Inhibition of Gonadotropin-Releasing Hormone Receptor Signaling by Expression of a Splice Variant of the Human Receptor

Robert Grosse, Torsten Schöneberg, Günter Schultz and Thomas Gudermann

Institut für Pharmakologie Freie Universität Berlin D-14195 Berlin, Germany


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GnRH binds to a specific G protein-coupled receptor in the pituitary to regulate synthesis and secretion of gonadotropins. Using RT-PCR and human pituitary poly(A)+ RNA as a template, the full-length GnRH receptor (wild type) and a second truncated cDNA characterized by a 128-bp deletion between nucleotide positions 522 and 651 were cloned. The deletion causes a frame shift in the open reading frame, thus generating new coding sequence for further 75 amino acids. The truncated cDNA arises from alternative splicing by accepting a cryptic splicing acceptor site in exon 2. Distinct translation products of approximately 45–50 and 42 kDa were immunoprecipitated from COS-7 cells transfected with cDNA coding for wild type GnRH receptor and the truncated splice variant, respectively. Immunocytochemical and enzyme-linked immunosorbent assay studies revealed a membranous expression pattern for both receptor isoforms. Expression of the splice variant, however, occurred at a significantly lower cell surface receptor density. In terms of ligand binding and phospholipase C activation, the wild type receptor showed characteristics of a typical GnRH receptor, whereas the splice variant was incapable of ligand binding and signal transduction. Coexpression of wild type and truncated proteins in transiently or stably transfected cells, however, resulted in impaired signaling via the wild type receptor by reducing maximal agonist-induced inositol phosphate accumulation. The inhibitory effect depended on the amount of splice variant cDNA cotransfected and was specific for the GnRH receptor because signaling via other Gq/11-coupled receptors, such as the thromboxane A2, M5 muscarinic, and V1 vasopressin receptors, was not affected. Immunological studies revealed that coexpression of the wild type receptor and the truncated splice variant resulted in impaired insertion of the wild type receptor into the plasma membrane. Thus, expression of truncated receptor proteins may highlight a novel principle of specific functional inhibition of G protein-coupled receptors.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The hypothalamic decapeptide, GnRH, plays a pivotal role in the neuroendocrine control of the mammalian reproductive system. GnRH is released into the hypophyseal portal circulation and acts on specific adenohypophyseal target cells to increase the secretion of the gonadotropins LH and FSH (1). GnRH, however, functions not only as a releasing factor in the pituitary but is distributed widely throughout the central and peripheral nervous system as well as in several extraneural and also in neoplastic tissues (2).

Cloning of the GnRH receptor cDNA from six mammalian species (2, 3, 4) has revealed that the receptor is a member of the large superfamily of heptahelical G protein-coupled receptors (GPCRs). The GnRH receptor is one of the smallest heptahelical receptors cloned so far and completely lacks a cytoplasmic C-terminal tail, which has been implicated in rapid desensitization (5). Upon hormone binding, the receptor activates phospholipase C ß-isoforms via pertussis toxin-insensitive G proteins, most probably belonging to the Gq family (6). Formal proof of direct activation of Gq/11 proteins by agonist-bound GnRH receptors, however, has not yet been provided.

The human GnRH receptor gene is localized on chromosome 4 and spans 18.9 kb (7). As the open reading frame is distributed between three distinct exons, the possibility for alternative splicing events and generation of receptor isoforms arises. Variant cDNAs (~30% of all clones analyzed) isolated from a gonadotrope cell line cDNA library entertain the notion of alternative processing of the mouse GnRH receptor gene (8). C-Terminal splice variants of GPCRs were found to differ from their wild type counterparts in terms of agonist-induced desensitization and down-regulation as well as G protein-coupling characteristics (9). Truncated receptor isoforms lacking the transmembrane domain were described for the LH receptor (10, 11). Apart from their hormone-binding ability, these variants were able to enhance LH-stimulable adenylyl cyclase activity when coexpressed with the full-length receptor (12), thereby indicating a physical interaction between the variant and wild type receptor.

In the present paper we report the molecular cloning of a splice variant of the human GnRH receptor. The cloned isoform and the wild type receptor were functionally characterized in transiently transfected COS-7 and stably transfected CHO-K1 cells. Our results emphasize a novel inhibitory mechanism of GnRH receptor signaling.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Molecular Cloning and Sequence Analysis of a Truncated Isoform of the Human GnRH Receptor
To study signal transduction processes initiated by the activated GnRH receptor, the human receptor was cloned by RT-PCR. Poly(A)+ RNA serving as template was extracted from human pituitaries. Oligo-dT as well as random hexamer priming led to the amplification of two specific bands of approximately 1000 and 850 bp. Both products were subcloned, and DNA sequences of at least two clones per ligation were determined for each product (Fig. 1Go). The larger product comprised 984 coding bases and was identified as the cDNA of the wild type GnRH receptor. Apart from a single silent transversion (G to A) at nucleotide (nt) position 150, the DNA sequence of the subcloned fragment was identical to the published human clone (13). The DNA sequence of the second, smaller product represented a truncated GnRH receptor cDNA displaying a 128-bp deletion between nt positions 522 and 651 (see Fig 1Go). The deletion caused a frameshift in the open reading frame, thus generating new coding sequence for 75 further amino acids. Both GnRH receptor cDNA sequences were identical up to nucleotide position 522 representing the 3'-boundary of exon 1 (7). The splicing acceptor site within intron 1 (5'-TACAG-3') differs only in one nucleotide position from the coding sequence of nucleotides 646 to 650 (5'-TTCAG-3') of the wild type GnRH receptor. The latter sequence fulfills all requirements to be designated a splicing acceptor consensus site. The use of this alternative acceptor site within exon 2 during the splicing process results in the generation of a truncated GnRH receptor cDNA (see Fig 1Go). The alternative splicing acceptor site is conserved in exon 2 of the human (13), rat (14), sheep (15), bovine (3), and ovine (4) GnRH receptor genes but is absent in the murine gene (16, 17). To rule out a cloning artifact, poly(A)+ RNA from a total of four different pituitaries was analyzed by RT-PCR, and the presence of wild type and truncated GnRH receptor mRNA was confirmed in each case. Therefore, alternative splicing of the GnRH receptor primary transcript in the human pituitary must be postulated. In a first approximation to understand whether splicing patterns of GnRH receptor mRNA are tissue- or cell-specific, we screened human tumor cell lines for the presence of both receptor isoforms. Although RT-PCR allowed us to confirm mRNA expression of the wild type GnRH receptor in a breast cancer (MCF-7) and a prostate tumor cell line (LNCaP), we consistently failed to detect the truncated splice variant in cells from extrapituitary origin.



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Figure 1. Nucleotide and Deduced Amino Acid Sequences of the Wild Type Human GnRH Receptor and the Truncated Splice Variant

Nucleotide and amino acid sequences of the GnRH receptor clones are numbered, starting with the initiating ATG and Met, respectively. Putative transmembrane domains are underlined and numbered by roman characters. Splicing consensus sites are marked by open boxes. Putative N-linked glycosylation sites are marked by open triangles. A silent transversion (G to A) is indicated by a lowercase a and an asterisk.

 
Hydropathy analysis of the amino acid sequence of GnRH receptor revealed structural characteristics of GPCRs: seven hydrophobic stretches of approximately 20 amino acids assumed to form an {alpha}-helical structure and to anchor the protein in the plasma membrane (Fig. 2AGo, upper panel). A hypothetical two-dimensional model of the full-length human GnRH receptor (Fig. 2AGo, lower panel) illustrates a highly conserved disulfide bridge between the first and second exoloop and the lack of a cytoplasmic C-terminal tail. The first exon/intron splice junction is located at the C-terminal end of the fourth transmembrane domain (TM IV). Hydropathy analysis of the splice variant of the human GnRH receptor (Fig. 2BGo, upper panel) revealed a hydrophilic stretch of amino acids forming a new, enlarged exoloop 2 (Fig. 2BGo, lower panel). The new sequence resulting from alternative splicing was found to terminate in a hydrophobic stretch of 21 amino acids potentially constituting a new fifth TM. Although the newly encoded sequence shows no similarity to corresponding regions of the full-length receptor, a cysteine residue was found in the N-terminal portion of exoloop 2. Thus, primary structural requirements to establish the conserved disulfide bridge appear to be fulfilled in the splice variant as well (see Fig 2BGo, lower panel).



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Figure 2. Hydropathy Profile and Putative Two-Dimensional Structure of GnRH Receptor Clones

Hydropathy analyses of the deduced amino acid sequence of the wild type GnRH receptor (A) and the splice variant (B) were performed. The wild type GnRH receptor shows a heptahelical structure typical for GPCRs. The splice variant is a C-terminally truncated protein lacking transmembrane domains VI and VII. Hypothetical two-dimensional structures of the GnRH receptor isoforms are shown in the lower panels.

 
Expression of GnRH Receptor cDNAs in COS-7 Cells
To address the issue as to whether both GnRH receptor cDNAs would be translated into proteins when introduced into eukaryotic cells, a nine-amino acid hemagglutinin (HA) epitope tag (18) was added to the N terminus of either receptor, and COS-7 cells were transiently transfected with expression plasmids encoding one or the other receptor isoform. The expression of proteins was analyzed by metabolic labeling of cells with [35S]methionine followed by specific immunoprecipitation (Fig. 3Go). When precipitates of the wild type GnRH receptor were resolved by SDS-PAGE, three closely spaced bands with apparent molecular masses between 45 and 50 kDa were detected, whereas one protein band of approximately 42 kDa represented the truncated splice variant (see Fig 3Go). None of these bands was detected in untransfected cells or in COS-7 cells expressing the V2 vasopressin or M3 muscarinic receptors (data not shown). The appearance of several protein bands migrating with molecular masses higher than the ones that can be deduced from cDNAs is generally presumed to represent heterogeneous processing of oligosaccharides and has been described previously for other GPCRs (e.g. Ref. 19). It should be noted, however, that, in spite of two identical consensus sites for N-linked glycosylation (see Fig. 1Go), the glycosylation patterns of the two GnRH receptor isoforms appeared to be remarkably distinct.



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Figure 3. Immunoprecipitation of GnRH Receptor Constructs

COS-7 cells were transfected with plasmids coding for the wild type GnRH receptor or the GnRH receptor splice variant and incubated with [35S]methionine for 18 h. Crude cell membranes were prepared and lysed. Immunoprecipitation using a monoclonal antibody directed against the N-terminal HA-tag and SDS-PAGE was performed as described under Materials and Methods. One experiment of two is shown.

 
To ascertain the expression and subcellular localization of the GnRH receptor isoforms, immunocytochemical studies were performed. Confocal fluorescence microscopy on nonpermeabilized (Fig. 4Go, A and C) and permeabilized (Fig. 4Go, B and D) COS-7 cells transfected with epitope-tagged receptor cDNAs revealed a similar subcellular distribution of full-length (Fig. 4Go, A and B) and truncated (Fig. 4Go, C and D) GnRH receptors. Expression of the two proteins could be detected in the endoplasmic reticulum/Golgi complex and in the plasma membrane. Plasma membrane insertion of the truncated splice variant, however, was consistently reduced when compared with immunoreactive wild type receptor in nonpermeabilized cells (cf. Fig. 4Go, A and C). To further characterize the expression of GnRH receptors in the plasma membrane, binding studies were carried out employing the agonist [125I]buserelin. Partially purified plasma membranes of COS-7 cells transfected with epitope-tagged wild type GnRH receptor cDNA displayed specific high-affinity binding sites (Kd = 3.7 nM; Bmax = 400 fmol/mg protein). However, in plasma membranes prepared from cells that had been transfected with identical amounts of HA-tagged splice variant cDNA (4 µg per 100-mm dish), we were unable to detect specific binding.



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Figure 4. Subcellular Localization of GnRH Receptor and Its Splice Variant

Immunofluorescence studies were carried out with transfected COS-7 cells grown on glass coverslips as described in Materials and Methods. Cells expressing wild type GnRH-receptor (A and B) or the splice variant (C and D) were probed with the monoclonal antibody 12CA5 directed against the N-terminal epitope tag present in these constructs. Experiments were carried out with nonpermeabilized (A and C) and permeabilized (B and D) cells, and fluorescence images were obtained with a confocal microscope, using a fluorescein-isothiocyanate-linked anti-mouse IgG secondary antibody. Each picture is representative of three independent experiments.

 
To investigate whether the cloned human GnRH receptor isoforms were capable of activating G proteins and whether the N-terminal insertion of an epitope tag would affect signal transduction processes, GnRH-induced inositol phosphate (IP) accumulation (Fig. 5Go) in transiently transfected COS-7 cells was determined. In cells expressing the epitope-tagged or the untagged wild type receptor, GnRH stimulated IP accumulation with comparable potencies (EC50 = 1.1 and 1.6 nM, respectively) and efficacies (see Fig. 5Go). Thus, the HA epitope tag did not adversely affect agonist-induced phosphoinositide hydrolysis. Cells expressing the truncated splice variant did not respond to challenge with agonist (see Fig 5Go).



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Figure 5. GnRH-Induced Phosphoinositide Hydrolysis of Wild Type GnRH Receptor, HA-tagged GnRH Receptor, and the Splice Variant

COS-7 cells were transfected with the constructs indicated (4 µg plasmid DNA/100-mm dish), and concentration-response curves to GnRH were obtained. IP accumulation was determined as described in Materials and Methods. Data represent means ± SD of one of three independent experiments each performed in triplicate.

 
Inhibition of GnRH Receptor Signaling by Coexpression of a Truncated Splice Variant
When increasing amounts of splice variant cDNA (1.0–3.75 µg per 100-mm dish) were cotransfected with constant amounts of wild type cDNA (0.25 µg per 100-mm dish), a progressively pronounced inhibition of agonist-induced IP accumulation was observed in cells stimulated with 1 µM GnRH (Fig. 6AGo). Basal values remained constant under these experimental conditions, whereas agonist-induced phosphoinositide hydrolysis was reduced by 30–50% when excess splice variant cDNA was cotransfected. The total amount of DNA transfected remained constant (4 µg per 100-mm dish) as complementary amounts of the expression vector (pCMV-5) were included in the transfection mixtures. Figure 6BGo illustrates that the inhibitory effect of the coexpressed splice variant can be observed at different agonist concentrations. The concentration-response curve to GnRH is not significantly shifted toward higher agonist concentrations, thereby ruling out any serious impairment of GnRH binding to the wild type receptor due to the coexpressed splice variant. The efficacy of GnRH to elicit IP accumulation was reduced by approximately 40% at saturating agonist concentrations (see Fig 6BGo).



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Figure 6. Inhibition of GnRH Receptor Signaling by Coexpression of the Splice Variant

A, COS-7 cells were transfected with constant amounts of wild type GnRH receptor cDNA (0.25 µg/100 mm dish). Additionally, the transfection mixture contained a variable amount of splice variant cDNA (0 to 3.75 µg) supplemented with expression vector to keep the total amount of plasmid DNA constant. IP accumulation in response to 1 µM GnRH was determined. Data represent means ± SEM of five independent experiments. B, COS-7 cells were cotransfected with GnRH receptor and splice variant cDNA (0.25 and 3.75 µg/100-mm dish, respectively), and concentration-response curves to GnRH were obtained. Means ± SEM of three independent experiments are shown. C, CHO cells (106 cells per 60 mm dish) stably transfected with wild type GnRH receptor cDNA were transiently transfected with expression vector or with splice variant cDNA (each 7.0 µg/dish), and IP accumulation in response to 1 µM GnRH was determined. Data represent means ± SEM of three independent experiments.

 
To exclude the possibility that the inhibitory effect of the coexpressed splice variant on wild-type GnRH receptor signaling was caused by competition of cotransfected expression plasmids for amplification in COS-7 cells, thereby leading to reduced copy numbers of individual plasmids, CHO-K1 cell lines that permanently express wild type human GnRH receptors were generated. From 15 stable cell lines, the clonal CHO-GnRH-6 cell line was selected for further functional studies because the extent of agonist-induced IP accumulation (~5-fold stimulation of basal IP production) most closely resembled those values that were normally obtained in transient transfection experiments with COS-7 cells. Transient expression of the truncated splice variant in CHO-GnRH-6 cells reduced GnRH-stimulated IP accumulation by 45% (Fig. 6CGo). These results prompted us to critically consider the possibility that the translation product of the alternatively spliced GnRH receptor cDNA was responsible for the functional effects observed.

To determine whether the inhibitory effect of splice variant expression on GnRH receptor signaling was a general phenomenon or whether it was specific for the GnRH receptor, other primarily Gq/11-coupled receptors were transiently expressed in COS-7 cells in the absence and presence of the truncated isoform (Fig. 7Go). Coexpression of the truncated GnRH receptor splice variant did not affect basal and agonist-induced IP accumulation in COS-7 cells expressing thromboxane A2, M5 muscarinic, and V1 vasopressin receptors, whereas GnRH receptor-mediated signaling was substantially suppressed (see Fig. 7Go). Thus, expression of a GnRH receptor splice variant exerted a specific inhibitory effect on signal transduction processes initiated by the activated wild type receptor. These results were compatible with the assumption of a direct intermolecular interaction between the two GnRH receptor isoforms.



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Figure 7. Specificity of Inhibition

To study the specificity of GnRH receptor-mediated inhibition, cells were cotransfected with constant amounts of splice variant cDNA (3.75 µg/dish) and wild type GnRH- (GnRH-R), thromboxane A2 (TXA2-R), M5 muscarinic- (M5-R), or vasopressin V1- (V1-R) receptor cDNAs (0.25 µg/dish). Cells were stimulated (solid bars) with 1 µM GnRH (GnRH-R), 1 µM U46619 (TXA2-R), 100 µM carbachol (M5-R), and 100 nM Arg-vasopressin (V1-R), respectively. Unstimulated cells (open bars) served as controls. Means ± SD of three independent experiments are shown.

 
Mechanism of Inhibition
It has been suggested that GPCRs behave structurally in a fashion analogous to multiple-subunit receptors (20, 21, 22). For muscarinic acetylcholine and adrenergic receptors, two independent folding units (the first containing TM I-V; the second containing TM VI and VII) that are linked by a fairly large third cytoplasmic loop are well characterized (20, 23). In addition, it has been shown that the association of the above mentioned N- and C-terminal folding units does not only occur intra- but also intermolecularly, indicating a molecular basis for receptor association (20). Therefore, we asked whether the GnRH receptor would also be composed of independent folding units thus offering a potential molecular model for wild type/splice variant interaction in analogy to results obtained with full length and truncated muscarinic receptors (20). An expression plasmid coding for a truncated GnRH receptor containing TM I-V and the N-terminal portion of the third cytoplasmic loop was cotransfected with a second plasmid encoding the corresponding C-terminal receptor fragment (Fig. 8Go). Solitary expression of any of these receptor fragments in COS-7 cells did not result in measurable GnRH-stimulated IP accumulation (data not shown). Coexpression of both receptor fragments, however, led to the reappearance of concentration-dependent GnRH-stimulated phospholipase C activation with a 10-fold shift of EC50 values toward higher agonist concentrations as compared with the wild type receptor (see Fig. 8Go). Receptor function of the truncated splice variant, however, could not be restored by coexpression of a C-terminal receptor fragment containing the C-terminal portion of the third cytoplasmic loop and TM VI and VII (see Fig. 8Go).



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Figure 8. Restoration of Functional GnRH Receptors by Coexpression of Two Receptor Fragments

Equal amounts of wild type GnRH receptor (•) plus vector DNA (2.0 µg/dish each), of cDNA coding for two complementary fragments of GnRH receptor split in the third cytoplasmic loop (GnRH-R-trunc and GnRH-R-tail) ({blacktriangleup}) or of splice variant and GnRH-R-tail cDNA ({diamond}) were cotransfected into COS-7 cells, and GnRH-induced IP accumulation was determined. One representative experiment of three is shown. Data represent means ± SEM.

 
We reasoned that our observation of a specific interaction between two GnRH receptor isoforms resulting in reduced efficacy, yet unaltered potency, of agonist acting at the wild type receptor could potentially be explained by a reduced wild type receptor density. Therefore, we set out to quantify the amounts of wild type receptor and splice variant in the plasma membrane. As COS-7 cells expressing the truncated splice variant were devoid of specific [125I]buserelin binding sites, we alternatively developed an indirect cellular enzyme-linked immunosorbent assay (ELISA) protocol to detect membranous GnRH receptors as employed for other GPCRs (22, 24). The HA epitope-tagged wild type receptor was expressed at different receptor densities by stepwise reduction of the amount of transfected plasmid DNA. In parallel, ELISA experiments were carried out with nonpermeabilized COS-7 cells derived from the same batch of cells as those used for binding studies. The inset of Fig. 9Go shows that the optical density (OD) values observed in ELISA studies were directly proportional to receptor densities (Bmax) determined in radioligand binding studies. Based on these findings, ELISA experiments were performed with nonpermeabilized COS-7 cells transfected with epitope-tagged splice variant cDNA. These experiments yielded OD readings that were significantly higher than background values. Assuming a linear relationship between OD readings and amount of protein present in the plasma membrane, the splice variant was expressed at a level of about 20–25% of the wild type receptor (Fig. 9Go). It should be noted, however, that the expression level obtained with a transfected HA-tagged human V2 vasopressin receptor, which had been cloned into the same eukaryotic expression vector and served as a positive control, were consistently 3.3-fold higher than those obtained with the full-length GnRH receptor. As expected, diluting the wild type receptor expression plasmid (1 µg per 100-mm dish) with vector (5 µg per 100-mm dish) resulted in a 50% reduction of receptor density. When expression plasmids coding for the truncated splice variant were used instead of vector plasmids, wild type receptor levels dropped to less than 30% of vector controls. In summary, these data show that, in a given cell, the GnRH receptor splice variant interferes with proper plasma membrane expression of the wild type receptor, resulting in impaired agonist efficacy.



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Figure 9. Reduction of Cell Surface Expression of GnRH Receptor by Coexpression with the Splice Variant

COS-7 cells were transfected with various GnRH receptor contructs (supplemented with vector DNA to keep the amount of transfected plasmid DNA constant at 6 µg/100-mm dish): 6 µg HA-tagged wild type GnRH receptor cDNA (HA-wild-type) alone, 6 µg HA-tagged splice variant cDNA (HA-splice variant) alone, 1 µg HA-wild type cDNA plus 5 µg vector DNA, and 1 µg HA-wild type cDNA plus 5 µg HA-splice variant cDNA. ELISA measurements were performed as described in Materials and Methods. Data are presented as percentage of OD values obtained with HA-tagged wild type GnRH receptor (0.628 ± 0.056). Inset, Relationship between wild type GnRH-receptor density and OD reading determined by an indirect cellular ELISA system. COS-7 cells were transfected with increasing amounts of HA-tagged wild type GnRH receptor cDNA (0.25–4 µg supplemented with vector DNA to keep the amount of transfected plasmid DNA constant at 4 µg/100-mm dish). ELISA measurements and saturation binding assays were performed in parallel. GnRH saturation binding studies were carried out as described in Materials and Methods. The assay was performed in triplicate. Data represent means of one of three independent experiments performed in triplicate.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The GnRH receptor plays a pivotal role in the regulation of the hypothalamic-pituitary-gonadal axis. Northern blots of human pituitary mRNA with a GnRH receptor-specific probe revealed a predominant hybridizing species of approximately 5 kb (13, 25) and two fainter bands of 2.5 and 1.5 kb (25). To study GnRH receptor function and to investigate whether alternative splicing of the primary GnRH receptor transcript occurs, the cDNA of the human GnRH receptor was amplified from poly(A)+ RNA isolated from human pituitaries. In addition to the full-length receptor cDNA, a second truncated clone was isolated and characterized by a deletion of 128 bp. A systematic sequence comparison between the two clones and the exon/intron organization of the human GnRH receptor (7) revealed a cryptic splicing acceptor site within exon 2. Therefore, the truncated GnRH receptor clone is postulated to have arisen from alternative splicing of the primary transcript.

The first genes for GPCRs that were cloned lacked introns in their coding regions. The assumption that this feature was characteristic for the entire receptor family was abandoned after isoforms of various receptors were described arising through alternative splicing of the primary transcript of a single-copy gene (9). Alternatively spliced receptor isoforms have been implicated in altered receptor function. To address the latter issue, GnRH receptor cDNAs were transiently transfected into COS-7 cells, and protein expression was monitored by selective immunoprecipitation of epitope-tagged GnRH receptor proteins from metabolically labeled cells. SDS-PAGE resolved the wild type GnRH receptor into several bands migrating with an apparent molecular mass (45–50 kDa) higher than predicted from the protein core encoded by the cDNA. Therefore, it is likely that the receptor is glycosylated because previous studies on pituitary GnRH receptors suggested a molecular mass between 50 and 60 kDa (26). Three distinct bands representing the wild type GnRH receptor could be resolved after immunoprecipitation. Photoaffinity labeling studies, however, showed a more homogeneous size distribution (26). Several closely spaced bands, assumed to result from differences in the extent of glycosylation, have previously been reported for other GPCRs, e.g. N-formyl peptide (27) and 5-HT1A serotonin receptors (19). The GnRH receptor splice variant was expressed as one sharp band of 42 kDa, indicating heterogenous posttranslational processing of GnRH receptor isoforms in spite of complete conservation of two potential N-linked glycosylation sites in both receptor species. In addition, immunoprecipitation of solubilized receptors does not differentiate between receptor species expressed at the plasma membrane and those retained intracellularly. Therefore, various GnRH receptor forms may also represent improperly folded/processed receptors that are not inserted into the plasma membrane.

In permeabilized cells, confocal laser scanning fluorescence microscopy showed a comparable reticular fluorescence pattern for both receptor isoforms. In nonpermeabilized cells, however, expression of the wild type receptor in the plasma membrane resulted in intense staining of the plasma membrane, whereas the expressed splice variant gave rise to faint, yet clearly discernible, surface staining. Thus, proper plasma membrane insertion of the GnRH receptor does not require the presence of the full-length receptor protein. Similar data have been obtained in expression studies on fragments of the M3 muscarinic receptor (22) and on rhodopsin (21). However, the full-length receptor protein may be required for optimal protein trafficking to the plasma membrane.

The expressed wild type receptor displayed functional characteristics ([125I]buserelin binding, IP production) similar to those observed in gonadotropic {alpha}T3–1 cells endogenously expressing the GnRH receptor (28). The C-terminally truncated isoform, however, neither bound hormone nor was capable of signal transduction, although plasma membrane insertion was proven. Similar results were obtained with truncated M3 muscarinic and V2 vasopressin receptors (20, 22, 24). The seven-transmembrane helices of GPCRs are assumed to be sequentially arranged in a ring-like, counterclockwise (as viewed from the extracellular membrane surface) fashion, thus forming a tightly packed transmembrane receptor core (29, 30). In the case of receptors for biogenic amines, the structural integrity of the ring-like receptor structure is an absolute requirement for high-affinity ligand binding and signal transduction (31, 32). Productive receptor-G protein interaction critically depends on the proper combination of multiple cytoplasmic receptor regions being arranged in a way to form a multiple-loop interactive conformation (9, 33). Experimental evidence for a close physical proximity between TM I and/or II and TM VII in the assumed ring-like structure of the GnRH receptor has been presented (34, 35, 36, 37). Disruption of such critical conformation, as realized by the deletion of C-terminal transmembrane domains including the pivotal third cytoplasmic loop (33), thus results in loss of signaling ability.

Coexpression of wild type and truncated GnRH receptors resulted in markedly suppressed signaling capability of the wild type receptor. In a recent series of experiments (38, 39, 40) it was demonstrated that signaling via GPCRs can be inhibited by overexpression of cytoplasmic receptor domains involved in G protein activation, and it has been postulated that the second intracellular loop of the GnRH receptor is involved in G protein coupling (41). The first and second cytoplasmic loops are unaltered in the GnRH receptor splice variant. Thus, in analogy to the conclusions drawn by Lefkowitz and co-workers (38, 39), a working hypothesis may be construed based on a competition between wild type and truncated, functionally inert GnRH receptors for cellular Gq-proteins, thereby reducing the agonist-stimulable G protein pool. This model, however, implies that splice variant coexpression affects signaling not only via GnRH receptors, but via other Gq-coupled receptors as well. However, we show in the present study that the dominant negative action of the coexpressed splice variant is highly specific for the GnRH receptor. This observation can only be accounted for by assuming a direct and specific physical interaction between wild type receptor and the splice variant.

Several studies suggest that GPCRs may exist in an oligomeric array. Coexpression of mutant muscarinic and adrenergic receptors provided conclusive evidence of intermolecular interactions between receptors (20). Similarly, coexpression of two binding-defective angiotensin II receptor mutants led to restoration of hormone binding (42). The functional rescue of mutant V2 vasopressin receptors by coexpressed receptor fragments modified the concept of receptor-receptor interaction by showing that not only full-length receptors, but also smaller receptor polypeptides, can specifically interact with GPCRs (24). The exact positioning of individual helices appears to be determined by specific intrahelical interactions (43, 44). Aggregation of GnRH receptor molecules in the plasma membrane has been suggested to be an integral event in hormone action (45). The molecular mechanism of intermolcecular receptor contacts, however, is unclear.

Muscarinic receptors as prototypical GPCRs have recently been shown to be composed of at least two independent folding domains, one containing TM I to V and the other containing TM VI and VII. Based on these findings, a molecular mechanism of receptor dimerization was proposed that involves the intermolecular exchange of N- (TM I-V) and C-terminal (TM VI and VII) receptor domains (20). To investigate whether the GnRH receptor was also patterned according to a modular architecture that may subserve receptor dimerization, we coexpressed two different putative receptor folding units (TM I-V and TM VI-VII) resulting in agonist-induced second messenger production only slightly less potent than signaling by the wild type GnRH receptor. Receptor function could not be restored by coexpressing the splice variant and the wild type C-terminus (TM VI-VII), probably due to incomplete restoration of the wild type receptor’s TM V and third cytoplasmic loop. These data suggest that the GnRH receptor is composed of at least two independent folding units potentially providing a mechanistic model of receptor association.

As outlined above, coexpression of wild type GnRH receptor and splice variant resulted in decreased maximal agonist-induced IP production, whereas the potency of GnRH was hardly affected. As shown by Zhou et al. (46), a diminished receptor density could well account for this phenomenon. Applying an ELISA approach to quantify the content of wild type GnRH receptor and splice variant in membranes of COS-7 cells, we corroborated results from immunocytochemical studies in that the splice variant was found to be inserted into the plasma membrane only as a small fraction of the wild type content. Interestingly, coexpression of wild type receptor and splice variant profoundly inhibited the appearance of wild type protein in the plasma membrane. Thus, impaired insertion of the wild type receptor into the plasma membrane may be the molecular mechanism underlying the specific dominant negative effect of the coexpressed splice variant. The recent observation, that a naturally occurring allele coding for a truncated CCR-5 chemokine receptor (a coreceptor for infection by primary M-tropic HIV-1 strains) exerts a dominant negative effect on the viral env protein-mediated cell fusion (47), lends further credence to our proposed mode of action. In addition, defective intracellular transport due to the formation of misfolded complexes between wild type and mutated rhodopsin in the endoplasmic reticulum is held responsible for the dominant effect of one mutated allele in cases of retinal degeneration in Drosophila (48). As exemplified for rhodopsin, folding and assembly of GPCRs are thought to be governed by a multistep process (49): individual {alpha}-helices are assumed to be inserted into membranes of the endoplasmic reticulum in proper orientation followed by tight packing that is mediated by specific interhelical interactions. During the latter process, mutant or truncated rhodopsin molecules presumably associate, and subsequently interfere, with the maturation of wild type molecules in the endoplasmic reticulum (48). It is imaginable that the stage for a similar scenario is set when wild type GnRH receptor and truncated splice variant are coexpressed. Suppression of wild type receptor signaling by coexpression of a variant receptor form has recently been shown for the EP1 prostaglandin E receptor (50). The underlying mechanism, however, differs from the one described by us in that the variant prostaglandin receptor competes with the wild type receptor for ligand binding, yet is incapable of signal transduction.

At present, it is unclear whether alternative splicing of GnRH receptor transcripts plays a physiological role. While preparing our manuscript, distinct alternative transcripts of the human GnRH receptor gene, sb1, sb2, and sb3, were independently described by Kottler et al. (50) with sb2 being identical to the isoform characterized by us. Splicing appears to regulated in a tissue-specific fashion and may serve a potential, as yet unknown, physiological role. Coexpression of truncated receptor isoforms could modulate the gonadotrope’s responsiveness to GnRH and thus contribute to the fine tuning of gonadotropin release.

In the present study we demonstrate that a truncated isoform of the human GnRH receptor produces receptor-specific inhibition of GnRH-mediated biological effects in the intact cell. Most probably, our findings can also be extended to other GPCRs, and it may be feasible to devise optimized receptor fragments that are likely to silence the constitutive activity of mutated GPCRs that cause human disease.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
cDNA Cloning and DNA Constructs
Polyadenylated RNA was extracted from human pituitaries using the Fast Track mRNA isolation kit (Invitrogen, Leek, The Netherlands). Portions of human pituitaries were obtained from four male subjects at the time of autopsy at the Institut für Pathologie (Universitätsklinikum Essen, Germany) and were immediately frozen in liquid nitrogen and then stored at -70 C until RNA isolation. First-strand cDNA was synthesized using the Strata Script RT-PCR kit (Stratagene, Heidelberg, Germany), and two products were amplified with primer 1 (nt positions 1–17 of the GnRH receptor wild type cDNA plus KpnI site) and primer 2 (nt positions 970–987 plus XbaI site). The two cDNAs were ligated into the mammalian expression vectors pCMV-5 (kindly provided by D. W. Russell, University of Texas Southwestern Medical Center, Dallas, TX) and pcDNA3 (Invitrogen). The two PCR products were further analyzed by dideoxy sequencing (52). To study the subcellular localization of the two GnRH receptor proteins by immunological techniques, a stretch of nucleotides coding for a nine-amino acid epitope (YPYDVPDYA) derived from the influenza virus hemagglutinin protein (HA-tag) (18) was inserted after the initiating methionine (Met) codon using a PCR-based procedure. The following primers were used: a sense primer consisting (5' to 3') of a KpnI site, the initiating ATG followed by the coding sequence for the HA-epitope tag, and nts corresponding to positions 4–24 (see Fig. 1Go) of the GnRH receptor wild type cDNA; and an antisense primer corresponding to nt positions 336 to 353 of the wild type GnRH receptor. The PCR product was subsequently cut with KpnI and PflM I and exchanged for the corresponding fragment of the wild type receptor and splice variant, respectively.

For receptor folding and assembly studies, two constructs named GnRH-R-trunc and GnRH-R-tail were generated using PCR-based mutagenesis techniques. For GnRH-R-trunc, a sense primer 1 was designed containing wild type sequence from nt position 723–753 including an endogenous EcoN I-site (nt positions 723–733) followed by a premature stop codon (TGA) and an annealing region for pCMV-5 corresponding to nt positions 3421–3438 of pCMV-5. The antisense primer 1 contained a SmaI site and covered nt positions 3465–3483 of the pCMV-5 expression vector. The resulting PCR fragment was digested with EcoN I and SmaI and subsequently ligated into the HA-tagged wild type GnRH receptor construct. For GnRH-R-tail generation, a sense primer 2 containing a KpnI site, an initiating ATG followed by nt corresponding to positions 754 to 771 of the wild type sequence, and antisense primer 1 were applied for PCR reactions using the wild type GnRH receptor construct as a template. After digestion with KpnI and SmaI, the PCR fragment was inserted into the mammalian expression vector pCMV-5.

Tissue Culture and Transfections
Cell Lines
The breast (MCF-7) and prostate tumor (LNCaP) cell lines were obtained from the American Type Culture Collection (ATTC, Rockville, MD). The cell lines were cultured and maintained strictly following the recommendations of ATTC.

Transient Expression of GnRH Receptors
COS-7 cells were cultured in DMEM containing 10% heat-inactivated FCS, penicillin (50 U/ml), and streptomycin (50 µg/ml) under 7% CO2 at 37 C. For transfections, 2 x 106 cells were seeded into 100-mm dishes. Twenty-four hours later, cells were transfected with various cDNA constructs (4 µg plasmid DNA per dish) by lipofection (Life Technologies, Eggenstein, Germany).

CHO-K1 Cells Permanently Expressing the Human GnRH Receptor
CHO-K1 cells were cultured in DMEM containing 10% heat-inactivated FCS, penicillin (50 U/ml), and streptomycin (50 µg/ml) under 5% CO2 at 37 C. Cells were transfected by lipofection with the expression vector pcDNA 3 containing the cDNA of the human GnRH receptor. Transfectants were selected by growing cells in medium containing 400 µg/ml G418 (Life Technologies). Several CHO-GnRH cell lines were then recloned from single cells by limiting dilution. Twelve of 15 two G418-resistant clones responded to GnRH with increased IP production. The CHO-GnRH-6 cells used in this study are derived from one such cloned cell line.

Radioligand-Binding Assays
For radioligand binding studies, plasma membranes of COS-7 cells were prepared as described (53). GnRH receptor-binding assays were performed with [125I]buserelin (in 1% BSA, 0.01 M formic acid, 20 µCi/ml) as described (54). The protein content of samples was determined by the method of Bradford (55). Binding data were analyzed by a nonlinear least squares curve-fitting procedure using the computer program LIGAND (56).

Immunoprecipitation
COS-7 cells were transfected with wild type and splice variant GnRH receptor constructs as described above. About 18 h later, cells were seeded into six-well plates (5 x 105 cells per well). On the following day, transfected cells were incubated with [35S]methionine (0.5 mCi/ml; Dupont-NEN, Brussels, Belgium) for and additional 18 h. Cells were then washed twice with PBS and treated with 120 µl lysis buffer [10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, 1% deoxycholate, 1% NP-40, 0.2 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin). After vigorous vortexing, followed by removal of cell debris by centrifugation, 20 µg/ml of the monoclonal anti-HA antibody (12CA5; Boehringer Mannheim, Mannheim, Germany) were added to the supernatants containing solubilized receptor protein. After incubation of samples at 4 C for 2 h at constant rotation, 60 µl of 10% (wt/vol) Protein A-Sepharose beads (Sigma, Deisenhofen, Germany) were added, and samples were incubated overnight at 4 C. Sepharose beads were pelleted (12,000 x g, 3 min) and washed twice with 1 ml of washing buffer A (600 mM NaCl, 50 mM Tris-HCl, pH 7.4, 0.1% Triton X-100, 1% NP-40) and twice with 1 ml washing buffer B (300 mM NaCl, 10 mM EDTA, 100 mM Tris-HCl, pH 7.4). Next, pellets were boiled with 40 µl SDS sample buffer, and SDS-PAGE (12.5%) was performed. Precipitated 35S-labeled membrane proteins were visualized by autoradiography of dried gels with Kodak X-OMAT AR-5 films.

Immunofluorescence Microscopy
Twenty to 24 h after transfection, COS-7 cells expressing HA-tagged wild type GnRH receptor or the truncated splice variant were transferred into six-well plates (1–2 x 105 cells per well) containing sterilized glass coverslips. Forty-eight hours later, cells were fixed in PBS containing 4% formaldehyde. After washing with PBS, unspecific binding sites were blocked with 1% BSA in PBS. Cells were then incubated for 90 min at room temperature with a monoclonal antibody directed against the HA-epitope tag (12CA5, 10 µg/ml in PBS). After washing with PBS, cells were incubated for another 90 min at room temperature with a 1:100 dilution of a fluorescein isothiocyanate-conjugated anti-mouse IgG antibody (Sigma). Unbound secondary antibody was removed by washing with PBS, and coverslips were mounted on microscope slides using a glycerol/PBS mixture (1:1, vol/vol). To permeabilize the cell membranes, cells were treated with 0.5% Triton X-100 in PBS for 10 min at room temperature. Images were obtained using a confocal laser-scanning fluorescence microscope (LSM 410, Carl Zeiss, Jena, Germany).

ELISA
ELISA measurements were carried out with nonpermeabilized cells essentially as described (22). COS-7 cells were transferred into 48-well plates (105 cells per well) 20–24 h after transfections. Forty-eight hours later, cells were fixed with 4% formaldehyde in PBS for 30 min at room temperature. After washing with PBS and blocking with DMEM containing 10% FCS, cells were incubated for 3 h at 37 C with the monoclonal antibody 12CA5 (20 µg/ml in DMEM/10% FCS). Plates were then washed with PBS and incubated with a 1:2500 dilution of a peroxidase-conjugated anti-mouse IgG antibody (Sigma) for 1 h at 37 C. H2O2 and o-phenylenediamine (2.5 mM each in 0.1 M phosphate-citrate buffer, pH 5.0) were then added to serve as substrate and chromogen, respectively. The enzymatic reaction (carried out at room temperature) was stopped after 30 min with 1 M H2SO4 containing 0.05 M Na2SO3, and the color development was measured bichromatically at 450 and 630 nm, using the ELISA reader (Titertek Multiscan MCC/340).

Measurement of IP Accumulation
IP accumulation assay was performed as described with minor modifications (57). Briefly, after transfection cells were seeded into six-well tissue culture plates containing DMEM/10% FCS supplemented with 2 µCi/ml myo-[3H]inositol (Amersham-Buchler, Braunschweig, Germany) and were grown for 24 h. After washing with PBS (containing 5.5 mM glucose, 0.5 mM CaCl2, and 0.5 mM MgCl2), 20 µl of 1 M LiCl/well were added to the remaining 0.5 ml PBS, and labeled cells were incubated for 10 min at 37 C. The incubation was continued for an additional 30 min at 37 C by adding 0.5 ml PBS/0.2% BSA to each well containing appropriate ligand concentrations. Incubations were stopped by adding ice-cold 20 mM formic acid to each well. IPs produced were separated from myo-inositol as described (55). The accumulation of IPs was normalized by dividing the counts for [3H]IPs by the sum of the counts for myo-[3H]inositol plus [3H]IPs.

Accession Number
The nucleotide sequence of the truncated splice variant of the human GnRH receptor was submitted to the EMBL data bank under the accession number Z81148 HSGTRHSV.


    ACKNOWLEDGMENTS
 
Expert technical assistance by Nicole Stresow and Monika Bigalke is gratefully acknowledged. [125I]Buserelin was generously supplied by Dr. W. von Rechenberg (Hoechst AG, Frankfurt/Main, Germany). We would like to thank Dr. F. Jockenhövel (Abteilung für Endokrinologie, Zentrum für Innere Medizin, Universitätsklinikum Essen) for help in obtaining human pituitaries. We are grateful to Dr. B. Wiedenmann (Freie Universität Berlin) for access to a confocal laser scanning microscope. The cDNA of the M5 muscarinic receptor was a generous gift from Dr. Lutz Birnbaumer (University of California, Los Angeles). We are grateful to Dr. M. J. Brownstein (NIH, Bethesda, MD) for providing the V1 vasopressin receptor and to Drs. P. V. Halushka and C. J. Allan (University of South Carolina, Charleston) for donating the thromboxane A2 cDNA.


    FOOTNOTES
 
Address requests for reprints to: Thomas Gudermann, Institut fur Pharmakologie, Freie Universitat Berlin, Thielallee 69–73, D-14195 Berlin, Germany.

This work was supported by the Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie.

This study was presented in part at the 40th Symposium of the German Society of Endocrinology, Marburg, Germany, February 28 to March 2, 1996.

Received for publication January 2, 1997. Revision received April 21, 1997. Accepted for publication April 24, 1997.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Hotchkiss J, Knobil E 1994 The menstrual cycle and ist neuroendocrine control. In: Knobil E, Neill JD, Greenwald GS, Markert CL, Pfaff DW (eds) The Physiology of Reproduction. Raven Press, New York, vol 2:711–750
  2. Stojilkovic SS, Reinhart J, Catt KJ 1994 Gonadotropin-releasing hormone receptors: structure and signal transduction pathways. Endocr Rev 15:462–487[Medline]
  3. Kakar SS, Rahe CH, Neill JD 1993 Molecular cloning, sequencing, and characterization of the bovine receptor for gonadotropin-releasing hormone (GnRH). Domest Anim Endocrinol 10:335–342[CrossRef][Medline]
  4. Campion CE, Turzillo AM, Clay CM 1996 The gene encoding the ovine gonadotropin-releasing hormone (GnRH) receptor: cloning and initial characterization. Gene 170:277–280[CrossRef][Medline]
  5. Davidson JS, Wakefield IK, Millar RP 1994 Absence of rapid desensitization of the mouse gonadotropin-releasing hormone receptor. Biochem J 300:299–302[Medline]
  6. Hsieh K-P, Martin TFJ 1992 Thyrotropin-releasing hormone and gonadotropin-releasing hormone receptors activate phospholipase C by coupling to the triphosphate-binding proteins Gq and G11. Mol Endocrinol 6:1673–1681[Abstract]
  7. Fan NC, Jeung EB, Peng C, Olofsson JI, Krisinger J, Leung PCK 1994 The human gonadotropin-releasing hormone (GnRH) receptor gene: cloning, genomic organization and chromosomal assignment. Mol Cell Endocrinol 103:R1–R6
  8. Zhou W, Sealfon SC 1994 Structure of the mouse gonadotropin-releasing hormone receptor gene: variant transcripts generated by alternative processing. DNA Cell Biol 13:605–614[Medline]
  9. Gudermann T, Kalkbrenner F, Schultz G 1996 Diversity and selectivity of receptor-G protein interaction. Annu Rev Pharmacol Toxicol 36:429–459[CrossRef][Medline]
  10. Loosfelt H, Misrahi M, Atger M, Salesse R, Vu Hai-Luu Thi MT, Jolivet A, Guichon-Mantel A, Sar S, Jallal B, Garnier J, Milgrom E 1989 Cloning and sequencing of porcine LH-hCG receptor cDNA: variants lacking transmembrane domain. Science 245:525–528[Medline]
  11. Aatsinki JT, Pietilä EM, Lakkakorpi JT, Rajaniemi HJ 1992 Expression of the LH/CG receptor gene in rat ovarian tissue is regulated by an extensive alternative splicing of the primary transcript. Mol Cell Endocrinol 84:127–135[CrossRef][Medline]
  12. Vu Hai-Luu Thi MT, Misrahi M, Houllier A, Jolivet A, Milgrom E 1992 Variant forms of the pig lutropin/choriogonadotropin receptor. Biochemistry 31:8377–8383[Medline]
  13. Chi L, Zhou W, Prikhozhan A, Flanagan C, Davidson JS, Golembo M, Illing N, Millar RP, Sealfon SC 1993 Cloning and characterization of the human GnRH receptor. Mol Cell Endocrinol 91:R1–R6
  14. Eidne KA, Sellar RE, Couper G, Anderson L, Taylor PL 1992 Molecular cloning and characterization of the rat pituitary gonadotropin-releasing hormone (GnRH) receptor. Mol Cell Endocrinol 90:R5–R9
  15. Illing N, Jacobs GFM, Becker II, Flanagan CA, Davidson JS, Eales A, Zhou W, Sealfon SC, Millar RP 1993 Comparative sequence analysis and functional characterization of the cloned sheep gonadotropin-releasing hormone receptor reveal differences in primary structure and ligand specificity among mammalian receptors. Biochem Biophys Res Commun 196:745–751[CrossRef][Medline]
  16. Reinhart J, Mertz LM, Catt KJ 1992 Molecular cloning and expression of cDNA encoding the murine gonadotropin-releasing hormone receptor. J Biol Chem 267:21281–21284[Abstract/Free Full Text]
  17. Tsutsumi M, Zhou W, Millar RP, Mellon PL, Roberts JL, Flanagan CA, Dong K, Gillo B, Sealfon SC 1992 Cloning and functional expression of a mouse gonadotropin-releasing hormone receptor. Mol Endocrinol 6:1163–1169[Abstract]
  18. Kolodziej PA, Young RA 1991 Epitope tagging and protein surveillance. Methods Enzymol 194:508–519[Medline]
  19. Butkerait P, Zheng Y, Hallak H, Graham TE, Miller HA, Burris KD, Molinoff PB, Manning DR 1995 Expression of the human 5-hydroxytryptamine1A receptor in Sf9 cells. Reconstitution of a coupled phenotype by co-expression of mammalian G protein subunits. J Biol Chem 270:18691–18699[Abstract/Free Full Text]
  20. Maggio R, Vogel Z, Wess J 1993 Coexpression studies with mutant muscarinic/adrenergic receptors provide evidence for intermolecular "cross-talk" between G-protein-linked receptors. Proc Natl Acad Sci USA 90:3103–3107[Abstract]
  21. Ridge KD, Lee SS, Yao LL 1995 In vivo assembly of rhodopsin from expressed polypeptide fragments. Proc Natl Acad Sci USA 92:3204–3208[Abstract]
  22. Schöneberg T, Liu J, Wess,J 1995 Plasma membrane localization and functional rescue of truncated forma of a G protein-coupled receptor. J Biol Chem 270:18000–18006[Abstract/Free Full Text]
  23. Kobilka BK, Kobilka TS, Daniel K, Reagan JW, Caron MG, Lefkowitz RJ 1988 Chimeric {alpha}2-ß2-adrenergic receptors: delineation of domains involved in effector coupling and ligand binding specificity. Science 240:1310–1316[Medline]
  24. Schöneberg T, Yun J, Wenkert D, Wess J 1996 Functional rescue of mutant V2 vasopressin receptors causing nephrogenic diabetes insipidus by a co-expressed receptor polypeptide. EMBO J 15:1283–1291[Abstract]
  25. Kakar SS, Musgrove LC, Devor DC, Sellers JC, Neill JD, Human gonadotropin releasing hormone (GnRH) receptor: further molecular characterization. Program of the 75th Annual Meeting of The Endocrine Society, Las Vegas, NV, 1993, p 339 (Abstract)
  26. Iwashita M, Catt KJ 1985 Photoaffinity labeling of pituitary and gonadal receptors for gonadotropin-releasing hormone. Endocrinology 117:738[Abstract]
  27. Quehenberger O, Prossnitz ER, Cochrane CG, Ye RD 1992 Absence of Gi proteins in the Sf9 insect cell. Characterization of the uncoupled recombinant N-formyl peptide receptor. J Biol Chem 267:19757–19760[Abstract/Free Full Text]
  28. McArdle CA, Schomerus E, Gröner I, Poch A 1992 Estradiol regulates gonadotropin-releasing hormone receptor number, growth and inositol phosphate production in {alpha}T3–1 cells. Mol Cell Endocrinol 87:95–103[CrossRef][Medline]
  29. Baldwin JM 1993 The probable arrangement of the helices in G protein-coupled receptors. EMBO J 12:1693–1703[Abstract]
  30. Liu J, Schöneberg T, Rhee M, Wess J 1995 Mutational analysis of the relative orientation of the transmembrane helices I and VII in G protein-coupled receptors. J Biol Chem 270:19532–19539[Abstract/Free Full Text]
  31. Strader CD, Fong TM, Tota MR, Underwood D, Dixon RAF 1994 Structure and function of G protein-coupled receptors. Annu Rev Biochem 63:101–132[CrossRef][Medline]
  32. Wess J 1996 Molecular biology of muscarinic acetylcholine receptors. Crit Rev Neurobiol 10:69–99[Medline]
  33. Gudermann T, Nürnberg B, Schultz G 1995 Receptors and G proteins as primary components of transmembrane signal transduction. II. G proteins: structure and function. J Mol Med 73:51–63[Medline]
  34. Zhou W, Flanagan C, Ballesteros JA, Konvicka K, Davidson JS, Weinstein H, Millar RP, Sealfon SC 1994 A reciprocal mutation supports helix 2 and helix 7 proximity in the gonadotropin-releasing hormone receptor. Mol Pharmacol 45:165–170[Abstract]
  35. Arora KK, Cheng Z, Catt KJ 1996 Dependence of agonist activation on an aromatic moiety in the DPLIY motif of the gonadotropin-releasing hormone receptor. Mol Endocrinol 10:979–986[Abstract]
  36. Awara WM, Guo C-H, Conn PM 1996 Effects of Asn318 and Asp87Asn318 mutations on signal transduction by the gonadotropin-releasing hormone receptor and receptor regulation. Endocrinology 137:655–662[Abstract]
  37. Davidson JS, McArdle CA, Davies P, Elario R, Flanagan CA, Millar RP 1996 Asn102 of the gonadotropin-releasing hormone receptor is a critical determinant of potency for agonists containing C-terminal glycinamide. J Biol Chem 271:15510–15514[Abstract/Free Full Text]
  38. Luttrell LM, Ostrowski J, Cotecchia S, Kendall H, Lefkowitz RJ 1993 Antagonism of catecholamine receptor signaling by expression of cytoplasmic domains of the receptors. Science 259:1453–1457[Medline]
  39. Hawes BE, Luttrell LM, Exum ST, Lefkowitz RJ 1994 Inhibition of G protein-coupled receptor signaling by expression of cytoplasmic domains of the receptor. J Biol Chem 269:15776–15785[Abstract/Free Full Text]
  40. Carlson SA, Chatterjee TK, Fisher RA 1996 The third intracellular domain of the platelet-activating factor receptor is a critical determinant in receptor coupling to phosphoinositide phospholipase C-activating G proteins. Studies using intracellular minigenes and receptor chimeras. J Biol Chem 271:23146–23153[Abstract/Free Full Text]
  41. 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]
  42. Monnot C, Bihoreau C, Conchon S, Curnow KM, Corvol P, Clauser E 1996 Polar residues in the transmembrane domains of the type 1 angiotensin II receptor are required for binding and coupling. Reconstitution of the binding site by co-expression of two deficient mutants. J Biol Chem 271:1507–1513[Abstract/Free Full Text]
  43. Elling CE, Nielsen SM, Schwartz TW 1995 Conversion of antagonist-binding site to metal-ion site in the tachykinin NK-1 receptor. Nature 374:74–77[CrossRef][Medline]
  44. Sheikh SP, Zvyaga TA, Lichtarge O, Sakmar TP, Bourne HR 1996 Rhodopsin activation blocked by metal-ion-binding sites linking transmembrane helices C and F. Nature 383:347–350[CrossRef][Medline]
  45. Janovick JA, Conn PM 1996 Gonadotropin releasing hormone agonist provokes homologous receptor microaggregation: an early event in seven-transmembrane receptor mediated signaling. Endocrinology 137:3602–3605[Abstract]
  46. Zhou W, Rodic V, Kitanovic S, Flanagan CA, Chi L, Weinstein H, Maayani S, Millar RP, Sealfon SC 1995 A locus of the gonadotropin-releasing hormone receptor that differentiates agonist and antagonist binding sites. J Biol Chem 270:18853–18857[Abstract/Free Full Text]
  47. Samson M, Libert F, Doranz BJ, Rucker J, Liesnard C, Farber C-M, Saragosti S, Lapouméroulie C, Cognaux J, Forceille C, Muyldermans G, Verhofstede C, Burtonboy G, Georges M, Imai T, Rana S, Yi Y, Smyth RJ, Collman RG, Doms RW, Vassart G, Parmentier M 1996 Resistance to HIV-1 infection in caucasian individuals bearing mutant alleles of the CCR-5 chemokine receptor gene. Nature 382:722–725[CrossRef][Medline]
  48. Colley NJ, Cassill JA, Baker EK, Zucker CS 1995 Defective intracellular transport is the molecular basis of rhodopsin-dependent dominant retinal degeneration. Proc Natl Acad Sci USA 92:3070–3074[Abstract]
  49. Khorana HG 1992 Rhodopsin, photoreceptor of the rod cell. An emerging pattern for structure and function. J Biol Chem 267:1–4[Free Full Text]
  50. Kottler ML, Bergametti F, Carre MC, Starzec A, Counis R, Alternative splicing and tissue distribution of transcripts for human gonadotropin-releasing hormone receptor. Program and Abstracts of the 10th International Congress of Endocrinology, San Francisco, CA, 1996, p 817 (Abstract)
  51. Okuda-Ashitaka E, Sakamoto K, Ezashi T, Miwa K, Ito S, Hayashi O 1996 Suppression of prostaglandin E receptor signaling by the variant form of EP1 subtype. J Biol Chem 271:31255–31261[Abstract/Free Full Text]
  52. Sanger F, Nicklen S, Coulson AR 1977 DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467[Abstract]
  53. Gudermann T, Birnbaumer M, Birnbaumer L 1992 Evidence for dual coupling of the murine luteinizing hormone receptor to adenylyl cyclase and phosphoinositide breakdown and Ca2+ mobilization. Studies with the cloned murine luteinizing hormone receptor expressed in L cells. J Biol Chem 267:4479–4488[Abstract/Free Full Text]
  54. Millar RP, Flanagan CA, Milton RC, King JA 1989 Chimeric analogues of vertebrate gonadotropin-releasing hormones comprising substitutions of the variant amino acids in positions 5, 7 and 8. J Biol Chem 264:21007–21013[Abstract/Free Full Text]
  55. Bradford MM 1976 A rapid and sensitive method for the quantitation of microgramm quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72:248–254[CrossRef][Medline]
  56. Munson PJ, Rodbard D 1980 LIGAND: a versatile computerized approach for characterization of ligand-binding systems. Anal Biochem 107:220–239[Medline]
  57. Zhu X, Gilbert S, Birnbaumer M, Birnbaumer L 1994 Dual signaling potential is common among Gs-coupled receptors and dependent on receptor density. Mol Pharmacol 46:460–469[Abstract]