Influence of a Species-Specific Extracellular Amino Acid on Expression and Function of the Human Gonadotropin-Releasing Hormone Receptor

Krishan K. Arora1, Hye-Ok Chung1 and Kevin J. Catt

Endocrinology and Reproduction Research Branch National Institute of Child Health and Human Development National Institutes of Health Bethesda, Maryland 20892


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The mammalian GnRH receptor is an atypical G protein-coupled receptor which lacks the C-terminal cytoplasmic tail that is present in all other seven-transmembrane domain receptors. The mouse and rat GnRH receptors contain 327 amino acids, whereas human, sheep, and bovine receptors have an additional residue in the second extracellular loop at position 191. Another notable species difference is that human receptors undergo agonist-induced internalization much more rapidly than the mouse receptor. In this report, the role of the additional amino acid (Lys191) in GnRH receptor function was studied in transiently expressed mutant and wild-type human and mouse GnRH receptors. Deletion of Lys191 from the human GnRH receptor caused a 4-fold increase in receptor expression in COS-1 and HEK 293 cells and a modest increase in binding affinity. The magnitude of the agonist-induced inositol phosphate response mediated by the {Delta}K191 human receptor was similar to that of the wild-type receptor, but the EC50 was decreased by about 5-fold. In addition, the rate of internalization of the {Delta}K191 human receptor was significantly reduced and was similar to that of the mouse receptor. In contrast to these effects of deletion of Lys191, its replacement by Arg, Glu, Gln, or Ala caused no significant change in receptor expression or function. These findings demonstrate that a specific residue in the extracellular region of the human GnRH receptor is a significant determinant of receptor expression, agonist-induced activation, and internalization.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The hypothalamic decapeptide, GnRH, controls the activity of the reproductive system by regulating the synthesis and release of LH and FSH from the anterior pituitary gland (1). Synthetic analogs of GnRH are widely used to treat a variety of clinical disorders that respond to stimulation or, more commonly, suppression of gonadotropin secretion (2). In pituitary gonadotrophs, GnRH binds to specific high-affinity receptors that mediate its actions by promoting G protein-dependent stimulation of phosphoinositide turnover and calcium mobilization (3). The cloned GnRH receptors of mouse, rat, human, sheep, cow, and pig exhibit more than 85% amino acid identity among species (4). The hydropathy analysis of the GnRH receptor-coding region is consistent with the seven-transmembrane domain structure that is characteristic of the G protein-coupled receptor superfamily. Mammalian GnRH receptors exhibit several unique structural features, including the absence of an intracellular carboxyl-terminal tail (3, 4). Recently, the cloned GnRH receptors from catfish and amphibia were found to contain a 50-amino acid cytoplasmic tail (5, 6).

In functional studies, the human receptor has been found to be more rapidly internalized than the mouse receptor and is less abundantly expressed in transfected mammalian cells. Among the structural differences between the two species is the slightly larger size of the human GnRH receptor. Whereas the mouse and rat GnRH receptors contain 327 amino acids, the human, cow, and sheep receptors contain 328 amino acids, due to the presence of an additional residue in the second extracellular loop. This residue is Lys191 in the human GnRH receptor and Glu191 in the ungulate receptors (4). The present study was performed to examine the role of the additional Lys191 residue in the function of the human GnRH receptor. For this purpose, the Lys191 residue was deleted or substituted by other amino acids. The mutant GnRH receptors were transiently expressed in COS-1 cells and analyzed for ligand binding, agonist-stimulated inositol phosphate (InsP) production, and agonist-induced internalization of the hormone-receptor complex. The results of this study have demonstrated that deletion of Lys191 from the human GnRH receptor significantly increases its expression at the cell surface and alters its signaling properties and internalization kinetics. However, receptors in which Lys191 was substituted with arginine, glutamine, glutamic acid, or alanine behaved in the same manner as the wild-type receptor. These findings indicate that the Lys191 residue in the second extracellular loop of the human GnRH receptor has an unexpectedly prominent role in several aspects of receptor function.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Expression of Human GnRH Receptors
COS-1 cells were transfected with cDNA encoding either the wild-type or the {Delta}K191 human GnRH receptor to compare the binding, signaling, and internalization properties of the two receptors. As shown in Fig. 1Go, cells expressing the {Delta}K191 human GnRH receptor bound more than 4 times as much [125I]GnRH-Ag as the wild-type (WT) receptor at 37 C. To determine the surface expression level and structural integrity of these receptors, radioligand binding to COS-1 cells expressing WT or mutant receptors was measured at 4 C. Scatchard analysis of the binding data revealed that the dissociation constant (Kd) of the WT receptor for the GnRH agonist was 10.5 ± 0.45 nM (n = 3), whereas that of the {Delta}K191 receptor was slightly but consistently lower (7.6 ± 0.40 nM, n = 3). The 4-fold greater degree of GnRH-Ag binding to cells expressing the {Delta}K191 receptor vs. the WT receptor was due to its higher expression level. The Bmax values were 72 ± 8 and 307 ± 30 fmol/well for the WT and {Delta}K191 receptors, respectively. The increase in expression level observed for the {Delta}K191 receptor was also evident when enzyme-linked immunosorbent assay (ELISA) measurements of receptor protein levels were performed (Fig. 2Go). Furthermore, when HEK 293 cells were used to transiently express human GnRH receptors, the {Delta}K191 receptors were nearly 8-fold better expressed than the WT receptor (data not shown). However, when Lys191 was replaced by Arg, Gln, Glu, or Ala, the mutant receptors were expressed at almost the same level as the WT human receptor (Fig. 1BGo). These results indicate that deletion of Lys191 from the human GnRH receptor significantly increases the expression level of the receptor, whereas its substitution by other residues maintains the basal level of receptor expression.



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Figure 1. Radioligand Binding to WT Human and Mouse GnRH Receptors

A, Effects of Lys191 deletion and insertion on [125I]GnRH-Ag binding to human and mouse receptors, respectively. B, Effects of mutations and deletion of Lys191 on [125I]GnRH-Ag binding to the human GnRH receptor. COS-1 cells expressing WT human, mouse, or Lys191-mutated (deleted, inserted, or substituted) GnRH receptors were incubated with 0.5 ml of binding medium containing [125I]GnRH-Ag at 37 C. Nonspecific binding was determined in the presence of a 1000-fold excess of unlabeled GnRH-Ag and was subtracted to obtain the specific binding. Values shown in the bar graphs are means ± SE and are representative of three similar experiments each performed in triplicate.

 


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Figure 2. Cell-Surface Expression of WT and Lys191-Mutated GnRH Receptors

ELISA measurements were performed on nonpermeabilized COS-1 cells expressing HA-epitope-tagged WT or mutant GnRH receptors as described in Materials and Methods. Cells transfected with pcDNA1/Amp vector only served as a negative control. Data represent means ± SE of one of three independent experiments performed in triplicate. OD, Optical density.

 
The surface density of the {Delta}K191 human receptors is similar to that of the WT mouse receptors, which are well expressed in this transient cell expression system (see Fig. 1Go). Interestingly, the insertion of a Lys residue at position 191 in the mouse receptor did not have a deleterious effect on its expression. In fact, the +K191 mouse receptor is equally well expressed and has the same binding affinity as the WT mouse receptor. The Kd values of the WT and +K191 mouse receptors were 2.4 ± 0.22 (n = 3) and 2.0 ± 0.13 nM (n = 3), respectively.

In addition to the presence of an extra amino acid (Lys191) in the second extracellular loop of the human receptor, this region has at least four other differences from the sequence of the mouse receptor. The human receptor contains His, Ser, Gln, and Ser at positions 182, 186, 189 and 203, whereas the mouse receptor has Tyr, Gly, Pro, and Pro as the analogous residues. When these residues in the human receptor were changed individually to the corresponding amino acids in the mouse receptor, radioligand binding to the mutant receptors (H182Y, S186G, Q 189P or S 203P) was similar to that of the WT human receptor (Fig. 3Go). These findings suggest that the other regional amino acid differences between two species do not account for the observed differences in receptor expression. As observed in Fig. 1Go, deletion of Lys191 caused a marked increase in the expression of the human receptor.



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Figure 3. Effect of Site-Directed Mutations in the Second Extracellular Loop on Radioligand Binding to GnRH Receptors

WT or H182Y, S186G, Q189P, S203P and {Delta}K191 mutant GnRH receptors were transiently expressed in COS-1 cells, and radioligand binding was measured as described in the legend to Fig. 1Go. Values shown in the bar graph are means of two experiments, and the range was within 10% of the means.

 
Signal Transduction by WT and Lys191-Mutated GnRH Receptors
The ability of the mutant receptors to couple to phospholipase C via Gq/G11 proteins was determined by measuring the InsP responses of transfected COS-1 cells stimulated with 10-10 to 10-6 M GnRH in the presence of 10 mM LiCl. Under these experimental conditions, the major accumulated products of phosphoinositide hydrolysis in GnRH receptor-transfected cells are InsP2 and InsP3 (7). The GnRH-induced InsP responses mediated by {Delta}K191 human receptors were similar to those of the WT receptors, but the EC50 for agonist stimulation was decreased by about 5-fold (Fig. 4AGo); EC50 values were 75.0 ± 10.0 pM (n = 3) and 350 ± 70 pM (n = 3) for the {Delta}K191 and WT human receptors, respectively. The InsP response profiles for the mouse, +K191 mouse, and K191R, K191E, K191Q, and K191A human GnRH receptors were virtually identical to that of the WT human receptor (Fig. 4Go, A and B) and had similar EC50 values. These results indicate that overexpressed {Delta}K191 receptors are functionally active and show significantly enhanced ability to elicit phosphoinositide hydrolysis.



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Figure 4. GnRH-Induced InsP Production in Cells Expressing WT Human, Mouse, or Lys191-Mutated GnRH Receptors

Panel A corresponds to the WT human and mouse, and Lys191 deleted or inserted receptors, and panel B shows the human WT and Lys191-substituted receptors. COS-1 cells expressing WT or mutant GnRH receptors were labeled for 24 h with [3H]inositol and stimulated with increasing concentrations of GnRH in the presence of 10 mM LiCl. InsPs were extracted and separated by anion exchange chromatography as described in Materials and Methods. Data are expressed as the combined radioactivity (cpm) of the InsP2 and InsP3 fractions and are representative of three similar experiments each performed in duplicate.

 
Internalization of WT and Lys191-Mutated GnRH Receptors
Mouse GnRH receptors expressed in COS cells undergo ligand-induced internalization that is similar to that of the native receptors in pituitary gonadotrophs and {alpha}T3–1 cells (7, 8, 9, 10). A comparison of internalization kinetics for the WT mouse and human GnRH receptors is shown in Fig. 5AGo. It is evident that human receptors have at least 2-fold higher internalization kinetics than the mouse receptor. The endocytotic rate constant, which defines the probability of an occupied receptor being internalized in 1 min at 37 C, was calculated from these data as described previously (8). These values were 0.007 for the mouse and 0.022 for the human receptor.



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Figure 5. Internalization of WT and Mutant GnRH Receptors

The internalization kinetics of WT and mutant GnRH receptors, measured as the acid-resistant uptake of [125I]GnRH-Ag, are shown. A, Effects of Lys191 deletion and insertion on time course of internalization by human and mouse receptors, respectively. B, Effects of deletion and mutations of Lys191 on internalization kinetics of human GnRH receptors. C, Percent internalization at 60 min for the WT and mutant human and mouse receptors from panels A and B. Values shown are representative of three similar experiments each performed in duplicate.

 
The effects of Lys191 mutation on receptor internalization were evaluated by measuring the kinetics of 125I-GnRH-Ag uptake over a period of 60 min at 37 C in cells expressing WT mouse, WT human, or mutant GnRH receptors. A direct comparison between the WT and mutant receptors was made by plotting the percentage of bound radioligand that was internalized with increasing time of incubation (Fig. 5Go, A and B). When Lys191 was deleted from the human receptor, the kinetics of internalization were much slower than those of the native human receptor (Fig. 5AGo), and the percent of radioligand sequestered at 60 min was similar to that of the mouse WT or +K191 receptors (Fig. 5CGo). However, the internalization rates of the four Lys191-substituted receptors were similar to that of the WT human receptor (Fig. 5BGo), with the same amounts of radioligand sequestered at 60 min (Fig. 5C).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The primary finding of the present study was that the deletion of Lys191 from the human GnRH receptor significantly increases its cell-surface expression, to a level comparable to that seen for the murine GnRH receptor that lacks this residue. The overexpressed human {Delta}K191 receptors bind GnRH agonist in a manner similar to the native WT receptor. The major functional difference between the two receptors is that the {Delta}K191 receptors internalize slowly, similar to the mouse receptor, whereas native human receptors have comparatively rapid internalization kinetics with an almost 3-fold higher endocytotic rate constant. The reason(s) for the low rate of {Delta}K191 receptor internalization are not apparent. The finding is intriguing in view of the extracellular location of the Lys191 residue, at a site distant from the transmembrane and intracellular regions that are believed to engage in interactions with the signaling and internalization machineries of the cell. It is remotely possible that the greater level of expression of the mouse and the {Delta}K191 receptors could saturate the endocytic machinery, leading to the relatively decreased internalization relative to the human receptor, which has lower expression but exhibits 2-fold more rapid internalization. However, this possibility is unlikely in view of our previous observations that several mutations in the mouse receptor caused increased internalization without any significant effect on receptor expression (7, 8).

The nature of the amino acid at position 191 does not appear to be critical, since its substitution by residues with different side-chain properties (Arg, Gln, Glu, or Ala) had no effect on receptor expression or function. It is possible that a motif containing Lys191 in the human receptor is not favorable to its folding and/or helices packing. Another possibility is that its presence in the human receptor augments the turnover rate, thus causing enhanced internalization of the receptor. If this is the case, then the mouse receptor bearing lysine at position 191 should have exhibited properties similar to that of the WT human receptor, but this was not observed. In the present study, no effect on receptor expression and internalization was seen after replacement of Lys191 with Glu, the amino acid residue that is usually present in the ungulate receptors. This implies that the expression and internalization patterns of ungulate receptors would resemble those of human rather than mouse receptors. These findings demonstrate that the presence of an additional residue at position 191 in the human receptor, and possibly in ungulates, is a critical determinant of receptor expression and internalization and that its deletion imparts the mouse phenotype.

The structural basis for the observation made in the present study is not yet known. As a first approximation, examination of the primary sequence in the second extracellular loop of the GnRH receptor revealed that, although this protein is highly homologous in human and mouse, there are a few differences upstream of residue Lys191. For example, the human receptor has His, Ser, and Gln at positions 182, 186, and 189, whereas the mouse receptor has Tyr, Gly, and Pro as the analogous residues. Downstream of Lys191, the human receptor contains Ser at position 203, and the corresponding residue in the mouse receptor is Pro. Individual mutations of these amino acids in the human receptor to the corresponding residues in the mouse had no significant effect on receptor expression, in contrast to the effect of deletion of Lys191 (Fig. 3Go). These amino acid differences presumably give rise to different local secondary structures in the second extracellular loop that are related to the observed interspecies differences in GnRH receptor expression and function. The secondary structure predicted by the algorithms of Chou and Fasman (11) in the region surrounding Lys191 of the human GnRH receptor indicates that there is no defined structure, whereas its deletion confers a higher order structure that could presumably stabilize the receptor protein (Fig. 6Go). The physiological relevance of these results is not clear at this time. One possibility is that the molecular factors governing the reproductive process at the level of the GnRH receptor differ in humans and mice. This could, in part, be related to the presence of the additional residue in the second extracellular loop, which influences its expression and internalization and possibly its desensitization mechanisms.



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Figure 6. Predicted Secondary Structure of Second Extracellular Loop of the GnRH Receptor

Secondary structure of the sequence of the second extracellular loop flanking the Lys191 residue (amino acids 186–196), as predicted by the algorithms of Chou and Fasman (11 ). The predicted turns and ß-strands are indicated by jagged lines and arrows, respectively, and the unpredicted/random structure by dots. In the native human receptor, the region around Lys191 has no defined structure. However, deletion of this residue confers a defined higher-order structure. In the mouse receptor, insertion of Lys191 does not affect the proposed structure.

 
In conclusion, these studies have demonstrated the importance of a species-specific residue (Lys191) in the extracellular loop of the human GnRH receptor in receptor expression and internalization. The deletion of Lys191 substantially increases the expression of the human GnRH receptor protein, with no significant effect on its agonist-binding affinity. In contrast to the prominent actions of deletion of Lys191, its replacement by Arg, Glu, Gln, or Ala do not affect receptor expression or function, implying that the nature of amino acid at position 191 is not critical. The enhanced expression of the {Delta}K191 human receptor in transfected mammalian cells could be of value in screening therapeutically useful GnRH analogs. The most interesting aspect of this finding, the mechanism whereby this single amino acid in the extracellular domain influences receptor expression and function, remains to be clarified.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
GnRH and its superagonist analog (des-Gly10-[D-Ala6]GnRH N-ethylamide, GnRH-Ag) were obtained from Peninsula Laboratories, Inc. (Belmont, CA). Fugene 6 Transfection Reagent was purchased from Boehringer Mannheim (Indianapolis, IN), cell-culture related products from Biofluids (Rockville, MD), and restriction and DNA-modifying enzymes from New England BioLabs, Inc. (Beverly, MA). Oligonucleotide primers for site-directed mutagenesis were synthesized in a Oligo 1000 DNA Synthesizer (Beckman Coulter, Inc., Fullerton, CA). The Muta-Gene phagemid in vitro mutagenesis kit (Version 2), AG-1-X8 resin (100–200 mesh formate form) and Poly-Prep Chromatography Columns for anion exchange chromatography were obtained from Bio-Rad Laboratories, Inc. (Hercules, CA). All other reagents were of analytical grade quality. myo-[3H]inositol (80–100 Ci/mmol) and Thermo Sequenase radiolabeled terminator cycle sequencing kits were from Amersham Pharmacia Biotech (Arlington Heights, IL). 125I-des-Gly10-[D-Ala6]GnRH N-ethylamide (125I-GnRH-Ag) was prepared by Covance Laboratories, Inc. (Vienna, VA).

Construction of WT and Mutant GnRH Receptors
The 1550-bp human GnRH receptor cDNA (kindly provided by Dr. Sham S. Kakar, University of Alabama at Birmingham, Birmingham, AL) was subcloned into pcDNA1/Amp at the EcoRI and XbaI sites, and a hemagglutinin (HA) epitope (YPYDVPDYA) was introduced at its amino terminus after the first methionine residue. The HA-tagged human GnRH receptor was used as a template for creating site-directed mutations according to the method of Kunkel et al. (12) using a Muta-Gene phagemid in vitro mutagenesis kit. Mutations were identified using the Thermo Sequenase radiolabeled terminator cycle sequencing kit. The construction of the plasmid expressing the mouse GnRH receptor has been described previously (7).

Receptor Expression in COS-1 Cells
WT and mutant GnRH receptors were transiently expressed in COS-1 cells. To measure InsP responses, [125I]GnRH-Ag binding to intact cells, and internalization kinetics, cultures were seeded in 24-well plates (Costar, Cambridge, MA) at a density of 0.15 x 106 cells per well 1 day before transfection. The plated cells were cultured in DMEM supplemented with 10% heat-inactivated FBS containing 100 U/ml of penicillin and 100 µg/ml streptomycin (Pen-Strep) at 37 C in an atmosphere consisting of 5% CO2/95% humidified air. Next day, the cells were transfected with WT or mutant plasmid DNA (1 µg/well) using Fugene 6 transfection reagent. The cultures were maintained for 48 h before analysis of receptor function by ligand binding and other assays.

Receptor Binding and Internalization Assays
The binding affinity and abundance of the mutant receptors were determined in transfected COS-1 cells incubated with 2 nM [125I]GnRH-Ag in binding medium (M199 containing 25 mM HEPES and 0.1% BSA) in the absence or presence of increasing concentrations of unlabeled peptide for 3–4 h at 4 C. The cells were then rapidly washed twice with ice-cold PBS (pH 7.4) and solubilized in 0.5 M NaOH/1% SDS solution. The cell-associated radioactivity was measured by {gamma}-spectrometry. All time studies were performed in duplicate on at least three occasions, and displacement curves were analyzed for binding affinity and capacity by the LIGAND program using a one-site model (13). The nonspecific binding of [125I]GnRH-Ag determined in the presence of unlabeled agonist (1 µM) for WT or mutant receptors was always less than 5% of the respective total binding.

For internalization studies, transfected COS-1 cells were washed once with binding medium before the addition of 2 nM 125I-labeled GnRH-Ag. After incubation at 37 C for the indicated times, the cells were washed twice with ice-cold PBS (pH 7.4) and incubated with 1 ml of 50 mM acetic acid/150 mM NaCl (pH 2.8) for 12 min to remove surface-bound tracer. The acid-released radioactivity was collected to determine the receptor-bound radioactivity, and the internalized (acid-resistant) radioactivity was quantitated after the cells were solubilized in NaOH/SDS solution. Radioactivities were measured by {gamma}-spectrometry, and the internalized radioligand at each time-point was expressed as a percent of the total (acid-resistant + acid-released) binding.

InsP Production
COS-1 cells were labeled 24 h after transfection by incubation in inositol-free DMEM containing 20 µCi/ml [3H]inositol as described previously (7). After 24 h, cells were washed with inositol-free M199 medium and preincubated in the same medium containing 10 mM LiCl for 30 min at 37 C, and then stimulated with 10-10 to 10-6 M GnRH for 20 min. Incubations were terminated by the addition of ice-cold perchloric acid [5% (vol/vol) final concentration]. The InsPs were extracted and separated by anion exchange chromatography as described previously (8), and their radioactivities were measured by liquid scintillation ß-spectrometry.

ELISA
An indirect ELISA protocol was used to quantify the expression of epitope-tagged WT or mutant GnRH receptors in the plasma membrane. COS-1 cells were seeded at a density of 90,000 cells per well in 48-well plates and transfected after 24 h with WT or mutant receptor cDNA. After 48 h, the cells were fixed with 4% paraformaldehyde in PBS for 30 min at room temperature. After washing with PBS three times, treatment with DMEM containing 10% FBS to block nonspecific binding sites, the cells were incubated at 37 C for 2 h with a monoclonal antibody directed against the HA-epitope tag (Babco, Richmond, CA) at a dilution of 1:500 in DMEM. Plates were then washed in DMEM and incubated with a 1:2000 dilution (in DMEM) of peroxidase-conjugated goat antimouse IgG antibody (Sigma Chemical Co., St. Louis, MO) for 1 h at room temperature. Hydrogen peroxide (0.03%) and o-phenylenediamine (5 mM) in 0.1 M phosphate-citrate buffer (pH 5.0), serving as substrate and chromogen, respectively, were then added, and the plates were kept in the dark for 30 min. The enzymatic reaction was terminated with 1 M H2SO4 containing 0.05 M Na2SO3, and the color development was measured at 495 nm using a Titertek Multiskan plate reader (EFLABOY, Helsinki, Finland).


    FOOTNOTES
 
Address requests for reprints to: Kevin J. Catt, Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, Building 49, Room 6A36, NIH, Bethesda, Maryland 20892-4516. E-mail: catt{at}helix.nih.gov

1 Both of these authors contributed equally to this work. Back

Received for publication November 30, 1998. Revision received March 8, 1999. Accepted for publication March 10, 1999.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Clayton RN, Catt KJ 1981 Gonadotropin-releasing hormone receptors: characterization, physiological regulation, and relationship to reproductive function. Endocr Rev 2:186–209[Medline]
  2. Barbieri RL 1992 Clinical applications of GnRH and its analogues. Trends Endocrinol Metab 3:30–34
  3. Stojilkovic SS, Reinhart J, Catt KJ 1994 Gonadotropin-releasing hormone receptors: structure and signal transduction pathways. Endocr Rev 15:462–499[Medline]
  4. Sealfon SC, Weinstein H, Millar RP 1997 Molecular mechanisms of ligand interaction with the gonadotropin-releasing hormone receptor. Endocr Rev 18:180–205[Abstract/Free Full Text]
  5. Tensen C, Okuzawa K, Blomenrohr M, Rebers F, Leurs R, Bogerd J, Schulz R, Goos H 1997 Distinct efficacies for two endogenous ligands on a single cognate gonadoliberin receptor. Eur J Biochem 243:134–140[Abstract]
  6. Troskie B, Sun Y, Hapgood J, Sealfon SC, Illing N, Millar RP, Mammalian GnRH receptor functional features revealed by comparative sequences of goldfish, frog, chicken receptors. Program of the 79th Annual Meeting of The Endocrine Society, Minneapolis, MN, 1997, p 167 (Abstract)
  7. Arora KK, Sakai A, Catt KJ 1995 Effects of second intracellular loop mutations on signal transduction and internalization of the gonadotropin-releasing hormone receptor. J Biol Chem 270:22820–22826[Abstract/Free Full Text]
  8. Arora KK, Cheng Z, Catt KJ 1997 Mutations of the conserved DRS motif in the second intracellular loop of the gonadotropin-releasing hormone receptor affect expression, activation, and internalization. Mol Endocrinol 11:1203–1212[Abstract/Free Full Text]
  9. Loumaye E, Catt KJ 1983 Agonist-induced regulation of pituitary receptors for gonadotropin-releasing hormone. Dissociation of receptor recruitment from hormone release in cultured gonadotrophs. J Biol Chem 258:12002–12009[Abstract/Free Full Text]
  10. Mason DR, Arora KK, Mertz LM, Catt KJ 1994 Homologous down-regulation of gonadotropin-releasing hormone receptor sites and messenger ribonucleic acid transcripts in {alpha}T3–1 cells. Endocrinology 135:1165–1170[Abstract]
  11. Chou PY, Fasman GD 1978 Prediction of the secondary structure of proteins from their amino acid sequence. Adv Enzymol 47:45–148[Medline]
  12. Kunkel TA, Roberts JD, Zakour RA 1987 Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol 154:367–382[Medline]
  13. Munson PJ, Rodbard D 1980 Ligand: a versatile computerized approach for characterization of ligand binding systems. Anal Biochem 107:220–239[Medline]