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Oregon Regional Primate Research Center and Department of Physiology and Pharmacology, Oregon Health & Science University, Beaverton, Oregon 97006
Address all correspondence and requests for reprints to: P. Michael Conn, Oregon Health & Science University, 505 NW 185th Avenue, Beaverton, Oregon 97006. E-mail: . connm{at}ohsu.edu
Abstract
In the present study, we demonstrate pharmacological rescue (assessed by ligand binding and restoration of receptor coupling to effector) of five naturally occurring GnRH receptor (GnRHR) mutants (T32I, E90K, C200Y, C279Y, and L266R), identified from patients with hypogonadotropic hypogonadism, as well as rescue of other defective receptors intentionally manufactured with internal or terminal deletions or substitutions at sites expected to be involved in establishment of tertiary receptor structure. The pharmacological agent used is a small, membrane-permeant molecule, originally designed as an orally active, nonpeptide receptor antagonist, but is believed to function as a folding template, capable of correcting the structural defects caused by the mutations and thereby restoring function. After rescue, this agent can be demonstrably removed. The rescued receptor, now stabilized in the plasma membrane, couples ligand binding to activation of the appropriate effector system. For comparison, low-, intermediate-, or high-affinity peptide antagonists of GnRHR (that do not penetrate the cell) were unable to effect rescue, as was a nonbinding peptidomimetic congener of the rescue agent; this latter effect demonstrates specificity of the rescue agent. Our findings, taken in concert with an earlier study showing rescue of a mutant by modifications to the receptor structure that enhance plasma membrane expression of the GnRHR, suggest that mutant GnRHRs have frequently not lost intrinsic functionality and are subject to rescue by techniques that enhance membrane expression. The present findings demonstrate the efficacy of an approach based on pharmacological rescue and suggest the basis of new approaches for intervention in this and similar diseases.
GONADOTROPIN-RELEASING HORMONE plays a central role in neural regulation of reproductive function. This decapeptide is produced by specialized neurons found in the mediobasal hypothalamus, the axons of which project to the median eminence. From there, GnRH enters the portal circulation and binds to a specific receptor on pituitary gonadotropes stimulating the synthesis and release of the gonadotropins, LH and FSH. Sequence analysis of the GnRH receptor (GnRHR) is consistent with the seven transmembrane domain motif, characteristic of the G protein-coupled receptors (GPCR) superfamily (1). The human GnRHR is coupled to the Gq/11 system; after GnRH binding, the activated GnRHR-Gq/11 protein complex activates the membrane-associated enzyme phospholipase Cß, leading to inositol 1,4,5-trisphosphate production and the release of intracellular calcium (2).
Some forms of congenital hypogonadotropic hypogonadism (HH), result from mutational defects in the synthesis or action of GnRH itself. Although a mutation in the GnRH gene has been reported in the hypogonadotropic hpg/hpg mouse (3), no similar mutation has been reported in humans. Kallmann's syndrome may be transmitted as autosomal-dominant, autosomal-recessive, or X-linked disorders. The latter form is caused by a migrational defect that involves the GnRH neuronal system (4). The gene for the X-linked form of Kallmann's syndrome has been mapped to chromosome Xp22.3 (5, 6), and several mutations have been described to date (7, 8, 9, 10). Mutations of the AHC gene (also in Xp22.3), which result in congenital adrenal hypoplasia and HH, have also been described (11). Mutations of the GnRHR are almost always autosomal recessive in inheritance; they account for about 40% of the existing inheritance of familial causes of HH and lack anosmia. Thus, the GnRHR gene is one of five mutational sites that can cause HH, three of which are associated with Kallman’s syndrome and two of which are associated with HH without anosmia.
Although mutational defects in the human GnRHR (hGnRHR) function constitute an apparently rare cause of HH (12, 13, 14, 15, 16, 17, 18), 13 different hGnRHR mutations causing either complete or partial HH have now been described. Of interest for the present study, these mutations are widely distributed along the entire receptor sequence (12, 13, 14, 15, 16, 17, 18). Such mutations were isolated from patients with a wide spectrum of phenotypes, from partial to complete hypogonadism. When expressed in heterologous expression systems, such as COS cells, these show greatly reduced or absent ligand binding, receptor expression at the cell surface, and/or signal transduction (12, 13, 14, 15, 16, 17, 18).
One mutant, E90K (Fig. 1
), was previously rescued (19) either by deleting K191 (which, when present, decreases expression of hGnRHR at the plasma membrane; Refs. 20 and 21) or by adding a carboxyl-terminal sequence. Either of these approaches alone supports high-membrane expression of GnRHR (20, 22). Rescue by these genetic approaches was of academic interest but not therapeutically significant, because this approach is not presently practical. Nonetheless, this caused us to consider that the E90K mutation itself, while resulting in protein misfolding, does not irreversibly destroy the intrinsic ability of the mutant to bind ligand or to couple effector (19). One explanation for this would be misrouting of the mutant receptor within the cell. On the basis of this hypothesis, we sought both to determine whether other means (with potential therapeutic value) for receptor rescue were available and to assess the range of defective receptors to which they were applicable.
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Natural sequence GnRH was provided by the National Institute of Diabetes and Digestive and Kidney Diseases, National Hormone and Peptide Program (Bethesda, MD). The GnRH agonists, buserelin (D-tert-butyl-Ser6, des-Gly10, Pro9, ethylamide-GnRH) and leuprolide (D-Leu6, Pro9, des-Gly10-ethylamide-GnRH) were kind gifts of Hoeschst-Roussel Pharmaceuticals (Somerville, NJ) and TAP Pharmaceuticals, Inc. (Deerfield, IL). The following GnRH antagonists were obtained as indicated: D-Phe2, D-Phe6-GnRH, Wyeth-Ayerst Laboratories (Andover, MA); ‘Nal-Arg,’ [Ac-D-Nal1, D-Cpa2, D-Pal3, Arg5, D-Arg6, D-Ala10]-GnRH, Dr. Jean Rivier (Salk Institute, La Jolla, CA); ‘Nal-Glu,’ [Ac-D-Nal1, D-Cpa2, D-3Pal3, Arg5, D-Glu6, D-Ala10]-GnRH), Dr. Jean Rivier, (Salk Institute); cetrorelix, Peninsula Laboratories, Inc. (San Carlos, CA); FE486 [Ac-D-2Nal1, D-4Cpa2, D-3Pal3, Ser4- 4Aph(L-hydroorotyl)3, D-4Aph (carbamoyl)6, Leu7, ILys8, Pro9, D-Ala10]-GnRH, Ferring Research Institute (Southampton, UK); azaline B, [Ac-D-Nal1, D-Cpa2, D-Pal3, Aph5 (atz), D-Aph6 (atz), ILys8, D-Ala10]-GnRH (Jean Rivier, Salk Institute, La Jolla, CA); acyline, [Ac-D-2Nal1, D-4Cpa12, D-3Pal3, Ser4Aph(Ac)35, D-4Aph(Ac)6, Leu7, ILys8, Pro9, D-Ala10-NH2], Dr. Jean Rivier (Salk Institute); and antide, Serono Laboratories (Aubonne, Switzerland). Irrelevant molecules GHRP6, octreotide, human galanin, human calcitonin, and TSH-releasing hormone were obtained from Phoenix Pharmaceuticals, Inc. (Belmont, CA), and vapreotide was obtained from Debiopharm (Lausanne, Switzerland). DMEM, OPTI-MEM, Lipofectamine and PCR reagents were purchased from Life Technologies, Inc. (Grand Island, NY). Restriction enzymes, modified enzymes and competent cells for subcloning were purchased from Promega Corp. (Madison, WI). The Endofree Maxi-prep kit was purchased from QIAGEN (Valencia, CA).
IN3, (2S)-2-[5-[2-(2-azabicyclo[2.2.2]oct-2-yl)-1,1-dimethyl-2-oxo-ethyl]- 2-(3,5-dimethylphenyl)-1H-indol-3-yl]-N-(2-pyridin-4-ylethyl) propan-1-amine, and IN4, (1-[2-(dimethylamino)ethyl]-2-(3,5-dimethylphenyl)-N,N-diethyl-3-{2-[(4-pyridin-4-ylbutyl)amino]ethyl}-1H-in-dole-5-carboxamide) were synthesized by Drs. Wallace T. Ashton and Mark Goulet (23, 24, 25) and obtained through the auspices of Merck \|[amp ]\| Co., Inc. (Rahway, NJ). Other reagents were of the highest degree of purity available from commercial sources, unless otherwise noted.
Vector construction
The wild-type (WT) hGnRHR cDNA in pcDNA3 was subcloned into pcDNA3.1 at KpnI and XbaI restriction enzymes sites. The E90
K90 (GAG to AAG) mutation was constructed by overlap extension PCR (26). Other mutants were prepared by analogous processes. Rodent (r) sequences (rGnRHR) were expressed in vector pTracer-cytomegalovirus; exchange experiments indicated that both vectors expressed at equivalent levels in COS-7 cells. The identity of all constructs and the correctness of the PCR-derived sequences were verified by Dye Terminator Cycle Sequencing (Perkin-Elmer, Foster City, CA), according to the manufacturer’s instructions. For transfection, large-scale plasmid DNAs were prepared using a QIAGEN Endofree Maxi-prep kit (QIAGEN). The purity and identity of the amplified plasmid DNAs were further verified by restriction enzyme analysis.
Transient transfection of COS-7 cells
WT hGnRHR and altered receptors were transiently expressed in COS-7 cells. Cells were maintained in growth medium (DMEM) containing 10% fetal calf serum (FCS; Life Technologies, Inc.) and 20 µg/ml gentamicin (Gemini Bioproducts, Calabasas, CA) in a 5% CO2-humidified atmosphere at 37 C. One hundred thousand cells per well were seeded in 24-well plates (Costar, Cambridge, MA). Twenty-four hours after plating, the cells were transfected with 0.1 µg DNA per well using 2 µl lipofectamine in 0.25 ml OPTI-MEM containing 1% dimethylsulfoxide (DMSO; vehicle) or 1 µg/ml IN3 prepared in vehicle. After 5 h (see Fig. 3, period A), 0.25 ml DMEM containing 20% FCS with or without IN3 (as indicated) was added to each well. The cells were incubated for an additional 18 h (period B) at 37 C, then washed; fresh growth medium with or without IN3 was added to the cells for another 28 h (period C) at 37 C. The cells were then washed two times with DMEM/0.1% BSA/gentamicin and were preloaded with 3H-inositol [for inositol phosphate (IP) assays] or DMEM (for internalization studies) for 18 h before stimulation with agonist. During this latter 18-h period, as well as the period of GnRH stimulation, IN3 was not present.
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Quantification of IP production was performed as described previously (27). Briefly, 51 h after the start of transfection, transiently transfected cells were washed twice with DMEM (no IN3) containing 0.1% BSA, and intracellular inositol lipids were incubated in inositol-free DMEM supplemented with 4 µCi/ml [3H]myo-inositol for 18 h at 37 C. After the preloading period, cells were washed twice with DMEM (inositol free) containing 5 mM LiCl (no IN3) and incubated for 2 h at 37 C in the absence or presence of the indicated doses of buserelin dissolved in 0.5 ml DMEM (inositol free)-LiCl. At the end of the incubation period, medium was removed, and 1 ml 0.1 M formic acid was added to each well. Cells were then frozen and thawed to disrupt the cell membranes. IP accumulation was measured by Dowex anion exchange chromatography and liquid scintillation spectroscopy, as previously described (27).
Saturation binding for hGnRHR
COS-7 cells were transiently transfected with human WT or mutant cDNA as described above. The culture medium contained either IN3 (1 µg/ml in DMSO) or DMSO alone and was present for 27 h. The cells were then washed twice with DMEM/BSA/gentamicin. DMEM was added for 18 h as a washout period for the IN3 drug. Cells were washed twice with warm DMEM/BSA before incubating with [125I]-buserelin (Ref. 28 ; specific activity, 700 µCi/µg; 230,000 cpm/0.5 ml), and nonspecific binding was measured in the presence of 1 µM GnRH. Cells were incubated at room temperature for 90 min. The medium was removed, plates containing the cells were placed on ice and washed twice with ice-cold PBS. Then, 0.2 M NaOH/0.1% SDS (29) was added to the wells to solubilize the cells. The sample was transferred to a glass tube 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).
Internalization
Cells were transfected as described above, except that 12-well plates were used and 2 x 105 cells were plated per well. Each plate served as one time point and contained either WT or mutant receptors transfected into cells using 2 µg plasmid DNA and 5 µl lipofectamine in 0.5 ml OPTI-MEM per well. IN3 (1 µg/ml) or vehicle was present for the same amount of time as described for the IP assay. Sixty-nine hours after transfection, a radioligand acid wash method (28, 30) was used to measure internalization of the mutant or WT hGnRHRs. This method has been used for GnRH (30) and other receptors and shown to distinguish internalized and noninternalized receptor. Briefly, the cells were washed twice with 0.5 ml DMEM containing 0.1% BSA. The cells were incubated with [125I]-buserelin (Ref. 28 ; specific activity, 700 µCi/mg; 2 x 105 cpm/0.5 ml) as noted. At the indicated time, the iodinated ligand was removed, and the plate was placed on ice. Cells were washed twice with 0.5 ml ice-cold PBS, then 0.5 ml acid wash solution (50 mM acetic acid and 150 mM NaCl, pH 2.8) was added to each well and incubated for 12 min on ice. To determine the surface-bound iodinated ligand, the acid wash was collected and counted on a counter (Packard Instruments). To determine the internalized radioligand-receptor complex, the cells were solubilized in 0.5 ml PBS containing 0.1% Triton X-100, collected, and counted. Nonspecific binding for all conditions was determined using the same procedure but in the presence of 10 µM unlabeled GnRH. Nonspecific binding was subtracted from the surface-bound and internalized radioligand, and the amount of internalized radioligand was expressed as the percentage internalized of the total bound at each time point.
Statistical analysis
The data shown are the means ± SEM from triplicate incubations. 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; the results of a single experiment are shown.
Results
Figure 1
shows a representation of the hGnRHR indicating five loci of mutation resulting in HH and examined in the present study. In addition, the representation shows the Lys191 that is found in hGnRHR but is not found in preprimate species.
Previously and in the present study (Fig. 2), when the hGnRHR E90K mutant was expressed in COS-7 cells, it did not even show this modest level of binding and, in sharp contradiction to WT, showed no GnRH agonist-stimulated IP production (Fig. 3). Addition of 1 µg/ml IN3 at the time of transfection, however, resulted in binding and IP coupling (Figs. 2 and 3), showing that the effect of the mutational error was functionally corrected. Note that IN3, an antagonist, is washed out for 18 h (even at the longest time present) before exposure to the GnRH agonist. Thus, it is not present at the time of the challenge.
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). This specificity suggests that interaction with the receptor is, as would be expected for a structure-stabilizing molecule, essential and, in concert with the time course, suggests that the action of IN3 is not on receptor (or general) protein synthesis.
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, periods A–C). More modest coupling of rescued mutant GnRHR (measured by IP production) was observed when IN3 was present only part of the time. Neither IN3 nor IN4 induced or inhibited measurable cell proliferation, assessed by cell counting.
To compare the rescued E90K receptors with the WT, we examined ligand specificity and affinity, receptor features associated with pharmacological disease management. Figures 4 and 5 indicate that ligand specificity and the ED50 value for the GnRH agonist (4 nM) was indistinguishable from published values for the WT human receptor (29, 33) and both GnRH agonists and antagonists were recognized with fidelity and the characteristic varying potencies (29, 30, 31, 32, 33). A selection of irrelevant compounds (not normally bound by the WT receptor) evoked no IP response (Fig. 4).
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(binding) and Table 1 (IP response) show the surprising finding that we were able to rescue five known (34, 35) genetic variants identified from different patients with HH and widely distributed on the receptor. T32I, E90K, C200Y, C279Y, and L266R, most of which did not express at measurable levels before treatment, were fully rescued, assessed by the ability to respond to GnRH agonist with IP production or by radioligand binding. C200 may form a stabilizing bridge to C14 (36).
To address the degree of receptor distortion from which rescue could be effected, we also examined a palette of mutations in the rGnRHR at sites in which a cysteine normally appears (Table 2), because this amino acid is associated with maintenance of tertiary receptor structure. When varied substitutions were made at the same locus (i.e. C278A, C278V, C278T, C278M), there were differences in the ability to effect rescue. The mutants resulting from the more bulky substituents could, apparently, not be formed into the conformation necessary for rescue. Consistent with this view, it was not surprising to see that the expression of such variants on the plasma membrane also appeared variable in the absence of IN3. Substitution of the bulky Trp at the amino acid 279 (adjacent to C278) with Ala (W279A) also produced an inactive mutant that was rescued by IN3 (Table 2).
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We also examined the ability to rescue a shortened carboxyl-terminal truncation mutant [des(325–327)] that showed virtually no response to GnRH when expressed in cultured cells. Deletion of the last three amino acids resulted in loss of IP production in response to the agonist; much of this activity could be recovered by IN3. Removing a larger (12 amino acid) sequence from the carboxyl- terminal [des(316–327)] produced a mutant that could not be rescued, although as many as four amino acids could be removed from the third intracellular loop [des(237–241)] and rescue still effected (Table 2).
The observation that the percentage of surface-bound receptor-ligand complex internalized by the rescued receptor (40% and 43% at 120 and 160 min, respectively), in response to the radioiodinated agonist buserelin, was comparable to WT (34% and 40% at 120 and 160 min, respectively) excluded the possibility that rescue resulted from stabilization of extant receptor. Likewise, IN3 rescued mutants that were not (previously) measurably expressed on the cell surface (and, therefore not available to be stabilized). Peptide antagonists (37, 38, 39), which were designed by chemical analogy to agonists and are, therefore, expected to bind to the same site but unlike IN3 cannot permeate cells, were unable to rescue HH, deletion mutants, or Cys mutants (Table 3). The antagonists selected are weak (D-Phe2, D-Phe6-GnRH), intermediate (‘Nal-Glu’), and high (cetrorelix) affinity binders (37, 38, 39).
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Discussion
At the beginning of these studies we were influenced by our earlier observation (19) that HH mutants could be rescued by further modifications of the defective proteins, which were known to enhance membrane expression. The observation that these modifications could rescue a hGnRHR mutant (19) suggested to us that such mutants retained intrinsic hormone binding and effector coupling activity, yet, due to mutation, were misfolded and potentially not appropriately positioned at the plasma membrane. We reasoned that correction of such folding defects might restore activity and rescue the mutant. We sought to identify a pharmacological agent that could serve as a template for the nascent receptor and, thereby, effect rescue. We also had the prejudice that agonists would be poor candidates for rescue agents, because these would be expected to provoke desensitization or down-regulation. In addition, we wished to determine the range of receptor defects that could be rescued by this technique.
In this study, we did not assess the locus to which mutant receptors might be misrouted; indeed, it would not be surprising if different mutants (or manufactured defective receptors) might be routed to entirely different compartments. We specifically also note that misrouting may even include the positioning of a mutant receptor in the plasma membrane in a position that is unavailable to the ligand for binding or to the effector or G proteins.
The surprising finding that all HH mutations attempted could be rescued, as assessed by radioligand binding or by the ability to activate IP production, despite widely disparate loci along the receptor, presented the possibility that HH receptor mutants may be frequently misrouted or mispositioned molecules, yet otherwise fully competent to bind ligand and couple to effectors. This was the suggestion of the prior study (19) and prompted us to perform the present work.
In addition to the HH mutant, we synthesized a series of other molecules that were terminally truncated, had internal deletions, or lacked the ability to form bridges believed to be significant in formation of tertiary structure. It was possible to rescue a number of such defective receptors shown in the unrescued state to be inactive.
In addition to genetic approaches (19, 40), other methods, including increased expression (41) and the use of such nonspecific protein stabilizing agents as polyols and sugars (42, 43, 44), have been used to produce larger numbers of receptors or to stabilize extant molecules rendered incompetent by genetic defects. The present approach does not require genetic manipulation and offers high specificity, because the ligand used is known to be a specific hGnRHR antagonist (23, 24, 25). This approach is also expected to be useful in the laboratory to assess ligand binding and effector coupling of mutants previously not expressed at measurable levels or to increase levels of the WT receptor without exposure to (up-regulating) hormone.
The expression level of human WT receptor itself could be enhanced by IN3, presenting potential therapeutic approaches for disease states in which the receptor is the proper (WT) gene product but is processed (destroyed, internalized) excessively.
Our data and that from another group (45) showing rescue of the V2 vasopressin receptor suggests a general approach to diseases resulting from misfolded molecules. There is recent evidence that forms of cystic fibrosis, nephrogenic diabetes insipidus, hypercholesterolemia, and retinitis pigmentosa are included among such diseases (46, 47). Accordingly, the approach described in the present study for HH GnRHR mutant rescue may prove generally applicable for the development of therapeutic approaches for incorrectly routed receptors.
Because rescue molecules need not a priori be either agonists or antagonists, it is likely that extant chemical archives already contain valuable therapeutic compounds that would have been overlooked by screens requiring such activities. It is our current view that agonists would be poor rescue molecules because once receptor rescue occurred, the development of the desensitized state would be promoted. One could, additionally, envision useful therapeutic molecules that would support scaffolding for the active configuration of the receptor without occupying the active site and, thus, would not need to be removed before agonist activation.
Acknowledgments
We thank Drs. Wallace Ashton and Mark Goulet (Merck \|[amp ]\| Co., Inc.); Dr. Ursula Kaiser (Harvard University, Cambridge, MA); Drs. Sergio Ojeda, James Parker, and William Thompson (Oregon Health & Science University, Beaverton, OR); and Dr. Pierre Riviere (Ferring Research Institute) for reading and commenting on this manuscript.
Footnotes
This work was supported by NIH Grants HD-19899, RR-00163, HD-18185, and TW/HD-00668.
Abbreviations: DMSO, Dimethylsulfoxide; FCS, fetal calf serum; GnRHR, GnRHR receptor; GPCR, G protein-coupled receptors; hGnRHR, human GnRHR; HH, hypogonadotropic hypogonadism; IP, inositol phosphate; r, rodent; WT, wild-type.
Received January 14, 2002.
Accepted April 4, 2002.
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S. Wuller, B. Wiesner, A. Loffler, J. Furkert, G. Krause, R. Hermosilla, M. Schaefer, R. Schulein, W. Rosenthal, and A. Oksche Pharmacochaperones Post-translationally Enhance Cell Surface Expression by Increasing Conformational Stability of Wild-type and Mutant Vasopressin V2 Receptors J. Biol. Chem., November 5, 2004; 279(45): 47254 - 47263. [Abstract] [Full Text] [PDF]
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V. Bernier, M. Lagace, M. Lonergan, M.-F. Arthus, D. G. Bichet, and M. Bouvier Functional Rescue of the Constitutively Internalized V2 Vasopressin Receptor Mutant R137H by the Pharmacological Chaperone Action of SR49059 Mol. Endocrinol., August 1, 2004; 18(8): 2074 - 2084. [Abstract] [Full Text] [PDF]
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A. U. Meysing, H. Kanasaki, G. Y. Bedecarrats, J. S. Acierno Jr., P. M. Conn, K. A. Martin, S. B. Seminara, J. E. Hall, W. F. Crowley Jr., and U. B. Kaiser GNRHR Mutations in a Woman with Idiopathic Hypogonadotropic Hypogonadism Highlight the Differential Sensitivity of Luteinizing Hormone and Follicle-Stimulating Hormone to Gonadotropin-Releasing Hormone J. Clin. Endocrinol. Metab., July 1, 2004; 89(7): 3189 - 3198. [Abstract] [Full Text] [PDF]
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D. Mizrachi and D. L. Segaloff Intracellularly Located Misfolded Glycoprotein Hormone Receptors Associate with Different Chaperone Proteins than Their Cognate Wild-Type Receptors Mol. Endocrinol., July 1, 2004; 18(7): 1768 - 1777. [Abstract] [Full Text] [PDF]
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S. P. Brothers, A. Cornea, J. A. Janovick, and P. M. Conn Human Loss-of-Function Gonadotropin-Releasing Hormone Receptor Mutants Retain Wild-Type Receptors in the Endoplasmic Reticulum: Molecular Basis of the Dominant-Negative Effect Mol. Endocrinol., July 1, 2004; 18(7): 1787 - 1797. [Abstract] [Full Text] [PDF]
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R. P. Millar, Z.-L. Lu, A. J. Pawson, C. A. Flanagan, K. Morgan, and S. R. Maudsley Gonadotropin-Releasing Hormone Receptors Endocr. Rev., April 1, 2004; 25(2): 235 - 275. [Abstract] [Full Text] [PDF]
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 A. Ulloa-Aguirre, J. A. Janovick, A. Leanos-Miranda, and P. M. Conn Misrouted cell surface GnRH receptors as a disease aetiology for congenital isolated hypogonadotrophic hypogonadism Hum. Reprod. Update, March 1, 2004; 10(2): 177 - 192. [Abstract] [Full Text] [PDF]
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S. P. Brothers, J. A. Janovick, and P. M. Conn Unexpected Effects of Epitope and Chimeric Tags on Gonadotropin-Releasing Hormone Receptors: Implications for Understanding the Molecular Etiology of Hypogonadotropic Hypogonadism J. Clin. Endocrinol. Metab., December 1, 2003; 88(12): 6107 - 6112. [Abstract] [Full Text] [PDF]
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A. Leanos-Miranda, A. Ulloa-Aguirre, T. H. Ji, J. A. Janovick, and P. M. Conn Dominant-Negative Action of Disease-Causing Gonadotropin-Releasing Hormone Receptor (GnRHR) Mutants: A Trait That Potentially Coevolved with Decreased Plasma Membrane Expression of GnRHR in Humans J. Clin. Endocrinol. Metab., July 1, 2003; 88(7): 3360 - 3367. [Abstract] [Full Text] [PDF]
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Y. Bo-Abbas, J. S. Acierno Jr., J. K. Shagoury, W. F. Crowley Jr., and S. B. Seminara Autosomal Recessive Idiopathic Hypogonadotropic Hypogonadism: Genetic Analysis Excludes Mutations in the Gonadotropin-Releasing Hormone (GnRH) and GnRH Receptor Genes J. Clin. Endocrinol. Metab., June 1, 2003; 88(6): 2730 - 2737. [Abstract] [Full Text] [PDF]
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J. A. Janovick, M. Goulet, E. Bush, J. Greer, D. G. Wettlaufer, and P. M. Conn Structure-Activity Relations of Successful Pharmacologic Chaperones for Rescue of Naturally Occurring and Manufactured Mutants of the Gonadotropin-Releasing Hormone Receptor J. Pharmacol. Exp. Ther., May 1, 2003; 305(2): 608 - 614. [Abstract] [Full Text] [PDF]
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G. Y. Bedecarrats, K. D. Linher, and U. B. Kaiser Two Common Naturally Occurring Mutations in the Human Gonadotropin-Releasing Hormone (GnRH) Receptor Have Differential Effects on Gonadotropin Gene Expression and on GnRH-Mediated Signal Transduction J. Clin. Endocrinol. Metab., February 1, 2003; 88(2): 834 - 843. [Abstract] [Full Text] [PDF]
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 P. M. Conn, A. Leanos-Miranda, and J. A. Janovick Protein Origami: Therapeutic Rescue of Misfolded Gene Products Mol. Interv., September 1, 2002; 2(5): 308 - 316. [Abstract] [Full Text] [PDF]
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