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Divisions of Neuroscience (S.P.B., A.C., J.A.J., P.M.C.) and Reproductive Biology (P.M.C.), Oregon National Primate Research Center and Departments of Physiology and Pharmacology (S.P.B., A.C., J.A.J., P.M.C.) and Cell and Developmental Biology (P.M.C.), Oregon Health and Science University, Beaverton, Oregon 97006
Address all correspondence and requests for reprints to: P. Michael Conn, Oregon National Primate Research Center/Oregon Health and Science University, 505 Northwest 185th Avenue, Beaverton, Oregon 97006. E-mail: connm{at}ohsu.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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This etiology was first reported in 1997 as compound heterozygous mutations in a family with HH (2); to date, 14 point mutations of the GnRHR have been described, as well as one truncation mutant. The point mutations are widely distributed across the entire sequence of the hGnRHR; whereas some mutants retain a modest degree of function (N10K, T32I, Q106R, R262Q and Y284C), others are totally nonfunctional (E90K, A129D, R139H, S168R, A171T, C200Y, S217R, L266R, C279Y, and L314X). When coexpressed in heterologous cell systems, nonfunctional mutants (E90K, A129D, R139H, S168R, C200Y, S217R, L266R, and C279Y) also inhibit radioligand binding and effector coupling by the human WT receptor [dominant-negative effect (3)].
Sequence modifications, such as the addition of the African catfish GnRHR intracellular carboxy-terminal 51-amino acid sequence (4), or the deletion of the extra K191 [found in humans, but not rodent (5, 6)], increase plasma membrane expression of the hGnRHR. Such modifications also rescue HH mutants when engineered into the HH mutant receptor proteins. This suggested that mutants did not lose the intrinsic ability to bind ligand or activate effector but, rather, were misrouted proteins that were not present at the plasma membrane (7). This view was confirmed with the identification of membrane-permeable pharmacological chaperones that could rescue the majority of HH mutants (12 of the 14 point mutations reported) by correcting folding errors and allowing the receptors to route to the plasma membrane, where they functioned similarly to WT hGnRHRs (3, 8, 9).
Protein misrouting is becoming a recognized disease etiology; this mechanism may prove to be more common than previously appreciated (10, 11, 12). Diseases that are caused by misfolded proteins and resultant misrouting range from cystic fibrosis (CFTR chloride channel) to retinitis pigmentosa (rhodopsin, carotenoid receptors), and nephrogenic diabetes insipidus (aquaporin-2, V2R) among others and have been the subject of reviews (8, 10, 11, 12, 13, 14, 15, 16). Because, like other GPCR family members (17, 18), the GnRHR oligomerizes (19, 20), the possibility that the dominant-negative effect could be explained by intracellular retention of the WT GnRHR with the HH hGnRHR mutant was assessed.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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, inset) was diminished when the total amount of transfected cDNA exceeded 100 ng/well, regardless of the amount of WT hGnRHR used (Fig. 1A). Accordingly, in all subsequent cell culture experiments, empty vector was added to maintain the cDNA concentration at 100 ng/well, so as to eliminate nonspecific actions caused by differences in cDNA concentrations.
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). All subsequent cell culture experiments used 5 ng WT hGnRHR cDNA per well, as this concentration provided an adequate signal, yet was well within the linear response range.
Radioligand Binding in COS7 Cells Cotransfected with WT and HH Mutant GnRHRs
Ligand binding was determined in cells cotransfected with 5 ng WT hGnRHR and 0, 5, 50, or 95 ng of hGnRHR(E90K), hGnRHR(L266R), or hGnRHR(S168R) cDNAs (Fig. 2A; empty vector was added to keep the total cDNA at 100 ng/well). When the WT hGnRHR was coexpressed with either 95 ng of hGnRHR(E90K), hGnRHR(L266R), or hGnRHR(S168R), these cells bound 24.2 ± 1.1%, 21.5 ± 0.6%, and 24.2 ± 0.6% as much radioligand as cells cotransfected with WT hGnRHR and empty vector (P < 0.05).
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), IP production in response to 100 nM Buserelin was measured in COS7 cells that were cotransfected with 5 ng WT hGnRHR and a range of either hGnRHR(E90K), hGnRHR(L266R), or hGnRHR(S168R) cDNAs (0–95 ng). There was a gradual decrease in the quantity of IP produced in cells transfected with greater amounts of the mutant cDNA, and a maximal decrease in IP production was seen when 50–95 ng of HH mutant hGnRHR cDNA were transfected with the 5 ng WT hGnRHR (ratios between 10 and 19) for each mutant (Fig. 3A). In cells cotransfected with 5 ng WT hGnRHR and 95 ng hGnRHR(E90K), hGnRHR(L266R), or hGnRHR(S168R), IP production was reduced to 31.4 ± 3.2%, 32.7 ± 2.5%, and 30.4 ± 1.1% of IP production in cells with empty vector, respectively (Fig. 3Aand Table 1; P < 0.05).
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) was coexpressed with HH hGnRHR mutants to determine whether the mutants had a dominant-negative effect on this construct. GFP-tagged WT hGnRHR cDNA (5 ng) was cotransfected with 0, 5, 50, or 95 ng of hGnRHR(E90K), hGnRHR(L266R), or hGnRHR(S168R) cDNA. Cells expressing GFP-tagged WT hGnRHR without mutant produced a slightly greater amount of total IP in the presence of GnRH agonist, compared with WT (no GFP; Fig. 3B).
Subcellular Localization of GFP-Tagged WT hGnRHR in COS7 Cells Cotransfected with HH Mutants
GFP-tagged WT hGnRHR cDNA (5 ng) was cotransfected with 95 ng empty vector, hGnRHR(E90K), hGnRHR(L266R), or hGnRHR(S168R) cDNA. After 41 h, cells were treated for 5 h with the protein synthesis inhibitor, cycloheximide, and then imaged by confocal microscopy (Fig. 4, A–H). The endoplasmic reticulum (ER) was stained blue using ER-tracker dye (Molecular Probes, Eugene, OR). Cotransfection of the GFP-tagged WT hGnRHR and empty vector showed a broad distribution of the receptor in the cell (Fig. 4A). When HH hGnRHR mutants were coexpressed with the GFP-tagged WT hGnRHR, there was retention in the cell that colocalized with the ER stain (Fig. 4, C, E, and G).
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and 4D), compared with untreated cells (Fig. 4C). IN3-treated cells coexpressing GFP-tagged WT hGnRHR and hGnRHR(L266R) had a small amount of the GFP localized at the plasma membrane, with the remainder of the GFP colocalized with the ER stain (Fig. 4F), compared with untreated cells (Fig. 4E). Cells coexpressing GFP-tagged WT hGnRHR and hGnRHR(S168R) did not have any apparent change in GFP localization (Fig. 4H), compared with untreated cells (Fig. 4G); the GFP remained colocalized with the ER stain. GFP and ER colocalization was demonstrated by showing occupancy of the same space in confocal sections and by using threedimensional (3D) reconstructions; online supplemental material of the 3D reconstructions for Fig. 4, C and D, are provided (published as supplemental Figs. S1 and S2, respectively, on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org). Lysozome- and Golgi-specific stains showed no colocalization with GFP in either compartment (data not shown). The effect of treating cells transfected with only WT hGnRHR, hGnRHR(E90K), hGnRHR(L266R), or hGnRHR(S168R) cDNA and then treated with vehicle and 1 µg/ml IN3 is shown in Fig. 4, I–L: the IP production increases somewhat and is fully, partially, and not rescued after treatment with IN3, respectively.
Pharmacological Rescue of IP Production in COS7 Cells Cotransfected with WT and HH Mutant GnRHRs
IN3 treatment of cells cotransfected with the WT hGnRHR and hGnRHR(E90K) cDNAs rescued receptor-mediated IP production (Fig. 5). This drug also partially rescued receptor-mediated IP production in cells coexpressing the WT hGnRHR and hGnRHR(L266R) but was unable to rescue WT hGnRHR or hGnRHR(S168R) when these two receptors were coexpressed (Fig. 5): the hGnRHR(S168R) mutant continued to have a dominant-negative effect on the WT hGnRHR.
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). In contrast, when 5 ng WT hGnRHR cDNA and 0 or 95 ng rGnRHR(C114A) or rGnRHR(C195A) [nonfunctional rat GnRHR mutants (21)] were cotransfected, both the rat GnRHR mutants caused reduced IP production (Table 1).
Ninety-five nanograms of the human WT forms of the angiotensin II receptor (A2R), neuropeptide Y1 receptor (NPYR), or 
opioid receptor (KOR) were cotransfected with 5 ng WT hGnRHR. A2R and NPYR had no measurable effect on WT hGnRHR signaling, whereas the KOR had a dominant-negative effect on the WT hGnRHR (Table 1).
Because the COS7 cell line may be deficient in the receptor activity-modifying proteins [RAMPs (22)] proposed to be responsible for trafficking of particular GPCRs (23), 45 ng RAMP1, RAMP2, or RAMP3 cDNAs and 50 ng of empty vector, hGnRHR(E90K), hGnRHR(L266R), hGnRHR(S168R), A2R, NPYR, and KOR cDNA with 5 ng WT hGnRHR cDNA were cotransfected in COS7 cells. Overexpression of RAMP1, RAMP2, or RAMP3 had no measurable effect on the expression or function of the WT or mutant hGnRHRs (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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After it was determined that 5 ng WT hGnRHR cDNA with 100 ng total cDNA per well produced an IP production response in the linear range for transfection in COS7 cells, cells were cotransfected with WT hGnRHR and each of the HH mutant hGnRHRs to determine whether the WT hGnRHR binds ligand or couples with effector when coexpressed with individual HH hGnRHR mutants. There was a gradual decrease in the quantity of IP produced in cells transfected with greater amounts of the mutant cDNA, and maximal decrease in IP production was seen when 50–95 ng of HH mutant hGnRHR cDNA were transfected with 5 ng of WT hGnRHR (ratios between 10 and 19), regardless of which mutant cDNA was used. Loss of IP production paralleled loss of radioligand binding.
When the GFP-tagged WT hGnRHR was coexpressed with HH hGnRHR mutants, the mutants had a dominant-negative effect on GFP-tagged WT hGnRHR IP production; however, cells expressing GFP-tagged WT hGnRHR without mutant produced a slightly greater amount of total IP in the presence of GnRH agonist, compared with WT. In the GFP-tagged hGnRHR chimera a spacer derived from the African catfish carboxyl terminus was necessary to maintain a functional chimera (4). When spacer alone was attached to the GnRHR (i.e. no GFP) the chimera showed a 5-fold increase in plasma membrane receptor expression (4). Addition of the GFP molecule to the GnRHR-spacer chimera reduced plasma membrane expression of the final construct to only slightly above WT levels (24). Accordingly, a slightly greater quantity of HH mutant hGnRHR cDNA was required to obtain the same level of inhibition of the GFP-tagged WT GnRHR compared with the WT hGnRHR.
For confocal microscopy, an excess of mutant compared with WT receptor was used to ensure that most cells received mutant (the presence of GFP-tagged WT hGnRHR could be confirmed visually) so that COS7 cells transfected with GFP-tagged WT hGnRHR were also likely transfected with hGnRHR(E90K), hGnRHR(L266R), or hGnRHR(S168R) cDNA. After being treated with protein synthesis inhibitor and stained with ER dye, cells were imaged by confocal microscopy (Fig. 4, A–H). The broad distribution of the GFP-tagged WT hGnRHR, when cotransfected with empty vector presumably reflects plasma membrane routing, because identically cotransfected cells responded to agonist stimulation (Fig. 3B). When HH hGnRHR mutants were coexpressed with the GFP-tagged WT hGnRHR, there was retention in ER (Fig. 4, C, E, and G). Retention paralleled loss in agonist-stimulated IP production from identically cotransfected cells (Fig. 3B), suggesting that the mechanism of action of HH mutant GnRHRs is sequestration of the WT hGnRHR in the ER, precluding routing to the plasma membrane of the cell (and ligand). Interestingly, each of the dominant-negative mutants had similar effects on the WT receptor in the cell, with regard to both the quantity of receptor required for comparable reductions in IP production (Fig. 3A) and the localization of the WT receptor in the cell when coexpressed with mutant (Fig. 4). This suggests that the mechanism for the dominant-negative effect of the mutant receptor on the WT receptor is similar for each mutant, regardless of whether the mutant receptor is responsive to IN3 rescue.
IN3 has been shown to rescue most misfolded hGnRHR mutants by allowing them to escape the quality control apparatus that ordinarily degrades them (8, 9). After IN3 removal, the rescued mutants are similar to WT in terms of radioligand binding, effector coupling, turnover, or ligand specificity (9). Other GnRH peptidomimetics from different chemical classes, also rescue mutants (25). In cells expressing only the GFP-tagged WT hGnRHR or coexpressing GFP-tagged WT hGnRHR and hGnRHR(E90K), IN3 caused both the WT and hGnRHR(E90K) receptors to route to the plasma membrane, whereas IN3 treatment of cells coexpressing GFP-tagged WT hGnRHR and hGnRHR(L266R) partially rescued these receptors, with the remainder of the GFP colocalized with the ER. IN3 treatment of cells coexpressing GFP-tagged WT hGnRHR and hGnRHR(S168R) did not have any apparent change in GFP localization, the GFP remained localized in the ER.
IN3 treatment of cells cotransfected with the WT hGnRHR and hGnRHR(E90K) cDNAs rescued receptor-mediated IP production. This drug also partially rescued receptor-mediated IP production in cells coexpressing the WT hGnRHR and hGnRHR(L266R) but was unable to rescue WT hGnRHR or hGnRHR(S168R) when these two receptors were coexpressed, suggesting that the hGnRHR(S168R) mutant continued to have a dominant-negative effect on the WT hGnRHR. Rescue of the WT hGnRHR from the dominant-negative effect of the mutants paralleled rescue of the mutants alone, suggesting that the material retained in the ER is a heterooligomer of the WT and mutant. There is likely a specific recognition motif and/or a (chaperone) protein responsible for the routing of the receptor signaling complex; considering the wide distribution of point mutations in the GnRHR that can cause the dominant-negative phenotype, it is reasonable to imagine that a change in conformation promotes retention of the mutants. Such a mutation might block interaction with protein chaperones or target the mutant protein toward degradation pathways. The determinant for routing (or retention) of the receptor is encoded in the GnRHR itself, allowing that posttranslational modification of the GnRHR may have significant influence on the routing of the receptor to the plasma membrane. Receptor mislocalization is reversed by the addition of the GnRHR antagonist IN3, which binds to the WT and mutant GnRH receptors and rescues function by allowing proper plasma membrane localization, likely as a result of conformational stabilization of the receptor. Because IN3 cannot rescue the dominant-negative effect of hGnRHR(S168R) on the WT hGnRHR, it is likely that all proteins in a multimeric complex must be in the correct conformation to be properly routed (or to avoid degradation).
Because only about half of the hGnRHR synthesized is expressed at the plasma membrane, whereas almost all the rodent GnRHR appears to be routed to the plasma membrane (8, 26), the effect of human mutants on the rat WT GnRHR and rat mutants on the human WT GnRHR was assessed. There was no measurable decrease in IP production when any of the HH hGnRHR mutants were cotransfected with the rat WT GnRHR, whereas nonfunctional rat GnRHR mutants had a dominant-negative effect on the WT hGnRHR (Table 1). The observation that plasma membrane expression of rat WT GnRHR was not affected by human mutants, whereas rat mutants showed a dominant-negative effect on the human WT, is particularly interesting in light of our understanding that only about half of the hGnRHR synthesized is routed to the plasma membrane, whereas almost all of the rodent GnRHR appears to be routed to the membrane and is likely due to increased sensitivity for regulation of the hGnRHR compared with rat GnRHR, through an as-yet-unidentified mechanism. Routing appears to be regulated, in part, by K191, which is present in the primate, but not rodent, receptor (5, 6), although other features of the receptor appear to be associated with diminished expression at the plasma membrane (26, 27). In evolving from fish and avians to mammals, there appears to have been a carboxyl-terminal truncation that is associated with diminished plasma membrane expression levels, presumably due to more rigorous control of reproduction in more evolved species.
To assess specificity, the angiotensin II receptor (A2R), neuropeptide Y1 receptor (NPYR) or -opioid receptor (KOR) were cotransfected with the WT hGnRHR. The KOR, surprisingly, had a dominantnegative effect on the WT hGnRHR, whereas the other two receptors had no effect (Table 1). The inhibition of IP production from cells cotransfected with the WT hGnRHR and KOR was similar to cells cotransfected with the same quantities of WT hGnRHR and the HH GnRHR mutant cDNA, suggesting a similar mechanism of action. Observation of this dominant-negative effect is interesting because both the KOR and GnRHR regulate placental hCG release and opioids can regulate pituitary LH release (28, 29); also, KOR and GnRHR use similar mechanisms to down-regulate (30, 31). Concerns regarding the specificity of the dominant-negative effect were laid to rest as a result of two observations. First, among non-GnRHRs, this effect was not generally observed; and second, among GnRHR mutants, retention of WT was consistent with the known order with which mutants are retained in the ER.
Because the COS7 cell line may be deficient in the receptor activity modifying proteins, there was a possibility that the lack of these proteins could limit the cellular routing of the overexpressed GnRHRs. However, coexpression of RAMP1, RAMP2, or RAMP3 had no measurable effect on the expression or function of the WT or mutant hGnRHRs.
In vivo, each cell in a human heterozygote (mutant-WT) would likely express those genes equally, a condition that cannot be assumed when equal amounts of vectors are transfected into cells in vitro. Further, because heterozygotic patients expressing highly dominant-negative mutants would be infertile, such mutations would have been selected against, and those that appear in the population would be among the least severe in this regard. Accordingly, it is not surprising that slightly higher than a 1:1 ratio (mutant-WT) is required to observe a dominant-negative effect in vitro.
A recent report suggested that a fly receptor mutant trapped the WT receptor in the ER (32); our study suggests that a similar mechanism can explain a dominant-negative effect associated with human disease. ER retention, probably as a result of heterologous oligomerization between WT and dominant-negative receptors, may be more common than previously recognized because a large number of proteins linked to human disease appear to be caused by mislocalized proteins (8, 10, 11, 12, 13, 14, 15, 16). Coupled with the observation that hGnRHRs have evolved to restrict the percentage of plasma membrane-expressed protein, the present data suggest that aberrant protein retention might be a common feature in human disease.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-opioid receptor, RAMP1, RAMP2, and RAMP3 (Guthrie cDNA Resource Center, www.cdna.org) were obtained as indicated. Other reagents were obtained from commercial sources and were of the highest degree of purity available. WT, HH mutant, and hGnRHR cDNA tagged with GFP (GFP-tagged WT hGnRHR) were prepared as reported previously (33, 34).
Transient Transfection and Cotransfection
Cells were cultured and plated in growth medium (DMEM, 10% fetal calf serum, 20 µg/ml gentamicin); growth conditions were 37 C and 5% CO2 in a humidified atmosphere, and all medium was warmed to 37 C before being added to the cells, unless otherwise noted. For transfection of WT or mutant receptors into COS7 cells, 5 x 104 cells were plated in 0.25 ml growth medium in 48-well Costar cell culture plates. Twenty-four hours after plating the cells were washed once with 0.25 ml OPTI-MEM and then transfected with 100 ng total cDNA (pcDNA3.1 without insert "empty vector" was included to bring the total cDNA to 100 ng/well, unless otherwise indicated) and 1 µl lipofectamine in 0.125 ml OPTI-MEM (room temperature), according to manufacturer’s instructions. For cotransfection experiments the cells were cotransfected with WT hGnRHR (5 ng/well) and empty vector or HH mutant hGnRHR (5–95 ng/well) or other GPCR cDNAs, as indicated, using 1 µl lipofectamine in 0.125 ml Opti-MEM. The total amount of DNA transfected remained constant, as complementary amounts of empty pcDNA3.1 (empty vector), were included in the transfection mixture. Five hours after transfection, 0.125 ml DMEM with 20% fetal calf serum and 20 µg/ml gentamicin was added to the wells. Twenty-three hours after transfection the medium was removed and replaced with 0.25 ml fresh growth medium. Where indicated, 1 µg/ml IN3 in 0.1% final dimethylsulfoxide (vehicle) was added in respective media to the cells, and then removed 18 h before agonist treatment, as described elsewhere (3, 9, 34).
Saturation Binding Assays
Saturation receptor binding was performed on live cells using 2 x 106 cpm/ml of [125I]Buserelin [specific activity, 700 µCi/mg; (3)]. Twenty-seven hours after transfection the growth serum was removed and replaced with DMEM/20 µg/ml gentamicin. Forty-six hours after transfection the cells were washed twice in DMEM/0.1% BSA/10 mM HEPES, and the radioactivity was added to the cells in 0.25 ml of the same medium. The cells were allowed to equilibrate for 90 min at room temperature, the tracer was removed, and the plates were placed on ice and washed twice with 0.5 ml ice-cold PBS. The cells were dissolved in 0.2 M NaOH/1% sodium dodecyl sulfate for 30 min, the liquid was aspirated into tubes, and radioactivity was determined using a Packard 
-counter (Packard Instruments, Downers Grove, IL).
Inositol Phosphate (IP) Assays
Cells were washed twice with 0.25 ml DMEM containing 0.1% BSA and 20 µg/ml gentamicin 27 h after transfection and then preloaded for 18 h with 0.25 ml of 4 µCi/ml myo-[2-3H(N)]inositol in DMEM (prepared without inositol). After preloading, cells were washed twice with 0.25 ml DMEM containing 5 mM LiCl (without inositol), and then treated for 2 h with in 0.25 ml of a concentration of Buserelin (100 nM) in the same medium (LiCl prevents IP degradation). The media were removed and the cells were frozen and thawed in the presence of 0.5 ml of 0.1 M formic acid (to rupture cells), and total IPs were determined as previously described (35).
Confocal Experiments
Two-well culture slides (Costar, Cambridge, MA) were soaked in 12 N HCl for 2 h to facilitate cell attachment. The slides were then rinsed four times with sterile water and once with growth medium before use. Cells in 1 ml DMEM/10% fetal calf serum/20 µg /ml gentamicin were plated (50,000 cells per well) in chambered slides and transfected as described above. The cell medium was changed to DMEM/0.1% BSA/10 mM HEPES/2.5 µg/ml cycloheximide (DBHC) 46 h after transfection. Cells were washed 4.5 h later with 1 ml DBHC, and 1 ml ER-Tracker dye, LysoTracker Red, or BODIPY TR C5-ceramide complexed to BSA (according to manufacturer’s instructions) in DBHC was added to stain the ER, lysozomes, and Golgi, respectively. After 30 min, cells were washed and then imaged in 1 ml DBHC at room temperature. Where indicated, 1 µg/ml IN3 (or vehicle) was included in the washing, staining, and imaging media; IN3 did not absorb or fluoresce in our excitation-detection range. Confocal images were acquired with a Leica TCS SP confocal microscope (Leica Microsystems, Exton, PA) using a x63 NA 1.2 PL APO water immersion objective sequentially with 2 sec between green and blue imaging to avoid fluorescence transfer. ER-Tracker and GFP were excited at 361 nm and 488 nm and detected at 430–580 nm and 500–560 nm, respectively. Images were processed using Volocity 1.4.3 (Improvision Inc., Lexington, MA).
Statistics
Data (n 
3) were analyzed with one-way ANOVA and then paired Student’s t test (SigmaStat 3.0, Jandel Scientific Software, Chicago, IL; P < 0.05 was considered significant).
Online Supplemental Material
Supplemental data are published on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org. Movies of 3D reconstructions from COS7 cells in Fig. 4, C and D, cotransfected with the GFP-tagged WT hGnRHR and hGnRHR(E90K) cDNA, treated with vehicle and IN3, respectively, are shown (supplemental Figs. S1 and S2). Cells were digitally transected to show intracellular contents. Green represents the GFP, blue represents the ER, and turquoise represents areas of overlap.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: A2R, Angiotensin II receptor; 3D, three-dimensional; DBHC, DMEM/0.1% BSA/10 mM HEPES/2.5 µg/ml cycloheximide; ER, endoplasmic reticulum; GFP, green fluorescent protein; GnRHR, GnRH receptor; GPCR, G protein-coupled receptor; hGnRHR, human GnRHR; HH, hypogonadotropic hypogonadism; IN3, (2S)-2-[5-[2-(2-azabicyclo[2.2.2]oct-2-yl)-1,1-dimethyl-2-oxoethyl]-2-(3,5-dimethylphenyl)-1H-indol-3-yl]-N-(2-pyridin-4-ylethyl) propan-1-amine; IP, inositol phosphate; KOR, -opioid receptor; NPYR, neuropeptide Y-1 receptor; RAMP, receptor activity modifying protein; WT, wild-type
Received for publication March 3, 2004. Accepted for publication April 15, 2004.
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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S. R. Hawtin Pharmacological Chaperone Activity of SR49059 to Functionally Recover Misfolded Mutations of the Vasopressin V1a Receptor J. Biol. Chem., May 26, 2006; 281(21): 14604 - 14614. [Abstract] [Full Text] [PDF]
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 P. M. Apaja, J. T. Tuusa, E. M. Pietila, H. J. Rajaniemi, and U. E. Petaja-Repo Luteinizing Hormone Receptor Ectodomain Splice Variant Misroutes the Full-Length Receptor into a Subcompartment of the Endoplasmic Reticulum Mol. Biol. Cell, May 1, 2006; 17(5): 2243 - 2255. [Abstract] [Full Text] [PDF]
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A. T. McElvaine and K. E. Mayo A Dominant-Negative Human Growth Hormone-Releasing Hormone (GHRH) Receptor Splice Variant Inhibits GHRH Binding Endocrinology, April 1, 2006; 147(4): 1884 - 1894. [Abstract] [Full Text] [PDF]
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 R. A. Bakker, A. F. Lozada, A. van Marle, F. C. Shenton, G. Drutel, K. Karlstedt, M. Hoffmann, M. Lintunen, Y. Yamamoto, R. M. van Rijn, et al. Discovery of Naturally Occurring Splice Variants of the Rat Histamine H3 Receptor That Act as Dominant-Negative Isoforms Mol. Pharmacol., April 1, 2006; 69(4): 1194 - 1206. [Abstract] [Full Text] [PDF]
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D. Calebiro, T. de Filippis, S. Lucchi, C. Covino, S. Panigone, P. Beck-Peccoz, D. Dunlap, and L. Persani Intracellular entrapment of wild-type TSH receptor by oligomerization with mutants linked to dominant TSH resistance Hum. Mol. Genet., October 15, 2005; 14(20): 2991 - 3002. [Abstract] [Full Text] [PDF]
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P. E. Knollman, J. A. Janovick, S. P. Brothers, and P. M. Conn Parallel Regulation of Membrane Trafficking and Dominant-negative Effects by Misrouted Gonadotropin-releasing Hormone Receptor Mutants J. Biol. Chem., July 1, 2005; 280(26): 24506 - 24514. [Abstract] [Full Text] [PDF]
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 C. Castro-Fernandez, G. Maya-Nunez, and P. M. Conn Beyond the Signal Sequence: Protein Routing in Health and Disease Endocr. Rev., June 1, 2005; 26(4): 479 - 503. [Abstract] [Full Text] [PDF]
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 A. Leanos-Miranda, A. Ulloa-Aguirre, J. A. Janovick, and P. M. Conn In Vitro Coexpression and Pharmacological Rescue of Mutant Gonadotropin-Releasing Hormone Receptors Causing Hypogonadotropic Hypogonadism in Humans Expressing Compound Heterozygous Alleles J. Clin. Endocrinol. Metab., May 1, 2005; 90(5): 3001 - 3008. [Abstract] [Full Text] [PDF]
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A. J. Pask, H. Kanasaki, U. B. Kaiser, P. M. Conn, J. A. Janovick, D. W. Stockton, D. L. Hess, M. J. Justice, and R. R. Behringer A Novel Mouse Model of Hypogonadotrophic Hypogonadism: N-Ethyl-N-Nitrosourea-Induced Gonadotropin-Releasing Hormone Receptor Gene Mutation Mol. Endocrinol., April 1, 2005; 19(4): 972 - 981. [Abstract] [Full Text] [PDF]
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