability to undergo agonist-induced internalization. These results suggest that the interconversion between the dimeric and monomeric forms plays a role in opioid receptor internalization.The opioid receptor family, a member of the superfamily of G-protein-coupled receptors (GPCRs), 1 consists of three receptor types: ␦, , and . The opioid receptors transmit the signals induced by binding of opioid peptides and opiate alkaloids, such as morphine. Continuous or repeated exposure to opioid ligands causes decreased sensitivity to the drug and reduced cellular response; this response is regulated by multiple mechanisms. Long-term exposure to opioid ligands causes receptor down-regulation as a result of the receptor degradation (1-4).Short-term treatment with opioid ligands causes rapid loss of receptors from the surface of the cell as a result of the receptor endocytosis (5-7). 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). 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). Possible events that would precede ...

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...

Prolonged exposure to abused drugs such as opiates causes decreased response to the drug; this reduced sensitivity is thought to be due to the loss of receptors, or down-regulation. The molecular mechanism of the opiate receptor down-regulation is not known. In order to address this, we generated a number of mutants of the ␦ opiate receptor COOH-terminal tail. When expressed in the Chinese hamster ovary cells, both the wild type and the receptor with a deletion of 37 COOH-terminal residues bind diprenorphine with comparable affinities and show similar decreases in cAMP levels in response to D-Ala 2 , D-Leu 5, enkephalin (DADLE). However, the truncated receptor does not show down-regulation from the cell surface upon prolonged exposure (2-48 h) to DADLE. In contrast, both the wild type receptor and the receptor with the deletion of only 15 COOH-terminal residues show substantial down-regulation upon long term DADLE treatment. These results suggest that the region located between 15 and 37 residues from the COOH terminus is involved in the receptor down-regulation. In order to identify residues that play a key role in down-regulation, point mutations of residues within this region were examined for their ability to modulate receptor down-regulation. The receptor with a mutation of Thr 353 to Ala does not down-regulate, whereas the receptor with a mutation of Ser 344 to Gly down-regulates with a time course similar to that of the wild type receptor. Taken together, these results suggest that the COOH-terminal tail is not essential for functional coupling but is necessary for down-regulation and that Thr 353 is critical for the agonist-mediated down-regulation of the ␦ opiate receptor.It has been well established that chronic exposure to opiates such as morphine causes a decrease in the sensitivity to opiates (1). This reduced sensitivity is thought to be due to the loss of receptors from the cell's surface. This phenomenon, termed "down-regulation," has been implicated in the opiate tolerance/ dependence that is seen in narcotic addicts. The molecular basis of the down-regulation phenomenon is not known.Cell lines that express high levels of opiate receptors have been used for studies with agonist-mediated receptor function such as down-regulation (2-4). A neuroblastoma ϫ glioma hybrid cell line (NG108-15) has been widely used to characterize the ␦ subtype of the opiate receptor (2-4). Upon exposure to agonist the receptor number on the NG108-15 cells rapidly decreases (as detected with radioligand binding assays). Treatment for up to 24 h leads to a decrease of about 50 -80% of the cell surface receptors (2, 3). This receptor down-regulation is thought to be due to internalization of the ligand-receptor complex followed by degradation of the receptor (4).The primary structure of the opiate receptors as deduced from the cDNA has revealed that all three subtypes of the opiate receptors are members of the G-protein-coupled receptor family (5-7). Many structural features that are conserved in other G-protein-coupled r...

Many extracellular signals are transmitted to the interior of the cell by receptors with seven membrane-spanning helices that trigger their effects by means of heterotrimeric guanine-nucleotide-binding regulatory proteins (G proteins). These G-protein-coupled receptors (GPCRs) control various physiological functions in evolution from pheromone-induced mating in yeast to cognition in humans. The potential role of the G-protein signalling system in the control of animal ageing has been highlighted by the genetic revelation that mutation of a GPCR encoded by methuselah extends the lifespan of adult Drosophila flies. How methuselah functions in controlling ageing is not clear. A first essential step towards the understanding of methuselah function is to determine the ligands of Methuselah. Here we report the identification and characterization of two endogenous peptide ligands of Methuselah, designated Stunted A and B. Flies with mutations in the gene encoding these ligands show an increase in lifespan and resistance to oxidative stress. We conclude that the Stunted-Methuselah system is involved in the control of animal ageing.