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1 Division of Neuroscience and Reproductive Sciences, Oregon National Primate Research Center,
2 Departments of Physiology and Pharmacology,
3 Cell and Developmental Biology, Oregon Health and Science University, ONPRC/OHSU, 505 NW 185th Avenue, Beaverton, Oregon 97006, USA
(Requests for offprints should be addressed to P Michael Conn; Email: connm{at}ohsu.edu)
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Abstract |
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 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Pharmacological chaperones (‘pharmacoperones’) correct protein misfolding and allow molecules that would otherwise be retained in the endoplasmic reticulum (ER) to route to the plasma membrane (Ulloa-Aguirre et al. 2004, Castro-Fernandez et al. 2005). The pharmacoperone, IN3, increases plasma membrane expression of most naturally occurring mutant GnRHRs, since these are frequently misfolded proteins and retained in the ER (Conn et al. 2002, Couzin 2002, Janovick et al. 2002, Brothers et al. 2003, 2004, Ulloa-Aguirre et al. 2004, Castro-Fernandez et al. 2005). IN3 also increases human WT GnRHR expression at the plasma membrane, an effect not observed for the rodent GnRHRs (Conn et al. 2002, Janovick et al. 2002, Brothers et al. 2003, 2004).
Quality control system (QCS) chaperones in the ER may be responsible for retention of the GnRHR. Calnexin is one such chaperone that binds to newly synthesized proteins for quality assessment (Vassilakos et al. 1998, Ellgaard & Frickel 2003). Calnexin is thought to regulate routing of proteins by preventing protein aggregation (Hebert et al. 1996), allowing properly folded proteins to reach sites associated with their function (Schrag et al. 2003, Kleizen & Braakman 2004), by retaining misfolded proteins in the ER and routing them to degradation pathways (Liu et al. 1999, Molinari et al. 2003, Oda et al. 2003). The calnexin–protein interaction may depend on the phosphorylation state of the calnexin cytoplasmic carboxyl terminus (Roderick et al. 2000).
Calnexin was expressed with WT and mutant GnRHRs to determine whether it is responsible for quality control of GnRHRs. We demonstrate a role for calnexin mediation of GnRHR retention and describe features in both the receptor and the calnexin that are associated with retention.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The GnRH analog, D-tert-butyl-Ser6-des-Gly10-Pro9-ethylamide-GnRH (Buserelin, Hoechst-Roussel Pharmaceuticals, Somerville, NJ, USA), (2S)-2-[5-[2-(2-azabi cyclo[2.2.2]oct-2-yl)-1,1-dimethyl-2-oxoethyl]-2-(3,5-di methylphenyl)-1H-indol-3-yl]-N-(2-pyridin-4-ylethyl) propan-1-amine (IN3, Merck & Co.), myo-[2-3H(N)]-inositol and Na[125I] (Perkin–Elmer, Boston, MA, USA; NET-114A and NEZ-033L), competent cells (Promega), PCR primers, Dulbecco’s Minimal Essential Medium (DMEM), OPTI-MEM, lipofectamine, PBS, and pcDNA3.1 (Invitrogen), endofree maxi-prep kits (Qiagen), rat calnexin (a kind gift from Dr Larry Tjoelker; Tjoelker et al. 1994) and human calnexin, cDNA (Open Biosystems, Huntsville, AL, USA, MHS1011-60083), SMARTpool human calnexin siRNA and siCONTROL non-targeting pool siRNA (Dharmacon Inc., Lafayette, CO, USA), mouse monoclonal anti-hemagglutinin epitope tag (HA) antibody (12CA5, Roche Applied Sciences), rabbit polyclonal anti-calnexin (H-70) antibody (sc-11397; Santa Cruz Bio-technology, Santa Cruz, CA, USA) were obtained as indicated. Other reagents were obtained from commercial sources and were of the highest degree of purity available. GnRHR and calnexin cDNA were prepared using site-directed mutagenesis, as reported (Brothers et al. 2004). Forward and reverse primers for subcloning calnexin cDNA into pcDNA3.1, obtained from the Mammalian Genes Collection(MGC) contained KpnI restriction enzyme site sequence, Kozak’s consensus sequence followed by the ATG start site and 18 nucleotides of the N-terminus of the protein coding sequence (forward primer) or XbaI followed by the reverse complement stop codon and 18 nucleotides of the C-terminus of the complementary protein coding sequence (reverse primer). The identity of all the cDNA mutants and the correctness of all PCR-derived coding sequences were verified by Dye Terminator Cycle Sequencing, according to the manufacturer’s instructions (Perkin–Elmer, Foster City, CA, USA).
Transient transfection and co-transfection
COS-7 cells were cultured, plated, and transfected as previously reported (Brothers et al. 2004). Cells were transfected with 25 ng (unless indicated) WT human or rat GnRHR, or mutant GnRHR and pcDNA3.1 without insert (‘empty vector’) or WT/mutant calnexin cDNA (75 ng/well), as indicated, and 1 µl lipofectamine in 0.125 ml OPTI-MEM (room temperature), according to the manufacturer’s instructions. Empty vector (pcDNA3.1, without insert) was included to bring the total cDNA to 100 ng/well. The concentration of cDNA that does not interfere with the transfection efficiency is 100 ng/well (Janovick et al. 2003, Brothers et al. 2004). Where indicated, 1.5 pMol siRNA/5 x 104 cells was included in the transfection mixture, according to the manufacturer’s instructions. Five hours after transfection, 0.125 ml DMEM with 20% fetal bovine serum and 20 µg/ml gentamicin was added to the wells.
Inositol phosphate (IP) assays
Cells received IN3 treatments as previously reported (Brothers et al. 2004), where indicated. Cells were washed, then ‘pre-loaded’ for 18 h with 1 µCi myo-[2-3H(N)]-inositol in 0.25 ml DMEM (prepared without inositol; Brothers et al. 2004). The cells were then washed twice with 0.25 ml DMEM containing 5 mM LiCl (without inositol), treated for 2 h with 0.25 ml of the indicated Buserelin concentration in the same medium (LiCl prevents inositol phosphate (IP) degradation). Total IPs were determined as described previously (Huckle & Conn 1987, Brothers et al. 2004). Extracellular (EC50) and maximal IP production were calculated using Sigma Plot 8.02 (Jandel Scientific Software, Chicago, IL, USA). Using similar affinities for unlabeled Buserelin and EC50 values averaged from at least three independent experiments, the spare receptors (the percentage of unoccupied receptors at half maximal response, in this case IP production) were calculated (Zhu 1993).
Binding assays
Cells were cultured and plated in growth medium as described previously, except that 105 cells in 0.5 ml growth medium were added to 24-well Costar cell culture plates (cell transfection and medium volumes were doubled accordingly). After 23 h transfection, the medium was removed and replaced with 0.5 ml fresh growth medium. After 27 h transfection, the cells were washed twice with 0.5 ml DMEM containing 0.1% BSA and 20 µg/ml gentamicin, then 0.5 ml DMEM was added. After 18 h, cells were washed twice with 0.5 ml DMEM/0.1% BSA/10 mM HEPES, and then a range of concentrations of [125I]-Buserelin (from 2.5 x 105 to 8 x 106 c.p.m./ml) in 0.5 ml of the same medium were added to the cells and allowed to incubate at room temperature for 90 min (Brothers et al. 2002). After 90 min, the media were removed and radioactivity was measured as described previously (Brothers et al. 2003). To determine non-specific binding, the same concentrations of radioligand were added to similarly transfected cells in the presence of 10 µM unlabeled GnRH. Saturation binding curve fits and calculations were computed using the SigmaPlot 8.02 ( Jandel Scientific Software), a non-linear one-site binding model was used to calculate Kd and Bmax values (Klotz 1982). Binding data was also transformed into Scatchard plots.
Co-immunoprecipitation and western blotting
Cells were cultured and plated in growth medium as described previously, except that 4 x 106 cells in 2 ml growth medium were added to six-well Costar cell culture plates (cell transfection and medium volumes were adjusted accordingly). After 23 h transfection, the medium was removed and replaced with 2 ml fresh growth medium. After 27 h transfection, the cells were washed twice with 2 ml DMEM containing 0.1% BSA and 20 µg/ml gentamicin then 2 ml DMEM was added. For immunoprecipitations, cells were washed twice with 2 ml ice-cold PBS containing protease inhibitors, then 0.5 ml radio immunoprecipitation buffer with protease inhibitors was placed on the cells for 30 min at 4 °C. Immunoprecipitations were carried out as described previously (Firestone & Winguth 1990, Castro-Fernandez et al. 2002). For siRNA treated cells, after 18 h, cells were washed twice with ice-cold PBS and frozen, then thawed in the presence of 0.5 ml 2 x sample buffer. SDS-polyacrylamide gels (8%) and western transfers of cell lysates to nitrocellulose paper (Hoefer Scientific Instruments, San Francisco, CA, USA) were performed as described previously (Conn et al. 1992, Castro-Fernandez et al. 2002). Polyclonal calnexin antibody was used at a 1:200 titer. Control cells for co-immunoprecipitations were transfected with untagged WT GnRH receptor cDNA. The molecular weights of the protein bands were calculated from standards that were color-stained proteins (‘rainbow markers’) with the following molecular weights: myosin, 220 kDa; phosphorylase b, 97.4 kDa; BSA, 66 kDa; ovalbumin, 46 kDa; carbonic anhydrase, 30 kDa; trypsin inhibitor, 21.5 kDa; lysozyme, 14.3 kDa (Amersham Pharmacia Biotech). Western blot band optical densities were quantified using National Institute of Health (NIH) image 1.62 (http://rsb.info.nih.gov/nih-image/).
Statistics
Each experiment was repeated a minimum of three times. Replicates of at least four data points for each treatment group within an experiment were analyzed with one-way ANOVA followed by paired Student’s t-test for individual comparisons (SigmaStat 3.0, Jandel Scientific Software; P < 0.05 was considered significant).
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Human or rat GnRH receptors were co-transfected with calnexin in order to assess the effect on plasma membrane expression (PME) using a radioligand binding assay. Co-transfecting calnexin cDNA with the human or rat GnRHR did not alter Kd of either receptor (Fig. 1A or B
). Human GnRHR PME (Bmax) decreased by 47 ± 3% (P < 0.05) in the presence of calnexin compared with cells co-transfected with empty vector (Fig. 1B). Rat GnRHR PME decreased by 40 ± 6% (P < 0.05) when calnexin was co-transfected. Non-linear saturation binding curves used to calculate Kd and Bmax were also transformed into Scatchard plots (Zhu 1993; Fig. 1C and D).
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Maximal IP production and EC50 values were determined for the human and the rat GnRHRs in the absence or presence of calnexin. There was a significant decrease in the maximal IP production from cells co-transfected with the human receptor and the calnexin compared with cells without added calnexin (P < 0.05). Despite the reduction in PME of the rat GnRH receptor, there was no observable effect of calnexin on maximal IP production (P < 0.05; Fig. 2A).
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). The EC50 of the human GnRHR increased from 400 ± 47 to 640 ± 22 pM (P < 0.05) when calnexin was co-transfected. The EC50 of the rat GnRHR increased from 119 ± 4 to 202 ± 20 pM (P < 0.05) when calnexin was co-transfected. The EC50 values were used to calculate the percentage of unoccupied receptors at half maximal IP production (Gavel & von Heijne 1990). The human GnRHR showed 38.4 ± 5.0% spare receptors at the EC50 ligand concentration, but when calnexin was co-transfected with the human receptor, the quantity of spare receptors fell to 16.5 ± 1.6% (P < 0.05). Similarly, the rat GnRHR had 76.4 ± 0.7% spare receptors at the EC50 ligand concentration, but only 63.1 ± 3.1% spare receptors with calnexin (P < 0.05). The rat GnRHR appears to express sufficient numbers of receptors at the plasma membrane to saturate the signal transduction machinery, even when there is a 40% reduction in PME.
Calnexin co-immunoprecipitates with the GnRHR
HA-tagged human and rat WT GnRH receptors, as well as the E90K mutant of the hGnRHR were co-transfected with calnexin. If anti-HA antibody was used to immunoprecipitate the tagged receptors, then the western blots of the immunoprecipitates were probed with anti-calnexin antibody. When the receptors were transfected alone or in the presence of calnexin cDNA, calnexin co-immunoprecipitated with the receptor, although when calnexin cDNA was co-transfected with the receptor, there appeared to be more calnexin protein bound to the receptors (Fig. 3A). When WT (i.e. untagged) GnRH receptors were transfected into cells immunoprecipitated with the anti-HA antibody, there was no visible calnexin staining (not shown). Cellular calnexin was then immunoprecipitated and subjected to western-blot analysis. When the blots were probed for the presence of the HA-tagged GnRH receptor, there was little specific staining. Presumably this is due to the fact that calnexin is a chaperone for a great number of proteins besides the GnRH receptor that compete for calnexin binding.
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To determine whether calnexin affected the IP production of rat GnRH receptors expressed at approximately the same level as human receptors, cells were transfected with 25 ng human GnRHR cDNA and 2 ng rat GnRHR cDNA. Cells transfected with 2 ng rat GnRHR had approximately the same IP production as 25 ng human GnRHR transfected cells (not shown). Therefore, a ~12.5-fold reduction in DNA was required to achieve a similar level of IP production with the rat GnRHR. As expected, when the 2 ng rat GnRHR was co-transfected with calnexin, there was a significant reduction in the IP response (Fig. 3B).
siRNA knockdown of calnexin and western blotting
To determine whether calnexin protein production (rather than a non-specific effect related to calnexin cDNA transfection) was required for the inhibitory effect on PME, calnexin siRNA was used to knock down calnexin mRNA. Western blotting showed that the siRNA directed against the human calnexin gene also knocked down endogenous calnexin protein expression but not to undetectable levels (Fig. 4A). When calnexin siRNA was co-transfected into cells with either the human or rat GnRHR, there was no measurable change in IP production, compared with cells transfected with control (non-targeting) siRNA (P < 0.05; Fig. 4B). This was a surprising result since we expected that the IP production from these cells would increase in the presence of calnexin siRNA. However, there was significantly increased IP production from calnexin siRNA-treated cells when calnexin siRA was co-transfected into cells with the human GnRHR and calnexin cDNA (P < 0.05).
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). When calnexin siRNA was transfected in cells, there was a significant decrease in calnexin protein levels with the siRNA, calnexin was knocked down by 85–90% (P < 0.05; Fig. 4C). Effect of calnexin on mutant receptors isolated from hypogonadotropic hypogonadism patients
Calnexin was co-transfected with mutant GnRH receptors isolated from patients with hypogonadotropic hypogonadism (HH). The HH mutants hGnRHR (N10K) (partially functional, fully rescuable by pharmacoperone), hGnRHR(E90K) (non-functional, fully rescuable by pharmacoperone), hGnRHR(L266R) (nonfunctional, partially rescuable by pharmacoperone), and hGnRHR(S168R) (non-functional, non-rescuable by pharmacoperone) were selected for study because of these different responses to pharmacoperones.
As with the human WT GnRHR, when calnexin was co-transfected with hGnRHR(N10K), IP production decreased compared with cells transfected with mutant alone (Fig. 5A). Interestingly, co-transfection of either the human WT GnRHR or hGnRHR(N10K) with calnexin and treatment with IN3, yielded greater IP production than in IN3-treated cells expressing hGnRHR(N10K) alone (Fig. 5A). This suggests that calnexin can increase PME of these pharmacoperone-stabilized receptors; however, the PME could not be confirmed with ligand-binding experiments after pharmacoperone treatment, presumably due to incomplete removal of the lipophilic competitive antagonist (not shown).
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). However, as with the human WT GnRHR, IP production from hGnRHR(E90K)-expressing cells increased in the presence of both calnexin and IN3 (Fig. 5B). Even in the presence of IN3, there was no change in the IP production by hGnRHR(S168R) or hGnRHR(L266R) with calnexin compared with without calnexin co-transfection (Fig. 5C and D). K191 modification and addition of the piscine carboxyl terminus (c-tail)
Pre-mammalian GnRH receptors, such as fish and bird GnRHRs, have an extended carboxyl terminus which is truncated in the mammalian GnRHRs. This extended carboxyl terminus (c-tail), when added to the mammalian GnRHR, enhances plasma membrane expression (Lin et al. 1998, Arora et al. 1999, Maya-Nunez et al. 2000). Similarly, rat receptors (327 residues) lack the primate K191 residue that, when deleted from the human sequence (normally 328 residues), also results in increased receptor expression (Lin et al. 1998, Arora et al. 1999, Maya-Nunez et al. 2000).
When the c-tail was added to, or the K191 deleted from the human GnRHR, co-transfection with calnexin no longer affected IP production, either in the absence or presence of IN3 (Fig. 6B). This was also observed when both deletion of K191 and addition of c-tail were made to the human GnRHR (Fig. 6C).
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) when the K191 was deleted, the c-tail added or both modifications made (Fig. 6A–C). Further, neither insertion of K191, nor addition of the c-tail to the rat GnRHR changed receptor-mediated IP production when co-transfected with calnexin (Fig. 6D–E). Addition of both the K191 and the c-tail also did not measurably change receptor-mediated IP production independent of calnexin co-transfection (Fig. 6F). Rat and human calnexin have similar effects on the human and rat GnRHR
Human or rat calnexin was co-transfected with human or rat WT GnRHR. Rat calnexin had a similar effect on IP production from those receptors when compared with receptors co-expressed with human calnexin (not shown).
Mutational analysis of consensus PKC sites in calnexin
Mutations were made at two reported carboxyl terminal protein kinase C (PKC) consensus phosphorylation sites in calnexin (S504A, S583A, or both S504A/S583A; 31). Mutation of S504 to Ala in calnexin (calnexin(S504A)) and co-transfection with the human GnRHR caused decreased GnRHR signaling in both the absence and the presence of IN3, compared with cells co-transfected with either empty vector or WT calnexin (Fig. 7). Neither mutation of S583 to Ala (calnexin(S583A)) nor mutation of both S504 and S583 to Ala (calnexin(S504A/S583A)) changed the observable calnexin effect on IP production (not shown).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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In the presence of a pharmacoperone, there is a calnexin-mediated increase in human GnRHR signaling, likely reflecting an increase in PME, although PME could not be directly measured after pharmacoperone treatment likely due to residual pharmacoperone. The pharmacoperone-stabilized receptors seemed to be more efficiently routed to the plasma membrane. Calnexin appears to act as a quality control protein for the GnRHR by retaining misfolded receptors and steering properly folded receptors to the plasma membrane.
Calnexin did not affect rat receptor-mediated maximal IP production either with or without the pharmacoperone when expressed with similar amounts of cDNA as the human receptor, an interesting observation when considering that calnexin mediated a 40% reduction in rat receptor PME. This effect was not due to the species difference between human and rat calnexin proteins. Nearly, all of the rat GnRHR is properly folded and expressed at the plasma membrane (Lin et al. 1998, Arora et al. 1999, Maya-Nunez et al. 2000); such very high expression is consistent with the observations that the rat receptor is not rescued with pharmacoperone. Only when the cDNA of rat receptor was decreased 12.5-fold, did the additional calnexin decrease maximal IP production.
Addition of the non-mammalian c-tail or deletion of K191 from the human GnRHR dramatically increases plasma membrane expression in both cases (Lin et al. 1998, Maya-Nunez et al. 2000, Leanos-Miranda et al. 2003). Calnexin co-expression with human GnRHRs with the c-tail or without K191 no longer affected signaling. Thus, either calnexin does not interact with these receptors, or, more likely, any reduction in PME did not diminish IP production, as is seen with the rat GnRHR. The c-tail is important in PME since its presence reduces internalization rates resulting in increased PME (Willars et al. 1999, Brothers et al. 2002). Here, the c-tail also functions to increase the stability of the receptor, allowing increased PME.
Calnexin(S504A) reduced the signaling output of the human GnRHR both in the presence and the absence of pharmacoperone. Phosphorylation of this residue may decrease the ability of calnexin to retain the GnRHR. The effect of calnexin(S583A) or calnexin (S504A/S583A) on the human GnRHR appeared not different from the WT calnexin. Roderick et al.(2000) found that de-phosphorylation of S583, mediated by IP-stimulated Ca+ + mobilization, allowed greater expression of sarco-ER calcium ATPase (Wong et al. 1998). In agreement with their results, removal of the PKC consensus phosphorylation site S583 (replaced with Ala) allowed greater expression of the human GnRHR, but only when S504 was also mutated to Ala. The two PKC phosphorylation sites in the carboxyl terminus of calnexin appear to have opposing roles in the retention of proteins, such as the GnRHR.
Calnexin is a lectin-like protein that binds to newly glycosylated proteins in the ER; therefore, it is likely that the calnexin–GnRHR interaction involves one or more of the glycosylated residues in the receptor (Asn18 and Asn102; Davidson et al. 1995). However, when the Asn18 was mutated, the receptor was not expressed at the plasma membrane, and when the Asn102 was mutated, that receptor appeared identical to the wild-type receptor. Therefore, it was not possible to address the action of calnexin on these mutants (data not shown). Likely, glycosylated Asn18 is needed to mediate the interaction.
Like virtually all cells, the COS-7 cells used in this study express endogenous calnexin (Allen et al. 2001). When siRNA was used to knock down the transfected calnexin, the human GnRHR signaling was restored. Calnexin siRNA had little effect on the already robust rat GnRHR signaling. The siRNA targeting human calnexin did not knock down calnexin protein to undetectable levels. Several reasons might explain this initially surprising result. First, since the transfection efficiency is less than 100% (i.e., some cells may not have received siRNA), cells that express the receptor may not have also gotten calnexin siRNA and may express the calnexin protein. Further, it is well known that there is redundancy in protein quality control systems in the ER. One example, calreticulin, has been reported to have a nearly identical action on protein quality control as calnexin (Schrag et al. 2003). It is possible that calreticulin, and potentially other chaperone proteins in the ER, also act on the GnRHR. Finally, there is not full knockdown of the calnexin protein, as seen after quantification of the western blots, 10–15% of calnexin remains in the cell population. Sequence variations between the human and the African green monkey (COS-7 cell) calnexin nucleotide sequences may preclude complete siRNA knockdown.
Though GnRHR expression varies widely throughout the menstrual cycle, it is possible, that receptor expression is lower in vivo than in our model. Nonetheless, we chose to use COS-7 cells in this study for a number of reasons, principally that these cells produce a large quantity of protein from transfected cDNA, thus increasing the burden on the ER and saturating endogenous calnexin.
The effect of pharmacoperone and the observation that misfolded human mutant GnRHRs are retained by calnexin suggesting that this protein chaperone recognizes misfolded proteins. In addition to showing that a proportion of the WT human GnRHR are retained by calnexin, our studies also suggest phosphorylation-dependant regulation of GnRHR PME by calnexin. The increased control over the human GnRHR signaling may be advantageous when regulating the complicated human reproductive cycle but prove disadvantageous when mutations are introduced, as in hypogonadotropic hypogonadism.
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Acknowledgements |
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References |
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Received 25 July 2006
Accepted 24 August 2006
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