Thr353, Located within the COOH-terminal Tail of the ´ Opiate Receptor, Is Involved in Receptor Down-regulation

Abstract

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...

Dimerization of the δ Opioid Receptor:

“…We have shown previously that the receptors expressed in these cells bind opioids with high affinity and efficiently couple to adenylyl cyclase (4,7). Immunoblotting analysis of cell lysates with the M1 anti-Flag antibody revealed a predominant protein of about 60 kDa (Fig.…”

“…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.…”

“…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).…”

δ and κ Opioid Receptors Are Differentially Regulated by Dynamin-dependent Endocytosis When Activated by the Same Alkaloid Agonist

“…The effect of exchanging the carboxyl-terminal cytoplasmic tail was examined first because this cytoplasmic domain diverges greatly between individual opioid receptors, and truncation and point mutations within this domain have been reported to influence receptor internalization and downregulation (15,27). Replacing the carboxyl tail of the ␦ receptor with the corresponding sequence from the receptor created a chimeric mutant receptor (Cr.…”