In Vitro Coexpression and Pharmacological Rescue of Mutant ...

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The Journal of Clinical Endocrinology & Metabolism 90(5):3001–3008 Copyright © 2005 by The Endocrine Society doi: 10.1210/jc.2004-2071

In Vitro Coexpression and Pharmacological Rescue of Mutant Gonadotropin-Releasing Hormone Receptors Causing Hypogonadotropic Hypogonadism in Humans Expressing Compound Heterozygous Alleles Alfredo Leanõs-Miranda, Alfredo Ulloa-Aguirre, Jo Ann Janovick, and P. Michael Conn Research Unit in Reproductive Medicine (A.L.-M., A.U.-A., P.M.C.), Instituto Mexicano del Seguro Social, Me´xico D.F. 10101, Mexico; Divisions of Neuroscience and Reproductive Biology (A.L.-M., J.A.J., P.M.C., A.U.-A.), Oregon National Primate Research Center, Beaverton, Oregon 97006; and Department of Physiology and Pharmacology and Cell and Developmental Biology, Oregon Health and Science University (P.M.C.), Portland, Oregon 97239 We analyzed the function of mutant GnRH receptor (GnRHR) pairs associated with compound heterozygous patients showing complete or partial forms of hypogonadotropic hypogonadism. We did this to examine potential interactions between misfolded mutants that may influence net receptor function and response to pharmacological rescue. Nine pairs of GnRHR mutants and an unreported combination (L314X(stop)/R262Q) were studied. Coexpression of each pair of mutants in COS-7 cells resulted in an active predominant effect (Q106R/L266R, A171T/Q106R, T32I/C200Y, and R262Q/A129D mutant GnRHR pairs), an additive effect (R262Q/Q106R, N10K/Q106R, and R262Q/ Y284C human GnRHR pairs), or a dominant-negative effect (L314X(stop)/Q106R, Q106RⴙS217R/R262Q, and L314X(stop)/R262Q

T

HE HUMAN GNRH RECEPTOR (hGnRHR) is among the smallest members of the family of G proteincoupled receptors. The general structure of these membrane-anchored proteins is a single-polypeptide chain that traverses the lipid bilayer seven times, forming characteristic transmembrane helices or domains (TMD) connected by alternating extracellular and intracellular loops (IL) (1, 2). Mutations in the hGnRHR gene may lead to deletions and/or point mutations that alter functional properties, causing sporadic or familial (autosomal-recessive) forms of isolated hypogonadotropic hypogonadism (HH) (3, 4). Since the first report in a family with HH (5), reported cases of inactivating mutations of the hGnRHR gene have become more common, with the list of the hGnRHR mutants increasing rapidly. To date, 18 inactivating mutations in the hGnRHR gene (N10K, T32I, E90K, Q106R, A129D, R139H, S168R, C200Y, S217R, R262Q, L266R, C279Y, Y284C, L314Xstop, A171T, Q11K, P320L, and a splice junction mutation at the intron 1-exon 2 boundary) have been described causing HH (Fig. 1; splice mutant not shown) (3, 4). These mutations are distributed along the entire coding sequence of the receptor, First Published Online February 22, 2005 Abbreviations: hGnRHR, Human GnRH receptor; HH, hypogonadotropic hypogonadism; IP, inositol phosphate; TMD, transmembrane domain; IL, intracellular loop; WT, wild type. JCEM is published monthly by The Endocrine Society (http://www. endo-society.org), the foremost professional society serving the endocrine community.

GnRHRs). For all combinations, addition of the pharmacoperone IN3 increased both agonist binding and effector coupling. The IN3 response was unpredictable because responses could be either similar, higher, or lower, compared with that exhibited by the less affected mutant. The clinical phenotype in patients expressing complex heterozygous alleles appears to be dictated by both the contribution from each mutant and a dominant-negative effect similar to that reported for mutants and wild-type receptor. Depending on the genotype, partial or full restoration of receptor function in response to pharmacological chaperones may be achievable goals in patients bearing inactivating mutations in the GnRHR gene. (J Clin Endocrinol Metab 90: 3001–3008, 2005)

including the NH2 terminus, TMDs 2–7,extracellular loops 1 and 2, and the IL3, and two truncation mutants (L314Xstop and the intron 1-exon 2 splice site mutation); two hot spots (i.e. frequently appearing mutations) have been identified as the Q106R and the R262Q mutations. Six homozygous and 10 compound heterozygous combinations of hGnRHR mutants have been described in individuals exhibiting either partial or complete forms of HH; expression of 17 mutated hGnRHRs in heterologous cell systems has shown that some mutants are completely nonfunctional (E90K, A129D, R139H, S168R, A171T, C200Y, S217R, L266R, C279Y, P320L, and L314Xstop), whereas others retain a variable degree of function (N10K, N10K⫹Q11K, T32I, Q106R, R262Q, and Y284C) (3, 4). In principle mutations may alter any of several functions of the molecule including ligand binding, effector coupling, or receptor expression at the cell surface (3). However, it is now apparent that many loss-of-function mutations of the hGnRHR, initially thought to impair ligand binding or effector coupling, actually result from protein misrouting of otherwise functional receptors (4, 6 –11). We have previously shown that 12 of 15 hGnRHR mutants examined can be functionally rescued in vitro by peptidomimetic, cell surface membrane permeable GnRH antagonists behaving as pharmacoperones (pharmacological chaperones) that act as folding templates for misfolded receptors, restoring correct hGnRHR routing (4, 9 –13). Because these studies were performed by individually expressing each mutant receptor (i.e. mimicking a homozygous state), it was

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J Clin Endocrinol Metab, May 2005, 90(5):3001–3008

Leanõs-Miranda et al. • Rescue of Defective Human GnRH Receptors

FIG. 1. Sequence of the hGnRHR and location of the inactivating mutations identified to date (black and gray circles). EC, Extracellular space; IC, intracellular space.

unclear whether the degree of function and pharmacological rescue of a given mutant might be affected by a companion hGnRHR mutant, as may occur in vivo in compound heterozygous HH patients. This concern was raised in light of the observation that naturally occurring mutant hGnRHRs may exert a dominant-negative effect on wild-type (WT) hGnRHR, a portion of which is inefficiently processed by the cell in natural conditions (12, 14). Furthermore, genotypephenotype correlation studies performed by separately expressing mutant hGnRHR constructs suggest that the phenotype and degree of loss of function of a given compound heterozygous combination is mainly dictated by the less severe allele (3), an assumption that does not take into consideration the potential dominant-negative effect of a given mutant hGnRHR variant on the other. To analyze the interactions among distinct mutant hGnRHRs that may affect the net response of the expressed receptors to agonist and pharmacoperone treatment, we compared the in vitro function of hGnRHRs in cells transiently expressing one or two distinct naturally occurring mutant hGnRHRs (Table 1) and explored the response to pharmacoperone treatment of the coexpressed receptors. These data suggest that identification of the hGnRHR mutants present in patients harboring compound heterozygous expression is useful to determine whether treatment with pharmacological chaperones will lead to a desirable therapeutic outcome. Materials and Methods Materials Natural sequence GnRH was provided by the National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases National Hor-

mone and Peptide Program through Dr. A. F. Parlow (Torrance, CA). The GnRH agonist [Buserelin (d-tert-butyl-Ser6, des-Gly10, Pro9, ethylamide-GnRH] was a gift from Hoeschst-Roussel Pharmaceutical (Somerville, NJ). The indole IN3 [(2S)-2-[5-[2-(2-azabicyclo[2.2.2]oct-2yl)-1,1-dimethyl-2-oxoethyl]-2-(3,5-dimethylphenyl)-1H-indol-3-yl]-N(2-pyridin-4-ylethyl)propan-1-amine], originally designed as a peptidomimetic antagonist of GnRH, was synthesized by Drs. Wallace T. Ashton and Mark Goulet (Merck Research Laboratories, Rahway, NJ) (23). The expression vector pcDNA3.1, DMEM, OPTI-MEM, lipofectamine, and PCR reagents were purchased from Invitrogen (Gaithersburg, MD). Restriction enzymes, modified enzymes, and competent cells for cloning were purchased from Promega (Madison, WI). Other reagents were of the highest degree of purity available from commercial sources.

Vector construction WT and mutant hGnRHR cDNAs were subcloned into pcDNA3.1 at KpnI and XbaI restriction enzymes sites. All hGnRHR mutants were constructed by overlap extension PCR (24); the double-hGnRHR Q106R/ S217R variant was synthesized using the Q106R mutant as template. For transfection, large-scale plasmid DNAs were prepared using an EndTABLE 1. Genotype/phenotype relationships of the compound heterozygous HH patients bearing the hGnRHR mutations included in the present study Genotype

R262Q/Q106R 262

284

R Q/Y C Q106R⫹S217R/R262Q R262Q/A129D L314Xstop/Q106R T32I/C200Y Q106R/L266R N10K/Q106R A171T/Q106R

Phenotype

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5, 15 16 17 18 19 20 15 15 21 22


ofree maxiprep kit (QIAGEN, Valencia CA). The identity of all constructs and the correctness of the PCR-derived sequences were verified as previously described (8).

Transient transfection of COS-7 cells WT hGnRHR and mutant receptors were transiently expressed in COS-7 cells as described (14). Fifty thousand cells per well were plated in 48-well plates (Costar, Cambridge, MA). Twenty-four hours later, the cells were transfected or cotransfected with 0.05 ␮g/well total cDNA (0.025 ␮g of each hGnRHR mutant cDNA or pcDNA3.1 empty vector, as indicated) for inositol phosphates (IP) production or with 0.2 ␮g total cDNA per well (0.1 ␮g of each hGnRHR mutant cDNA or empty vector, as indicated) for [125I]-Buserelin binding, using 1 ␮l lipofectamine in 0.125 ml OPTI-MEM. After 5 h, 0.125 ml of DMEM containing 20% fetal calf serum was added to each well. The cells were incubated for an additional 18 h at 37 C and then washed with fresh growth medium and incubated 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. IP production was measured after exposure of the cells to the GnRH agonist, Buserelin for 2 h. For experiments with the cell permeant GnRH antagonist IN3, cultured COS-7 cells were transiently transfected with hGnRHR cDNA solutions containing either 1% dimethylsulfoxide (vehicle) or 1 ␮g/ml IN3 prepared in vehicle, as described (9, 10). 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 by Dowex anion exchange chromatography and liquid scintillation spectroscopy was performed as described previously (25).

Receptor binding assay COS-7 cells were transiently transfected as described above. Twentyseven hours after the start of transfection, the cells were washed twice with warm DMEM/BSA/HEPES and cultured in DMEM for 18 h before the addition of [125I]-Buserelin [specific activity, 700 ␮Ci/␮g, ⬃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 ␮m) GnRH plus 125I-Buserelin. Thereafter, the medium was removed, the plates containing the cells were placed on ice and washed twice with ice-cold PBS, and then the cells were solubilized by the addition of 0.2 m NaOH/0.1% sodium dodecyl sulfate. 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 ␮m GnRH) from total binding (no GnRH added).

Statistical analysis IP data shown are the means ⫾ sem from three or more independent experiments or representative experiments in triplicate incubations. One-way ANOVA (for n⬎3) followed by the Tukey (for paired samples) or Dunnet (for independent samples) post hoc tests as well as the Student’s t test (for n ⫽ 2) were employed for comparisons between groups, as indicated. Correlation statistics were used to measure the degree of association between the responses in terms of agonist-stimulated IP production and [125I]-Buserelin binding. In all experiments, the sd was typically less than 10% of the corresponding mean, except at basal levels in which the counts per minute were low. A two-tailed P ⬍ 0.05 was considered statistically significant.

Results Expression of WT and naturally occurring mutant hGnRHRs in COS-7 cells

Nine naturally occurring hGnRHR mutants previously detected in compound heterozygous HH patients were studied


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(Table 1). An unreported combination, L314X(stop)/R262Q, was also included because in preliminary experiments, the dominant-negative effect of L314X(stop) on the R262Q mutant (a hot spot mutation) was evident, compared with that of the truncated receptor on the hot spot Q106R mutation (see below). Consistent with previous reports (7, 8), GnRH agonist-stimulated IP production in COS-7 cells expressing individually the WT or the mutant hGnRHRs varied, depending on the particular mutant transfected. The N10K, T32I, Q106R, R262Q, and Y284C hGnRHR mutants partially responded to Buserelin stimulation, whereas in the Q106R⫹S217R, A129D, A171T, C200Y, and L314X(stop) mutants, agonist-stimulated IP production was virtually abolished (Figs. 2 and 3, A–C). As previously reported (8, 9, 12), addition of 1 ␮g/ml IN3 (a pharmacologic chaperone) resulted in variable degrees of functional rescue; cells expressing N10K, T32I, R262Q, and Y284C mutants showed a significant increase in Buserelinstimulated IP production, which was similar to that exhibited by the WT receptor in the absence of IN3. A modest pharmacological rescue was observed for Q106R, C200Y, and L266R mutant receptors, whereas lower, albeit significant, rescue was shown for the A171T hGnRHR variant. IN3 had virtually no effect on the A129D mutant, the double Q106R⫹S217R mutant, and the L314X(stop) truncated receptor. Buserelin-stimulated IP production by cells bearing the WT hGnRHR was increased by the addition of IN3. Similar results were found by radioligand binding assays (Fig. 3, D–F). Coexpression of naturally occurring hGnRHR mutants

COS-7 cells were cotransfected with equal amounts of each mutant hGnRHR cDNA as presented (and presumably expressed) by compound heterozygous HH patients. In parallel experiments and for comparative purposes, we also evaluated the response to GnRH agonist stimulation in cells cotransfected with each single mutant and the empty vector. As shown in Figs. 2 and 3, coexpression of each pair of mutants in COS-7 cells led to variable Buserelin-provoked IP effects. Three types of responses were identified in the absence of IN3 (Fig. 2): (1) active predominant effect, i.e. the combination of mutants yielded similar responses to agonist stimulation as did the more active of the two mutants cotransfected individually with the empty vector alone (Q106R/L266R, A171T/Q106R, T32I/C200Y, and R262Q/A129D mutant hGnRHR); (2) additive effect, i.e. the combination of each pair of mutants produced an additive response when compared with the single mutants cotransfected with the empty vector (R262Q/Q106R, N10K/Q106R, and R262Q/Y284C hGnRHR pairs); and (3) dominant-negative effect, i.e. the response of a single mutant was attenuated by the companion mutant (L314X(stop)/Q106R, Q106R⫹S217R/R262Q, and L314X(stop)/R262Q GnRHRs). For all combinations tested, the addition of IN3 resulted in a significant (P ⱕ 0.05) increase in Buserelin-stimulated IP production and agonist binding (Fig. 2). The response to IN3 was unpredictable however; in different cases responses were similar (A171T/Q106R, T32I/C200Y, R262Q/Q106R, and N10K/Q106R,), higher (Q106R/L266R and R262Q/Y284C), or lower (R262Q/A129D, L314X(stop)/Q106R, Q106R⫹S217R/R262Q, and L314X(stop)⫹R262Q), compared with the response exhibited by the less affected mutant

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FIG. 2. Buserelin-stimulated (10⫺7 M) IP production in COS-7 cells cotransfected with different heterozygous hGnRHR mutants or the empty vector at a 1:1 ratio and cultured in the absence or presence of IN3. Data are expressed as the percent of maximal IP production relative to that exhibited by control cells transfected with 0.050 ␮g of the WT hGnRHR cDNA (set as 100%). Data are the means ⫾ SEM from at least three independent experiments in triplicate incubations. *, P ⬍ 0.05; **, P ⬍ 0.01; N.S., Nonsignificant (Tukey and Dunnet post hoc tests).

in the presence of the chaperone. Radioligand binding positively correlated with IP production when mutants were expressed either individually [r ⫽ 0.92 and r ⫽ 0.94 in the absence and presence of IN3, respectively (P ⬍ 0.01 for both correlations)] or in combination [r ⫽ 0.90 (P ⬍ 0.01) and r ⫽ 0.75 (P ⫽ 0.02) in the absence and presence of the chaperone, respectively], thus confirming the presence of functional GnRHRs at the cell surface membrane. Representative examples of agonist-stimulated IP production and [125I]-Buserelin binding in response to IN3 are shown in Fig. 3. Figure 4 compares the total IP production of the WT hGnRHR with the different pairs of mutant hGnRHRs in the absence or presence of IN3 and grouped according to the clinical phenotypes reported in HH patients (Table 1). In the case of the T32I/C200Y and R262Q/Y284C combinations (both leading to complete forms of HH), functional rescue of the hGnRHR was completely achieved by IN3 treatment, whereas in the A171T/Q106R and R262Q/A129D mutations (also leading to complete HH), agonist-provoked IP production (after IN3) was below 2 sd of the mean activity, compared with that evoked by

the WT hGnRHR coexpressed with the empty vector, but above 2 sd of the mean activity exhibited by N10K/Q106R and R262Q/ Q106R combinations (which lead to partial HH) (Fig. 4, shaded area). The L314X(stop)/Q106R receptor pair was poorly rescued by IN3, reflecting both the severity of the truncation in receptor function imposed by the truncated hGnRHR and its negative effect on the Q106R receptor mutant. Likewise, pharmacological rescue of the mutant Q106R⫹S217R/R262Q combination (which leads to sex-dependent complete or partial forms of HH) was similar to that presented by the A171T/Q106R; in the former combination, the Q106R⫹S217R exerted a dominant-negative effect on the R262Q mutant in both the absence or presence of the pharmacoperone (Fig. 4), whereas in the case of the latter mutant, it is known that the A171T mutation leads to complete receptor inactivation (21). Finally, in the case of the three hGnRHR mutant combinations known to cause partial forms of HH in compound heterozygous, IN3 effected complete (N10K/ Q106R and R262Q/Q106R receptor mutants) or nearly complete (Q106R/L266R mutant) rescue.



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FIG. 3. Buserelin-stimulated (10⫺7 M) maximal IP production and specific [125I]-Buserelin binding (mean ⫾ SEM) in COS-7 cells transiently expressing the R262Q, Q106 R, and R262Q/Q106R (A and D), R262Q, Y284C, and R262Q/Y284C (B and E), and L314X(stop), R262Q, and L314X stop/R262Q naturally occurring mutant hGnRHRs. Cells were cultured in the absence or presence of IN3 as described in Material and Methods. IP production and ligand binding shown by cells transfected with 0.050 ␮g of the WT hGnRHR (WT) is also shown. The results are representative of at least three independent experiments (A–C) or the mean of triplicate determinations (D–F).

Discussion

Mutations in the hGnRHR isolated from patients with HH are widely distributed across the entire sequence of the pro-

tein (3, 4). As observed for other cell surface membrane receptors and channels (11, 26 –29), mutations in the hGnRHR frequently occur in regions of the protein not in-

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FIG. 4. Maximal Buserelin-stimulated IP production in COS-7 cells coexpressing different hGnRHR mutant combinations separated according to the clinical phenotype reported in HH patients (maximal IP production in cells transfected with 0.050 ␮g of the WT hGnRHR cDNA is taken as 100%). Cells were cultured in the absence (black bars) or presence (gray bars) of IN3 as described in Material and Methods. Data represent the mean ⫾ SEM of at least three independent experiments. The upper discontinuous horizontal line is set at ⫺2 SD of the maximal Buserelinstimulated IP production from cells cotransfected with the WT hGnRHR and the empty vector and cultured in the absence of IN3, whereas the lower horizontal line denotes ⫺2 SD of the mean maximal IP production exhibited by the coexpression of the heterozygous hGnRHR mutants leading to complete or partial (N10K/Q106R) or partial (R262Q/Q106R) forms of HH. The shaded area delimits the functional level of hGnRHR mutants that on pharmacological rescue would predict partial correction of the HH; levels above this area would lead to complete recovery and restoration of a normal phenotype, whereas levels below the area would predict complete or nearly complete failure to pharmacological rescue. Values above gray bars indicate the level of significance. *, P ⬍ 0.05; **, P ⬍ 0.01. N.S., Nonsignificant vs. WT hGnRHR/pcDNA3.1 no IN3 (Student’s t test).

volved in ligand binding or effector activation; such malformed receptors may fail to pass the cellular quality control apparatus and be degraded or recycled (11, 12). In principle, misfolded, trafficking-defective mutants are good candidates for rescue by pharmacological chaperones that act as molecular scaffolding, promoting proper folding, maturation, and correct targeting of the mutants from the endoplasmic reticulum to the cell surface (6 –12, 30). Conceivably, some mutants unable to recover function by pharmacoperone treatment may bear structural defects critical for receptor activation [e.g. the R139H mutation, which affects the highly conserved DRS motif located in the junction of the third TMD and the second IL of the hGnRHR (31)] or substitutions that may potentially disrupt interactions between adjacent helices and severely impair agonist binding (e.g. the A129D and S217R mutations in the third and fifth TMDs, respectively) (2, 4, 18, 19). An alternative possibility is that these unresponsive mutants cannot be refolded and exported from the endoplasmic reticulum to the cell surface, even in the presence of pharmacoperones Compound heterozygous HH patients bear two mutant

hGnRHR alleles. When expressed individually, these mutants show distinct agonist-stimulated responses, sensitivity to pharmacological rescue, and ability to exert dominantnegative effects on the WT receptor in vitro (9, 10, 14, 30). Nevertheless, it was still unclear how structural defects in each mutant receptor would affect the net functional response to agonist exposure when coexpressed in the host cell with a second hGnRHR mutant or whether combinations of trafficking-defective receptors would alter the predicted response to in vitro pharmacological treatment shown by individual mutant receptor species (4, 10, 11). To address this question, we recreated nine naturally occurring mutations in the hGnRHR as detected in compound heterozygous HH patients and tested their response to agonist stimulation and sensitivity to pharmacological rescue. Coexpression of each pair of hGnRHR mutants resulted in three different GnRH agonist-provoked IP responses: (1) active predominant effect, where the combination of mutants yielded similar responses as did the more active of the two mutants; (2) additive effect (i.e. the combination of each pair of mutants produced an effect similar to the sum of the single mutants);


and (3) dominant-negative effect, in which the response of a single mutant was attenuated by the companion mutant. Thus, GnRH exposure of cells coexpressing compound heterozygous alleles led to a variety of responses that were not solely determined by the mutant receptor variant with the less severe loss of function. These findings may explain the failure to correlate the degree of functional impairment of a given mutant hGnRHR with the clinical and/or biochemical phenotypes (3, 15). Although active or additive effects may account for the phenotypes found in patients with the Q106R/ L266R, N10K/Q106R, and R262Q/Q106R genotypes (which lead to partial forms of hypogonadism) as well as the T32I/C200Y genotype (which provoke complete HH), the phenotypes shown by HH individuals bearing other combinations (Table 1) are inconsistent with these effects, particularly when a partially functioning hGnRHR mutant (e.g. the R262Q and Q106R receptor mutants) is presumably coexpressed in vivo with a complete loss-of-function receptor mutant (such as the L314X(stop), Q106R⫹S217R, and A129D hGnRHR defective variants) that may potentially exert dominant-negative effects (14, 30). Because when expressed individually, the majority (eight of 11) of mutants examined in this study are sensitive to functional rescue by pharmacoperone treatment (Refs. 4,6,10 –13 and present study), addition of IN3 allowed a more clear distinction of potential interactions between the coexpressed mutants. We inferred that such interactions might in fact occur and affect hGnRHR function in light of experimental evidence, suggesting that the WT GnRHRs may form oligomers and be retained in the endoplasmic reticulum (30) and/or oligomerize upon agonist stimulation at the cell surface membrane level (32–34). In fact, addition of IN3 to cotransfected COS-7 cells revealed different responses that did not always correlate with the response observed in the absence of the pharmacoperone. For example, for some mutant combinations the level of IP production was similar to that provoked by the activation of the less functionally affected individual mutant in the presence or absence of IN3, whereas in others the response to pharmacoperone treatment resulted in higher or lower responses than that measured for the more active mutant. Thus, the functional effects of the coexpression and response to pharmacological treatment cannot be easily predicted or explained in all cases based only on the responses to agonist exhibited by each single mutant. In this regard, there are some possible explanations for this disparity. First, the mutants coexpressed did not interfere with one another’s function (e.g. T32I/C200Y, N10K/Q106R, and R262Q/Y284C), despite the potentially strong negative effects that one of the mutants [e.g. hGnRHR C200Y (14)] may exert on its pair. Furthermore, the coexpressed mutant hGnRHRs might even interact with each other to counteract any intrinsic functional impairment of the mutants, allowing the complex to reach levels of function similar to those found for the WT receptor in response to IN3 treatment (e.g. the R262Q/Y284C mutant pair). Second, the mutants interact with each other, with one of the mutants exerting dominant-negative effects on the other when the net response was less than the expected under conditions of pharmacological rescue (A171T/Q106R and Q106R/L266R mutants). In this case, interaction between the mutant receptors may potentially impede the complex to attain normal function in response to IN3.


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Last, the mutants interact with each other and exert dominant-negative effects on hGnRHR function, significantly reducing the function of the complexed receptors either in the absence and/or presence of IN3 (e.g. the L314X(stop)/Q106R, Q106R⫹S217R/R262Q, R262/A129D pairs and the unreported L314X(stop)/R262Q combination) or precluding, in some instances, that a given pair with one mutant highly sensitive to pharmacological rescue may reach the expected response to pharmacoperone treatment (e.g. the R262Q/Q106R receptor combination). The recent observation that mutant hGnRHRs insensitive to pharmacological rescue block intracellular trafficking of the green-fluorescent protein-tagged WT hGnRHR counterpart leading to retention of the labeled receptor within the endoplasmic reticulum (30), supports the notion that hGnRHRs may interact via formation of intracellular complexes and that the nature of such interactions (i.e. the particular conformation adopted by the complexed proteins) may eventually determine the functional fate of the formed complexes. Our study suggests that the dominant-negative effect exerted by one of the mutants may account for the majority of the inconsistencies in genotype/phenotype correlations, the exception being the R262Q/Y284C combination for which the coexistence of a ring chromosome 21 in patients bearing this genotype has been suggested as a potential modifier of the HH phenotype (17). In this scenario, it is reasonable to expect that in addition to the potential differential effects that some hGnRHR mutants may exert on signal transduction (35), the dominant effects imposed by one of the defective heterozygous receptors will also have an impact on the extent of clinical response to pharmacological rescue. This would be the case of the hGnRHR genotypes bearing the A171T, A129D, S217R, or L314X(stop) alleles; in these HH patients, treatment with pharmacoperones may result in a less than expected clinical response, whereas in others the potential interactions between mutant receptors would not affect, or might even favor, the outcome to pharmacoperone treatment. In these latter cases, a complete clinical response may be an achievable goal (Fig. 4). IN3 also increased the expression level of WT hGnRHR. This observation confirms previous findings (11, 12) and suggests that a large portion (nearly 50%) of the WT hGnRHR is intentionally inefficiently processed by the cell, retained in the endoplasmic reticulum and eventually degraded by the proteasome. In this setting, incompletely processed receptors may function as a reserve pool of molecules sensitive to functional rescue by natural [e.g. by overexpression during high demanding conditions (36)] or pharmacological means. The intrinsically lower expression of the hGnRHR may additionally reflect a relatively new evolutionary development for this receptor, as either deletion of the primate-specific K191 or addition of the piscine GnRHR carboxyl-terminal tail increases plasma membrane expression yet produces a modified receptor whose plasma membrane expression is not altered by pharmacological means (37). In summary, in vitro recreation of nine naturally occurring mutations in the hGnRHR found in compound heterozygous patients with HH led to a variety of functional responses to agonist exposure and pharmacological rescue that depended on the particular receptor combination coexpressed and ability of the heterozygous defective receptor to interact and exert dominant-negative effects on hGnRHR function. De-

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pending on the particular genotype, partial or full restoration of receptor function by pharmacoperone treatment may be achievable goals in HH patients bearing inactivating mutations in the hGnRHR gene. Recognition of the generality of diseases that result from misfolded and misrouted proteins (7, 11, 12) suggests that evaluation of protein oligomerization will become an increasingly important consideration in therapeutic approaches. Acknowledgments Received October 20, 2004. Accepted February 15, 2005. Address all correspondence and requests for reprints to: P. Michael Conn, Oregon National Primate Research Center/Oregon Health and Science University, 5050 NW 185th Avenue, Beaverton, Oregon 97006. E-mail: [email protected]; or Alfredo Leanõs-Miranda, Research Unit in Reproductive Medicine, Instituto Mexicano del Seguro Social, Apdo. Postal 99 – 065, Unidad Independencia, Me´xico D.F., C.P. 10101, Me´xico. E-mail: [email protected]. This work was supported by Fondo para el Fomento de la Investigacioń Me´dica- Instituto Mexicano del Seguro Social Grants FP2003/078 (to A.L.-M) and FP2003/007 (to A.U.-A.), Concejo Nacional de Ciencia y Tecnologa Grant 38056-M (to A.U.-A.), and National Institutes of Health Grants HD-19899, RR-00163, HD-18185, and TW/HD-00668 (to P.M.C.).

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