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 ␦-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 ␦-opioid receptor precursors were able to bind [ 3 H]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 ␦-opioid receptors by binding and stabilizing receptor precursors, thereby promoting their release from the stringent ER quality control. Endoplasmic reticulum (ER)2 quality control, involving molecular chaperones and folding factors, scrutinizes newly synthesized proteins and allows only correctly folded and assembled ones to proceed through the secretory pathway (1). Proteins that do not fulfill the criteria of the quality control are targeted for retrotranslocation and degradation in the cytosol by the ER-associated degradation pathway (2). Since ER quality control relies on conformational rather than functional criteria, even minor changes in the primary structure of a protein can lead to intracellular retention, thus preventing the affected protein from reaching its correct location in the cell. Thus, even salvageable proteins that might be functionally active can be incorrectly directed for degradation. Such an etiology is the underlying cause for a growing number of congenital and acquired conformational diseases, including those that affect G protein-coupled receptors (GPCRs), cell surface seven-transmembrane domain proteins that mediate extracellular messages into intracellular responses. Examples include nephrogenic diabetes insipidus, retinitis pigmentosa, and familial obesity that are caused by mutant forms of the V2 vasopressin receptor, rhodopsin, and melanocortin receptor 4, respectively (3).Since many of the disease-causing proteins are not inherently nonfunctional, attempts to correct their folding and trafficking have attracted considerable attention. Several different ways to alleviate their incorrect cellular localization have ...

The allele frequencies were 0.84 (A) and 0.16 (C). Females with the genotype AC or CC had longer QTcmax (477 +/- 99 ms) and Tpeak-Tend intervals (143 +/- 95 ms) than females with the genotype AA (441 +/- 69 ms and 116 +/- 65 ms, p = 0.005 and p = 0.025, respectively). In males, the QTcmax and the Tpeak-Tend intervals did not differ between the genotypes. After adjustment for echocardiographic and various laboratory variables, the HERG K897T polymorphism remained as an independent predictor of QTcmax (p = 0.009) and the Tpeak-Tend intervals (p = 0.026) in females. CONCLUSIONS; The common K897T polymorphism of the HERG channel is associated with the maximal duration and transmural dispersion of ventricular repolarization in middle-aged females.

The luteinizing hormone receptor (LHR) is a G protein-coupled receptor that is expressed in multiple RNA messenger forms. The common rat ectodomain splice variant is expressed concomitantly with the full-length LHR in tissues and is a truncated transcript corresponding to the partial ectodomain with a unique C-terminal end. Here we demonstrate that the variant alters the behavior of the full-length receptor by misrouting it away from the normal secretory pathway in human embryonic kidney 293 cells. The variant was expressed as two soluble forms of M r 52,000 and M r 54,000, but although the protein contains a cleavable signal sequence, no secretion to the medium was observed. Only a very small fraction of the protein was able to gain hormone-binding ability, suggesting that it is retained in the endoplasmic reticulum (ER) by its quality control due to misfolding. This was supported by the finding that the variant was found to interact with calnexin and calreticulin and accumulated together with these ER chaperones in a specialized juxtanuclear subcompartment of the ER. Only proteasomal blockade with lactacystin led to accumulation of the variant in the cytosol. Importantly, coexpression of the variant with the full-length LHR resulted in reduction in the number of receptors that were capable of hormone binding and were expressed at the cell surface and in targeting of immature receptors to the juxtanuclear ER subcompartment. Thus, the variant mediated misrouting of the newly synthesized full-length LHRs may provide a way to regulate the number of cell surface receptors. INTRODUCTIONNewly synthesized proteins destined for the secretory pathway enter the endoplasmic reticulum (ER) and after correct folding and assembly are either secreted from the cell or transported to their site of action in other cellular compartments. Folding of the nascent proteins is constantly monitored by the ER quality control apparatus. It has an important task in maintaining the cellular homeostasis by preventing premature export of incompletely folded and assembled proteins and removing misfolded and unassembled ones via the ER-associated degradation (ERAD) pathway, presumably by recognizing structural signals that are enriched in incompletely folded proteins (Ellgaard and Helenius, 2003;Helenius and Aebi, 2004). Recent evidence suggests that the ER quality control does not only target permanently misfolded proteins to ERAD but may also dispose some folding competent ones, possibly due to their slow folding kinetics. This applies also to several polytopic membrane proteins, including G protein-coupled receptors (GPCRs; Petäjä-Repo et al., 2000;Imai et al., 2001;Lu et al., 2003;Wü ller et al., 2004;Pietilä et al., 2005). Thus, export from the ER is the limiting step in their expression and probably provides a mean to regulate the number of functional receptors at the cell surface. This is supported by our recent finding that final maturation of the rat luteinizing hormone receptor (rLHR) was found to be developmentally regulated in targ...

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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 M r 73,000, containing high mannose-type N-linked glycans. In metabolic pulse-chase studies, only ϳ20% of these receptor precursors were found to gain hormone binding ability and matured to a form of M r 90,000, containing bi-and multiantennary sialylated N-linked glycans. The rest had a propensity to form disulfide-bonded complexes with a M r 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.