Opioid receptor pharmacological chaperones act by binding and stabilizing newly synthesized receptors in the endoplasmic reticulum

Abstract Accumulating evidence has indicated that membrane-permeable G protein-coupled receptor ligands can enhance cell surface targeting of their cognate wild-type and mutant receptors. This pharmacological chaperoning was thought to result from ligand-mediated stabilization of immature receptors in the endoplasmic reticulum (ER). In the present study, we directly tested this hypothesis using wild-type and mutant forms of the human δ-opioid receptor as models. ER-localized receptors were isolated by expressing the receptors in HEK293 cells under tightly controlled tetracycline induction and blocking their ER export with brefeldin A. The ER-retained δ-opioid receptor precursors were able to bind [³H]diprenorphine with high affinity, and treatment of cells with an opioid antagonist naltrexone led to a 2-fold increase in the number of binding sites. After removing the transport block, the antagonist-mediated increase in the number of receptors was detectable at the cell surface by flow cytometry and cell surface biotinylation assay. Importantly, opioid ligands, both antagonists and agonists, were found to stabilize the ER-retained receptor precursors in an in vitro heat inactivation assay and the treatment enhanced dissociation of receptor precursors from the molecular chaperone calnexin. Thus, we conclude that pharmacological chaperones facilitate plasma membrane targeting of δ-opioid receptors by binding and stabilizing receptor precursors, thereby promoting their release from the stringent ER quality control

Inefficient maturation of the rat luteinizing hormone receptor:a putative way to regulate receptor numbers at the cell surface

Abstract Increasing evidence suggests that the folding and maturation of monomeric proteins and assembly of multimeric protein complexes in the endoplasmic reticulum (ER) may be inefficient not only for mutants that carry changes in the primary structure but also for wild type proteins. In the present study, we demonstrate that the rat luteinizing hormone receptor, a G protein-coupled receptor, is one of these proteins that matures inefficiently and appears to be very prone to premature degradation. A substantial portion of the receptors in stably transfected human embryonic kidney 293 cells existed in immature form of Mr 73,000, containing high mannose-type N-linked glycans. In metabolic pulse-chase studies, only ∼20% of these receptor precursors were found to gain hormone binding ability and matured to a form of Mr 90,000, containing bi- and multiantennary sialylated N-linked glycans. The rest had a propensity to form disulfide-bonded complexes with a Mr 120,000 protein in the ER membrane and were eventually targeted for degradation in proteasomes. The number of membrane-bound receptor precursors increased when proteasomal degradation was inhibited, and no cytosolic receptor forms were detected, suggesting that retrotranslocation of the misfolded/incompletely folded receptors is tightly coupled to proteasomal function. Furthermore, a proteasomal blockade was found to increase the number of receptors that were capable of hormone binding. Thus, these results raise the interesting possibility that luteinizing hormone receptor expression at the cell surface may be controlled at the ER level by regulating the number of newly synthesized proteins that will mature and escape the ER quality control and premature degradation