Carboxyl-terminal Splicing of the Rat μ Opioid Receptor Modulates Agonist-mediated Internalization and Receptor Resensitization

Abstract

The rat opioid receptor is alternatively spliced into two isoforms (MOR1 and MOR1B) which differ in length and amino acid composition at the carboxyl terminus. ]enkephalin (DAMGO) with similar affinity and exhibit functional coupling to adenylyl cyclase with similar efficiency. However, the shorter isoform, MOR1B, desensitized at a slower rate during prolonged DAMGO exposure (4 h) but resensitized at a faster rate than MOR1 during agonist withdrawal (20 min). Immunocytochemical analysis revealed that DAMGO-induced internalization of MOR1B proceeded much faster than that of MOR1 followed by rapid recycling of the receptor to the cell surface. In addition, the greater resistance of MOR1B to homologous desensitization compared with MOR1 as well as MOR1B resensitization was abolished when receptor reactivation/recycling was blocked with monensin, an inhibitor of endosomal acidification. It is concluded that the sequence at the cytoplasmic tail of MOR1B facilitates clathrin-coated vesicle-mediated endocytosis which, in turn, promotes accelerated receptor reactivation. Taken together, our findings suggest that carboxyl-terminal splicing of the rat opioid receptor modulates agonist-induced internalization and receptor resensitization.Prolonged exposure of G protein-coupled receptors to agonists results in a rapid decrease of receptor responsiveness. It is now generally accepted that agonist-induced desensitization involves phosphorylation of intracellular receptor domains. Several kinases have been implicated in opioid receptor desensitization, including cAMP-dependent protein kinase (PKA), 1 protein kinase C (PKC), and calcium/calmodulin-dependent protein kinase II (CaM kinase II) (1-5). Specific phosphorylation sites have been localized in the third intracellular domain and at the carboxyl terminus, which play a critical role in homologous desensitization of the opioid receptor (6 -10). Following phosphorylation, the receptor is being targeted to the endocytotic machinery. A large body of evidence suggests that the main route of internalization of G protein-coupled receptors is via clathrin-coated pits and vesicles into early endosomes. Within the acidic environment of the endosomes, the ligand is effectively separated from the receptor which becomes dephosphorylated and thus resensitized. As a final step, the receptor recycles back to the cell surface (11).For the opioid receptor, desensitization seems to be regulated by CaM kinase II-mediated phosphorylation of two serine residues (Ser 261 /Ser 266 ) in the third intracellular loop (3, 9). Another important phosphorylation site of the opioid receptor is the threonine at position 394 in the carboxyl terminus. Indeed, we and others have recently observed that site-directed mutagenesis of Thr 394 to alanine profoundly delays DAMGOinduced desensitization, suggesting that this site may be a primary target for phosphorylation by GRKs upon agonist binding to the MOR1 (9, 10).We have previously shown, that the cytoplasmic tail of the rat opioid receptor undergoes alternati...

Functional characterization of human variants of the mu-opioid receptor gene

“…Functional differences have been reported between Cterminal splice variants of the OPRM1 gene in mice (45) and rats (17). When we compared MOR1 receptors to MOR1A receptors lacking the 12 aa coded by exon 4, we found that the 2 receptors had similar binding, activation, and trafficking profiles.…”

“…Also, splice variants of MOR have been reported for rodents and humans (16), and can result in isoforms with varying trafficking and signaling properties (17). Also, the MOR has been shown to oligomerize with other opioid receptors (18,19), and with receptors in other classes (20,21), which can significantly alter receptor function (10,22).…”

The orphan G protein-coupled receptor, Gpr161 , encodes the vacuolated lens locus and controls neurulation and lens development

“…Gpr161 is expressed at all examined stages of lens development: lens pit (E10.5), lens vesicle (E11.5), primary lens Vl Mutation Affects Gpr161 Receptor-Mediated Endocytosis. Receptor-mediated endocytosis is a common mechanism by which GPCR signaling is attenuated and is regulated by C-terminal tail phosphorylation (15,16,18,19). To investigate the effects of the mutation on Gpr161 plasma membrane targeting and intracellular localization, WT and vl-myc-Gpr161 constructs were transfected into HEK293T cells.…”

Chronic Morphine Treatment Inhibits Opioid Receptor Desensitization and Internalization

“…HEK293 cells have been widely used during the past to study receptor internalization Koch et al, 1998). We thus examined whether attenuation of DOR internalization can be reproduced in morphine-treated HEK293 cells stably expressing the mouse DOR.…”

“…Because each of these factors may contribute to the mechanism of agonist-induced desensitization of receptor activity, the present study was initiated to investigate whether chronic morphine treatment could possibly affect the regulatory properties of highefficacy opioids to desensitize OR function. Neuroblastoma x glioma (NG108-15) hybrid cells were used, because they endogenously express high levels of DORs that are better substrates for agonist-induced desensitization than MORs (Koch et al, 1998;Law et al, 2000). In addition, these cells are known to produce cellular correlates of morphine tolerance (Johnson and Fleming, 1989), an essential requirement for the present study.…”