Association of calnexin with wild type and mutant AVPR2 that causes nephrogenic diabetes insipidus.

Over 155 mutations within the V2 vasopressin receptor (AVPR2) gene are responsible for nephrogenic diabetes insipidus (NDI). The expression and subcellular distribution of four of these was investigated in transfected cells. These include a point mutation in the seventh transmembrane domain (S315R), a frameshift mutation in the third intracellular loop (804delG), and two nonsense mutations that code for AVPR2 truncated within the first cytoplasmic loop (W71X) and in the proximal portion of the carboxyl tail (R337X). RT-PCR revealed that mRNA was produced for all mutant receptor constructs. However, no receptor protein, as assessed by Western blot analysis, was detected for 804delG. The S315R was properly processed through the Golgi and targeted to the plasma membrane but lacked any detectable AVP binding or signaling. Thus, this mutation induces a conformational change that is compatible with endoplasmic reticulum (ER) export but dramatically affects hormone recognition. In contrast, the W71X and R337X AVPR2 were retained inside the cell as determined by immunofluorescence. Confocal microscopy revealed that they were both retained in the ER. To determine if calnexin could be involved, its interaction with the AVPR2 was assessed. Sequential coimmunoprecipitation demonstrated that calnexin associated with the precursor forms of both wild-type (WT) and mutant receptors in agreement with its general role in protein folding. Moreover, its association with the ER-retained R337X mutant was found to be longer than with the WT receptor suggesting that this molecular chaperone also plays a role in quality control and ER retention of misfolded G protein-coupled receptors.

Newly synthesized human delta opioid receptors retained in the endoplasmic reticulum are retrotranslocated to the cytosol, deglycosylated, ubiquitinated, and degraded by the proteasome.

We have previously shown that only a fraction of the newly synthesized human delta opioid receptors is able to leave the endoplasmic reticulum (ER) and reach the cell surface (Petäjä-Repo, U. E, Hogue, M., Laperrière, A., Walker, P., and Bouvier, M. (2000) J. Biol. Chem. 275, 13727-13736). In the present study, we investigated the fate of those receptors that are retained intracellularly. Pulse-chase experiments revealed that the disappearance of the receptor precursor form (M(r) 45,000) and of two smaller species (M(r) 42,000 and 39,000) is inhibited by the proteasome blocker, lactacystin. The treatment also promoted accumulation of the mature receptor form (M(r) 55,000), indicating that the ER quality control actively routes a significant proportion of rescuable receptors for proteasome degradation. In addition, degradation intermediates that included full-length deglycosylated (M(r) 39,000) and ubiquitinated forms of the receptor were found to accumulate in the cytosol upon inhibition of proteasome function. Finally, coimmunoprecipitation experiments with the beta-subunit of the Sec61 translocon complex revealed that the receptor precursor and its deglycosylated degradation intermediates interact with the translocon. Taken together, these results support a model in which misfolded or incompletely folded receptors are transported to the cytoplasmic side of the ER membrane via the Sec61 translocon, deglycosylated and conjugated with ubiquitin prior to degradation by the cytoplasmic 26 S proteasomes.

Pharmacological chaperones: a new twist on receptor folding.

Protein misfolding is at the root of several genetic human diseases. These diseases do not stem from mutations within the active domain of the proteins, but from mutations that disrupt their three-dimensional conformation, which leads to their intracellular retention by the quality control apparatus of the cell. Facilitating the escape of the mutant proteins from the quality control system by lowering the temperature of the cells or by adding chemicals that assist folding (chemical chaperones) can result in proteins that are fully functional despite their mutation. The discovery that ligands with pharmacological selectivity (pharmacological chaperones) can rescue the proper targeting and function of misfolded proteins, including receptors, might help to develop new treatments for 'conformational diseases'.

Export from the endoplasmic reticulum represents the limiting step in the maturation and cell surface expression of the human delta opioid receptor.

Synthesis and maturation of G protein-coupled receptors are complex events that require an intricate combination of processes that include protein folding, post-translational modifications, and transport through distinct cellular compartments. Relatively little is known about the nature and kinetics of specific steps involved in these processes. Here, the human delta opioid receptor expressed in human embryonic kidney 293S cells is used as a model to delineate these steps and to establish the kinetics of receptor synthesis, glycosylation, and transport. We found that the receptor is synthesized as a core-glycosylated M(r) 45,000 precursor that is converted to the fully mature M(r) 55,000 receptor with a half-time of about 120 min. In addition to trimming and processing of two N-linked oligosaccharides, maturation involves addition of O-glycans containing N-acetylgalactosamine, galactose, and sialic acid. In contrast to N-glycosylation, which is initiated co-translationally and is completed when the protein reaches the trans-Golgi network, O-glycosylation was found to occur only after the receptor exits from the endoplasmic reticulum (ER) and was terminated as early as the trans-Golgi cisternae. Once the carbohydrates are fully processed and the receptor reaches the trans-Golgi network, it is transported to the cell surface in about 10 min. The exit from the ER was found to be the limiting step in overall processing of the receptor. This indicates that early events in the folding of the receptor are probably rate-limiting and that receptor folding intermediates are retained in the ER until they can adopt the correct conformation. The overall low efficiency of receptor maturation, less than 50% of the precursor being processed to the fully glycosylated protein, further suggests that only a fraction of the synthesized receptors attain properly folded conformation that allows exit from the ER. This indicates that folding and ER export are key events in control of receptor cell surface expression. Whether or not the low efficiency of the ER export is a general feature among G protein-coupled receptors remains to be investigated.

Structure and functional significance of the carbohydrates of the LH/CG receptor.

The LH/CG appears to contain 1-4 bi- or multi-antennary complex-type N-linked oligosaccharide side chains, which appear to locate apart from the hormone-binding regions. The exact sites to which the N-linked chains are attached remain to be delineated. The carbohydrates of the mature membrane-inserted receptor do not contribute to either specific high-affinity ligand-binding or signal transduction of the receptor. Thus, the polypeptide core of the receptor is responsible for both high affinity binding and dictating the hormone specificity. Moreover, the deglycosylated receptor, once inserted to the plasma membrane in a functionally mature form, retains its functional conformation or permits the conformational change that is required for coupling of the receptor to effector enzymes. Addition of oligosaccharides to the nascent LH/CG receptor but not their subsequent conversion to complex-type ones appears to be required for acquiring the hormone-binding conformation. On the other hand, neither addition of oligosaccharides to the nascent receptor, nor their further maturation are needed for the transport of the receptor to the plasma membrane. Thus, one function of the N-linked oligosaccharides in the LH/CG receptor appears to be to direct the proper folding of the receptor.

Structural characterization of the carbohydrates of the rat ovarian luteinizing hormone/chorionic gonadotropin receptor.

The numbers and types of oligosaccharide present on the rat ovarian luteinizing hormone (LH)/chorionic gonadotropin (CG) receptor were determined by treating radiolabelled purified receptors with glycosidases and examining the changes in electrophoretic mobility and number of radiolabelled bands on SDS/PAGE. The purified receptor was also transferred to nitrocellulose after SDS/PAGE and probed with digoxigenin-labelled lectins. The following conclusions were drawn: (1) the rat ovarian LH/CG receptor contains at least two complex-type N-linked oligosaccharide chains, of which one is biantennary and the rest multiantennary. (2) The N-linked chains terminate in either unsubstituted galactose or sialic acid linked alpha 2-3 or alpha 2-6 to the penultimate galactose. (3) The N-linked oligosaccharides also contain internal poly(N-acetyl-lactosamine) sequences and fucose-linked alpha 1-6 to the proximal N-acetylglucosamine. (4) No O-linked carbohydrate moieties are present on the receptor molecule.

Significance of the extracellular domain and the carbohydrates of the human neutrophil N-formyl peptide chemotactic receptor for the signal transduction by the receptor.

The N-formyl peptide chemotactic receptor of human neutrophils possesses a 2-kDa papain-removable extracellular domain that contains two N-linked oligosaccharide side chains and is not required for the high-affinity ligand binding. In the present study, the significance of the extracellular domain and the carbohydrates for signal transduction was elucidated by measuring the N-formyl hexapeptide-induced intracellular free calcium ([Ca2+]i) and the change in myeloperoxidase secretion in the control and papain-treated human neutrophils. [Ca2+]i was monitored both in cell suspension and individual cells with intracellularly trapped Fura 2 acetoxymethyl ester, using spectrofluorometric analysis and fluorescence ratio image analysis, respectively. The exposure of the cells in suspension to N-formyl hexapeptide resulted in an immediate, dose-dependent burst of elevated [Ca2+]i, which was virtually identical in both control and papain-treated cells with respect to the extent and kinetics. The maximum burst was 1.6-fold and was obtained at 10(-6) M hexapeptide. The individual control and papain-treated cells responded to 10(-6) M hexapeptide in a similar manner with several successive transients of [Ca2+]i, the maximum level being 3.0-3.5 microM. In both groups the [Ca2+]i transient began initially in the cell periphery, expanding rapidly throughout the cells. Concomitantly, the cells became polarized, and their chemokinesis increased. The secretion of myeloperoxidase was monitored as a physiological end response to N-formyl chemotactic peptides. The exposure of the control and papain-treated cells in suspension to hexapeptide resulted in a dose-dependent secretion of myeloperoxidase. The maximum secretion after exposure to 10(-8)-10(-6) M hexapeptide was equal in control and papain-treated neutrophils. These results indicate that the functional properties of the membrane-inserted N-formyl peptide chemotactic receptor are inherent to the receptor's transmembrane and intracellular domains, as far as binding of the ligand and subsequent receptor activation are concerned.

Significance of the carbohydrate moiety of the rat ovarian luteinizing-hormone/chorionic-gonadotropin receptor for ligand-binding specificity and signal transduction.

The contribution of the carbohydrate moiety of the rat ovarian luteinizing-hormone (LH)/chorionic-gonadotropin (CG) receptor to ligand-binding specificity and signal transduction was investigated by using glycosidases. Purified membranes from pseudo-pregnant rat ovaries were treated with neuraminidase or peptide N-glycosidase F, to remove terminal sialic acids and N-linked oligosaccharides of the receptor, respectively. Ligand blotting and densitometric scanning of the autoradiograms showed that 90-95% of the receptors were deglycosylated, and that desialylation was virtually complete. Neither the desialylated nor the deglycosylated receptors were able to bind human follicle-stimulating hormone or bovine thyroid-stimulating hormone, as revealed by competition binding experiments. The 50% effective dose of hCG for adenylate cyclase activation, as determined by measuring the formation of cyclic [32P]AMP from [alpha-32P]ATP for 15 min at 30 degrees C, was similar in the control and deglycosylated membranes: 10.2 +/- 3.3 nM and 12.2 +/- 3.8 nM respectively. The same was true for the time course of the basal, hCG- and forskolin-stimulated enzyme activity. In addition, removal of oligosaccharides from the receptor did not restore the ability of desialylated hCG, nor of the deglycosylated hormone, to stimulate adenylate cyclase. In conclusion, the carbohydrate moiety of the native membrane-inserted rat ovarian LH/CG receptor does not contribute to the ligand-binding specificity, and it is not required for the functional coupling of the occupied receptor and the adenylate cyclase system. These functions are associated with the polypeptide portion of the receptor.

Rat and human neutrophil N-formyl-peptide chemotactic receptors. Species difference in the glycosylation of similar 35-38 kDa polypeptide cores.

Rat and human neutrophil N-formyl-peptide chemotactic receptors were subjected to glycosidase and proteinase treatments to determine the extent and species differences of glycosylation and the carbohydrate requirement in the high-affinity ligand binding. N-Formyl-Nle-Leu-Phe-Nle-125I-Tyr-Lys was attached to rat and human neutrophils either before or after glycosidase and proteinase treatments, and the labelled receptors were solubilized after glutaraldehyde cross-linking and analysed by SDS/PAGE and autoradiography. Both the rat and human N-formyl-peptide chemotactic receptors contain only N-linked oligosaccharides, as demonstrated by their sensitivity to peptide N-glycosidase F (PNGase F) and resistance to O-glycanase treatment. The N-linked oligosaccharides seem to be of the complex type rather than the high-mannose or hybrid type and lack terminal sialic acid, as demonstrated by their resistance to endoglycosidases D and H and neuraminidase treatments. This sensitivity pattern was similar in both species, and the shift in the molecular size of the receptors to 35-38 kDa after PNGase F treatment occurred through one intermediate product, suggesting that both receptors contain a similar 35-38 kDa polypeptide core with two N-linked complex-type oligosaccharides, the heterogeneity of which is responsible for the species difference in receptor size. Papain treatment alone or followed by PNGase F produced in both species a 33-36 kDa membrane-bound fragment that was still able to bind the ligand, suggesting that the oligosaccharides are located on the approx. 2 kDa papain-cleavable polypeptide fragment of the receptors. The cleavage sites for both papain and PNGase F were hidden in occupied receptors, suggesting a conformational or topographical change in these upon ligand binding. Scatchard analyses and cross-linking experiments demonstrated that carbohydrates are not required for high-affinity ligand binding and that the 33-36 kDa membrane-bound papain fragment of both receptors contains the ligand-binding site.

Significance of the glycan moiety of the rat ovarian luteinizing hormone/chorionic gonadotropin (CG) receptor and human CG for receptor-hormone interaction.

The role of the glycan moiety of the rat ovarian LH/CG receptor and human CG (hCG) in high-affinity receptor-hormone interaction was investigated by cross-linking and quantitative binding experiments. hCG and its derivatives, desialylated hCG and deglycosylated hCG were labeled either to the alpha-subunit (125I) or the beta-subunit (3H). The ligands were attached to ovarian membrane particles, which were treated with neuraminidase or peptide-N-glycosidase F to remove terminal sialic acids or N-linked oligosaccharides of the receptor, respectively, and the complexes formed were solubilized, cross-linked with glutaraldehyde, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All of the ligands produced similar autoradiographic patterns with the native or glycosidase-treated receptor, and only the receptor-(alpha)hCG and receptor-(alpha, beta)hCG complexes were detected. Moreover, quantitative binding studies indicated that all of the hormone derivatives had similar affinities for the native or glycosidase-treated receptor. In addition, the orientation of the carbohydrate side chains on the receptor-hormone complex was studied by digesting the complex with the glycosidases. The molecular weight of the receptor, evidenced by ligand blotting, was reduced to the same extent, whether the membrane-bound free receptor or receptor-hormone complex was treated with the glycosidases, suggesting that the oligosaccharide side chains of the receptor are apart from the hormone binding region. As peptide-N-glycosidase F treatment reduced the size of the Mr 90,000 receptor first to about Mr 67,000 and finally to about Mr 62,000, there may possibly be 2 N-linked carbohydrate chains per receptor polypeptide. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the glycosidase-treated receptor-[125I]hCG complex also revealed that neuraminidase was able to remove the sialic acids from both subunits of the receptor-bound hormone. In conclusion, the results suggest that hCG interacts with the polypeptide backbone of its ovarian receptor mainly through the peptide core of its alpha-subunit. Moreover, the carbohydrate side chains of both subunits of hCG are positioned on the outward face of the receptor-hormone complex.

Subunit interaction of human chorionic gonadotropin (hCG) with rat ovarian luteinizing hormone (LH)/CG receptor.

The subunit interaction of hCG with its rat ovarian LH/CG receptor was studied by cross-linking the solubilized receptor-hormone complex with glutaraldehyde (GA), disuccinimidyl suberate (DSS) or dithiobis(succinimidyl propionate) (DSP) and analyzing the complexes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. The hormone was labeled either in its alpha-subunit (125I-hCG) or in its beta-subunit (3H-hCG) or the label (3H) was introduced into the receptor molecule instead of the hormone. All of the labeling procedures led to the detection of only the receptor-(alpha,beta)hCG and receptor-(alpha)hCG complexes on the autoradiograms. The sizes of these complexes were 137,000 and 106,000, respectively, under reducing conditions. These results suggest that the receptor binds one hormone molecule, and that hCG interacts with the receptor mainly through its alpha-subunit. In addition, polyclonal antibodies directed against the LH/CG receptor and the alpha- and beta-subunits of hCG were used to detect the non-reduced receptor-(alpha,beta)hCG complex in immunoblotting. As antibodies directed against both the alpha-subunit and the beta-subunit were able to detect the Mr 130,000 complex, it is conceivable that both of the subunits are at least partially exposed on the receptor-hormone complex. 125I-hCG was also cross-linked to the membrane-bound receptor. The membrane-bound complex had an Mr of 144,000 under reducing conditions, i.e. approximately 7000 higher than that of the solubilized complex (Mr 137,000). This may indicate that the membrane-bound receptor is covalently modified or differs in conformation from the solubilized receptor.