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Oregon Health and Science University (A.L.-M., A.U.-A., J.A.J., P.M.C.), Portland, Oregon 97239-3098; Oregon National Primate Research Center (A.L.-M., A.U.-A., J.A.J., P.M.C.), Beaverton, Oregon 97006; Research Unit in Reproductive Medicine, Instituto Mexicano del Seguro Social (A.L.-M., A.U.-A., P.M.C.), México D.F., C.P. 10101, México; and Department of Chemistry, University of Kentucky (T.H.J.), Lexington, Kentucky 40506-0055
Address all correspondence and requests for reprints to: Dr. Michael Conn, Oregon Health and Science University, 3181 SW Sam Jackson Park Road, Portland, Oregon 97239-3098. E-mail: connm{at}ohsu.edu.
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Introduction |
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-amino-butryic acid B1 and B2 receptors) (10, 11, 12). Similarly, splice variants of the GnRH and D3-dopamine receptors impair cell surface expression of their corresponding wild-type (WT) receptors, presumably due to association in the endoplasmic reticulum (13, 14). These data suggest that although early GPCR dimerization in the endoplasmic reticulum may play a role in receptor folding and trafficking, it may also perturb the quality control system for the intracellular trafficking of GPCRs and attenuate the expression of WT receptor.
In the present study, we analyzed the effects of eight naturally occurring human (h) GnRHR mutants and an hGnRHR truncated within the second extracellular loop on the function of WT hGnRHR. We have previously shown that when expressed individually, none of these hGnRHR mutants or the truncated variant (Fig. 1
) stimulated measurable amounts of inositol phosphate (IP) production in response to GnRH agonist or bound [125I]Buserelin (15, 16, 17). We additionally tested the effects of these mutants on the function of two genetically modified hGnRH receptors, in which either deletion of the primate-specific Lys191 (hGnRHR
K191) or addition of the piscine carboxyl-terminal sequence of the catfish (cf). GnRHR [hGnRHR/catfish carboxyl-terminal tail chimera (hGnRHR-cfCtail)] is known to increase both plasma membrane expression and efficiency for G
q/11 activation (18, 19).
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Materials and Methods |
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Natural sequence GnRH was provided by the NIDDK National Hormone and Peptide Program (Bethesda, MD). The GnRH agonist, Buserelin (D-tert-butyl-Ser6,des-Gly10,Pro9-ethylamide-GnRH), was a gift fromHoechst-Roussel Pharmaceutical (Somerville, NJ). The expression vector pcDNA3.1, DMEM, Opti-MEM, Lipofectamine, and PCR reagents were purchased from Life Technologies, Inc. (Gaithersburg, MD). Restriction enzymes, modified enzymes, and competent cells for cloning were purchased from Promega Corp. (Madison, WI). Other reagents were of the highest degree of purity available from commercial sources.
Receptor construction
Wild-type hGnRHR cDNA in pcDNA3 was subcloned into pcDNA3.1 at KpnI and XbaI restriction enzyme sites. All hGnRHR mutants were constructed by overlap extension PCR (20), and sequence was confirmed as previously described (18). A truncated hGnRHR mutant was created by substituting a stop codon instead of the codon for amino acid 205 (W205X) using overlap extension PCR.
Transient transfection of COS-7 cells
WT hGnRHR and mutant receptors were transiently coexpressed in COS-7 cells as previously reported (15, 17). One hundred thousand cells per well were plated in 24-well plates (Costar, Cambridge, MA). Twenty-four hours later, the cells were cotransfected with hGnRHR-WT (0.0125 µg/well), hGnRHR-cfCtail, or hGnRHRK191 (3.25 ng/well for IP or 0.025 µg cDNA/well for [125I]Buserelin binding) and increasing concentrations (0.0125–0.1 µg/well) of hGnRHR mutant cDNAs, as indicated, using 2 µl Lipofectamine in 0.25 ml Opti-MEM. The total amount of DNA transfected remained constant as complementary amounts of the empty expression vector, pcDNA3.1 were included in the transfection mixture. After 5 h, 0.25 ml DMEM containing 20% fetal calf serum was added to each well. The cells were incubated for an additional 18 h at 37 C, then washed. Fresh growth medium was added to the cells for another 4 h at 37 C. The cells were then washed twice with DMEM/0.1% BSA/gentamicin and preloaded with [3H]myo-inositol (for IP assays) or DMEM (for binding studies) as described below.
For experiments with the cell-permeant GnRH antagonist 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; synthesized by Drs. Wallace T. Ashton and Mark Goulet, Merck Research Laboratories (21), Rahway, NJ], cultured COS-7 cells were transiently transfected with hGnRHR cDNA solutions containing either 1% dimethylsulfoxide (vehicle) or 1 µg/ml IN3 prepared in the vehicle, as previously described (16, 17). Cells were continuously exposed to the antagonist during the period of transfection and thereafter until the start of the [3H]myo-inositol or DMEM 18-h preloading periods.
Measurement of IP production
Quantification of IP production was performed by Dowex anion exchange chromatography and liquid scintillation spectroscopy, as described previously (22).
Saturation binding assay
COS-7 cells were transiently cotransfected with WT and mutant hGnRHR cDNAs as described above. Twenty-seven hours after the start of transfection, the cells were washed twice with warm DMEM/BSA and cultured in DMEM for 18 h before the addition of [125I]Buserelin (specific activity, 700 µCi/µg; 
106 cpm/0.5 ml; pH 7.4). Cells were incubated at room temperature for 90 min in the presence or absence of excess (1 µg) GnRH. Thereafter, the medium was removed, the plates containing the cells were placed on ice and washed twice with ice-cold PBS, and the cells were solubilized by the addition of 0.2 M NaOH/0.1% sodium dodecyl sulfate (23). Aliquots of samples were then transferred to glass tubes and counted in a -counter (Packard Instruments, Downers Grove, IL). Specific binding was calculated by subtracting nonspecific binding (binding measured in the presence of 1 µM GnRH) from total binding (no GnRH added).
Statistical analysis
The data shown are the mean ± SEM from triplicate (IP) or quadruplicate (binding) determinations. Unpaired t tests or one-way ANOVA followed by unpaired t tests were applied, as appropriate, to assess differences between means. In all experiments, the SD was typically less than 10% of the corresponding mean, except at basal levels, for which the counts per minute were extremely low. Each experiment was repeated three or more times with similar results; unless specified, the results of a single experiment are shown. P < 0.05 (two-tailed) was considered statistically significant.
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Results |
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COS-7 cells were cotransfected with constant amounts of WT receptor cDNA and increasing concentrations of each hGnRHR cDNA mutant. Cotransfection of hGnRHR mutants and WT receptor cDNAs at mutant/WT ratios of 1:1 to 8:1 progressively reduced maximal GnRH agonist-provoked IP responses compared with cells cotransfected with the WT receptor and pcDNA3.1 (empty vector; Fig. 2). The inhibitory effect of the hGnRHR mutant on WT hGnRHR function occurred at increasing GnRH agonist concentrations (Fig. 2, A–C). Maximal responses declined by 16–67% in cells expressing a relative excess (3- to 8-fold) of hGnRHR mutant cDNAs (Fig. 2, D–F). Further, the inhibition was most pronounced when provoked by hGnRHR mutants bearing substitutions located across the transmembrane domain 3 to 6 segment of the receptor, particularly the S168R, S217R, and L266R mutants.
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). In these experiments, substitutions spanning the transmembrane domain 3-intracellular loop 3 region of the receptor decreased receptor density on the cell more markedly.
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To determine whether the inhibitory effect of the hGnRHR mutants on WT receptor function was specific for receptor modifications that evolved in mammals or primates receptor species, hGnRHR-cfCtail and hGnRHRK191, were each transiently coexpressed with each hGnRH mutant. These modified receptors exhibit higher absolute levels of cell membrane expression compared with WT receptors (Fig. 5) (16, 17). COS-7 cells were transfected with hGnRHR-cfCtail or hGnRHR191 cDNAs (3.12 and 25 ng/well for IP production and radioligand binding, respectively) and increasing concentrations of hGnRHR mutant cDNAs. In contrast to the results observed with WT hGnRHR, coexpression of hGnRHR-cfCtail or hGnRHRK191 with each hGnRHR mutant did not alter agonist-provoked IP production or binding, even under conditions of high hGnRHR mutant to hGnRHR-cfCtail or hGnRHRK191 concentration ratios (Fig. 6).
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We examined the effect of IN3 on WT hGnRHR, hGnRHRK191 and hGnRHR-cfCtail function. In these experiments hGnRH receptor cDNAs were transfected in the presence or absence of 1 µg/ml IN3, as described in Materials and Methods. The expression level of human WT GnRHR itself was enhanced by exposure to IN3; IN3 treatment increased IP production by approximately 55% in response to Buserelin and specific [125I]Buserelin binding by about 120% (Fig. 5
). On the other hand, IN3 had measurable effect on hGnRHR-cfCtail function and impaired hGnRHRK191-mediated IP production and [125I]Buserelin binding, probably due to residual occupancy by IN3. A similar effect was found when higher concentrations (0.025–0.1 µg/well) of receptor cDNAs were transfected (not shown), suggesting that newly synthesized WT hGnRHR is inefficiently transported to the cell membrane, unlike the modified receptors.
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Discussion |
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The importance of the cell’s quality control system in regulating expression is increasingly recognized (24). In many cells, 30–60% of newly synthesized proteins never attain their correct native structure and are targeted for degradation (25, 26, 27). The human 
-opioid receptor, for example, normally expresses as a low efficiency endoplasmic reticulum (ER) export, with only about 40% of the receptor reaching the cell surface (26, 27). The ability to double the expression of hGnRH at the plasma membrane with efficient pharmacological chaperones suggests that about half of the synthesized receptor never arrives at the plasma membrane. This appears to be a relatively new evolutionary development for the GnRHR receptor, as deletion of the primate-specific Lys191 or addition of the piscine carboxyl-terminal tail increases plasma membrane expression yet produces a modified receptor that does not increase plasma membrane expression in the presence of a pharmacological chaperone. Similarly, plasma membrane expression of the rodent GnRHR is not elevated by pharmacological chaperones (28).
In the present study, coexpression of naturally occurring hGnRHR mutants inhibited both WT hGnRHR-mediated agonist binding and intracellular signaling in a dose-dependent manner and with specificity for individual mutant cDNAs. Several mechanisms potentially explain this effect. As the expression of receptor sequences that compete for G proteins may sequester them (29, 30, 31, 32, 33), the negative effect of hGnRHR mutant on WT receptor function may reflect competition. This seems unlikely based on the observation that coexpression of the naturally occurring hGnRHR mutants with genetically modified hGnRHR (hGnRH-cfCtail and K191 receptors) bearing intracellular domains involved in G protein coupling had no measurable effect on maximal IP production. Further, maximal IP production and agonist binding attenuation are graded in the presence of hGnRHR mutants, all of them bearing sequences or domains presumptively involved in G protein activation. An alternative possibility is that impaired WT hGnRHR trafficking may be due to intermolecular interactions between the WT receptor and the naturally occurring hGnRHR mutants. This view is supported by previous studies showing that a number of mutant GPCRs or splice variants may potentially act as negative or positive regulators of WT receptor expression and function via the formation of receptor oligomers that hamper or facilitate proper delivery and/or insertion of the native receptor to the cell surface (7, 8, 9, 10, 11, 12, 13, 14, 34, 35, 36). The ability of the mutant receptor to provoke a given effect (positive or negative) on WT receptor function may reside in the nature of the interaction between the receptor and the resulting conformation of the complex. Although it is desirable to do so, we have been unable to use microscopic techniques to monitor the intracellular routing of GnRH receptors because the use of green fluorescent protein derivatives of this particular receptor (37) requires the presence of a catfish tail spacer (19), which itself significantly influences the routing. Recent data (S. Brothers et al., manuscript submitted) also suggest that hemagglutinin tagging rescues particular conformationally defective hGnRHR mutants. Furthermore, efforts in many laboratories, including our own, to produce direct antisera capable of precipitating the GnRHR to allow identification of intracellular receptor complexes have not been successful.
In particular experiments we observed dissociation between the extent of attenuation in IP production and the inhibition of agonist binding. For example, cotransfection of the C200Y mutant with the WT receptor at relatively high mutant/WT receptor ratios (4:1) led to approximately 20% and 60% reduction in IP production and maximal agonist binding, respectively; a similar functional dissociation was also observed in cells coexpressing the W205X truncated mutant and the WT receptor. This suggests that some WT-mutant receptor complexes might, in fact, reach the cell surface membrane in the complexed state, with the degree of functional efficiency being compromised by the mutated molecule. This disproportionate decrease in agonist binding exhibited by the WT-C200Y receptor complex may be explained by failure of the complex to allow access of the binding pocket to the agonist (38).
In coexpression experiments, receptor binding and IP responses to agonist stimulation were inversely proportional to the quantity of mutant cDNA cotransfected with the WT receptor. This effect has also been observed with V2-vasopressin (7), hGnRH (13), and D2-dopamine-truncated receptor proteins (39), suggesting that the coexistence of mutant and WT receptors may yield multimeric complexes that are impeded from attaining a conformation consistent with cell surface transport. In fact, treatment of cells coexpressing the hGnRH mutant and WT receptors with a pharmacological chaperone led to complete or partial recovery of receptor function, suggesting that defects in folding predispose the formation of heterocomplexes between protein receptors and lead to degradation. In this regard it is not known whether IN3 stabilized and rescued receptor function by interacting with the mutant, the WT, or both receptor species. The finding that the function of the WT receptor complexed with the E90K, C200Y, or L266R mutants (all sensitive to rescue by IN3 (16, 17), recovered to levels above those observed for the WT receptor alone, suggests that the pharmacological agent interacted with and rescued both receptor species, whereas in the case of the S168R and S217R mutants (both insensitive to rescue by IN3 (17), the improved function was mainly due to rescue of the sequestered WT receptor.
Although the precise mechanism(s) of the intermolecular interactions between GPCRs is unknown, it has been proposed that association between receptors may occur in the membranes of the endoplasmic reticulum during the process of specific interhelical interactions that lead to tight -helical packing (34). In this regard, it was striking to find that the substitution of a single amino acid of the hGnRHR led to a dominant-negative phenotype and that the extent of inhibition of WT receptor function by the GnRHR mutants, followed an inverted bell-shaped pattern, in which the S168R, S217R, and L266S mutants exhibited the most pronounced negative effects. Thus, multiple regions of the receptor, including mainly transmembrane domains 3–5 and the carboxyl-terminal portion of the third intracellular loop, may be primarily involved in the formation of GnRH receptor complexes.
The hGnRHR mutants displayed dominant-negative effects when coexpressed with the WT receptor, but not with two genetically modified hGnRH receptors intrinsically exhibiting high maturation efficiency. This finding suggests that conformational variants of receptors are prone to associate and form complexes whose fate ultimately depends on the particular conformation adopted by the associated proteins. In this regard, it would be interesting to determine whether other WT GPCRs sensitive to negative or positive regulation by mutant congeners also exhibit intrinsically low maturation efficiencies, as documented here for the hGnRHR and previously for the -opioid receptors (27, 40).
In hypogonadotropic hypogonadism due to GnRH resistance, affected individuals are either compound heterozygous or homozygous for the mutation. Carriers of a mutant allele usually exhibit normal responsiveness to exogenous GnRH stimulation as well as normal gonadotropin levels and reproductive competence (41, 42). It is possible that these carriers express both WT and mutant receptor proteins at levels (e.g. 1:1 hGnRH mutant to WT hGnRHR ratios) compatible with the expression of a normal phenotype. Alternatively, the expression levels of WT hGnRHR, albeit reduced by the negative effect of the mutant receptor, may be otherwise sufficient to mediate physiological effects.
We conclude that a significant fraction of the WT hGnRHR is incompletely processed to the cell surface membrane. This appears to have occurred recently in evolution, perhaps to accompany the more complicated requirements for primate cyclicity and reproduction. Coexpression of hGnRHR mutants bearing folding defects may further aggravate the intrinsic functional deficit of the suboptimally expressed WT receptor population, probably due to the formation of heterocomplexes that cannot escape the cellular quality control apparatus. Defective intracellular transport or interference with proper maturation due to the formation of misfolded complexes between the receptor species appears to explain the observed dominant-negative effect of the mutant hGnRHR.
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Acknowledgments |
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Footnotes |
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Abbreviations: cfCtail, Catfish carboxyl-terminal tail chimera; GPCR, G protein-coupled receptor; hGnRHR, human GnRH receptor; IP, inositol phosphate; WT, wild type.
Received January 16, 2003.
Accepted April 1, 2003.
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32. J Biol Chem 272:30603–30606 opioid receptor. J Biol Chem 275:13727–13736 opioid receptors retained in the endoplasmic reticulum are retrotranslocated to the cytosol, deglycosylated, ubiquitinated, and degraded by the proteasome. J Biol Chem 276:4416–4423 opioid receptor maturation. EMBO J 21:1628–1637[CrossRef][Medline]
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, P < 0.05 vs. WT and pcDNA3.1; ¶, P < 0.01 vs. WT and pcDNA3.1; 
, P < 0.001 vs. WT and pcDNA3.1.
