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Institut für Pharmakologie Freie Universität Berlin D-14195 Berlin, Germany
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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).
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 1). 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 1). 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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-helical structure and to anchor the protein in the plasma membrane
(Fig. 2A, upper panel). A hypothetical
two-dimensional model of the full-length human GnRH receptor (Fig. 2A, 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. 2B, upper panel) revealed a hydrophilic stretch of
amino acids forming a new, enlarged exoloop 2 (Fig. 2B, 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 2B, lower panel).
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). 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 3). 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. 1
), the glycosylation patterns of the two GnRH receptor isoforms
appeared to be remarkably distinct.
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, A
and C) and permeabilized (Fig. 4, B and D) COS-7 cells transfected with
epitope-tagged receptor cDNAs revealed a similar subcellular
distribution of full-length (Fig. 4, A and B) and truncated (Fig. 4, 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. 4, 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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) 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. 5). 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 5).
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). 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 6B 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 6B).
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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. 6C). 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. 7). 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. 7).
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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). 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. 8). 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. 8).
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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. 9). 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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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 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 -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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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) 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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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2-ß2-adrenergic receptors: delineation
of domains involved in effector coupling and ligand binding
specificity. Science 240:1310–1316T3–1 cells. Mol Cell Endocrinol 87:95–103[CrossRef][Medline]
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 C. Y. Wang, Y. Wang, A. H. Y. Kwok, and F. C Leung Identification of two novel chicken GHRH receptor splice variants: implications for the roles of aspartate 56 in the receptor activation and direct ligand receptor interaction J. Endocrinol., December 1, 2007; 195(3): 525 - 536. [Abstract] [Full Text] [PDF]
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 S. H. Long, M. J. Berna, M. Thill, A. Pace, T. K. Pradhan, K. M. Hoffmann, J. Serrano, and R. T. Jensen Secretin-Receptor and Secretin-Receptor-Variant Expression in Gastrinomas: Correlation with Clinical and Tumoral Features and Secretin and Calcium Provocative Test Results J. Clin. Endocrinol. Metab., November 1, 2007; 92(11): 4394 - 4402. [Abstract] [Full Text] [PDF]
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 B. Faurholm, S. Cochrane, R. R Millar, and A. A Katz Gene structure and promoter functional analysis of the marmoset type II GnRH receptor J. Mol. Endocrinol., August 1, 2007; 39(2): 91 - 104. [Abstract] [Full Text] [PDF]
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 K. L. Egerod, B. Holst, P. S. Petersen, J. B. Hansen, J. Mulder, T. Hokfelt, and T. W. Schwartz GPR39 Splice Variants Versus Antisense Gene LYPD1: Expression and Regulation in Gastrointestinal Tract, Endocrine Pancreas, Liver, and White Adipose Tissue Mol. Endocrinol., July 1, 2007; 21(7): 1685 - 1698. [Abstract] [Full Text] [PDF]
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T. Ikemoto and M. K. Park Comparative analysis of the pituitary and ovarian GnRH systems in the leopard gecko: signaling crosstalk between multiple receptor subtypes in ovarian follicles J. Mol. Endocrinol., February 1, 2007; 38(2): 289 - 304. [Abstract] [Full Text] [PDF]
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 E. L. Baldwin, I. N. Wegorzewska, M. Flora, and T. J. Wu Regulation of Type II Luteinizing Hormone-Releasing Hormone (LHRH-II) Gene Expression by the Processed Peptide of LHRH-I, LHRH-(1-5) in Endometrial Cells Experimental Biology and Medicine, January 1, 2007; 232(1): 146 - 155. [Abstract] [Full Text] [PDF]
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 M. Shimizu and G. Y. Bedecarrats Identification of a Novel Pituitary-Specific Chicken Gonadotropin-Releasing Hormone Receptor and Its Splice Variants Biol Reprod, November 1, 2006; 75(5): 800 - 808. [Abstract] [Full Text] [PDF]
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 M. M. C. Kong, T. Fan, G. Varghese, B. F. O'Dowd, and S. R. George Agonist-Induced Cell Surface Trafficking of an Intracellularly Sequestered D1 Dopamine Receptor Homo-Oligomer Mol. Pharmacol., July 1, 2006; 70(1): 78 - 89. [Abstract] [Full Text] [PDF]
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 P. M. Apaja, J. T. Tuusa, E. M. Pietila, H. J. Rajaniemi, and U. E. Petaja-Repo Luteinizing Hormone Receptor Ectodomain Splice Variant Misroutes the Full-Length Receptor into a Subcompartment of the Endoplasmic Reticulum Mol. Biol. Cell, May 1, 2006; 17(5): 2243 - 2255. [Abstract] [Full Text] [PDF]
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A. T. McElvaine and K. E. Mayo A Dominant-Negative Human Growth Hormone-Releasing Hormone (GHRH) Receptor Splice Variant Inhibits GHRH Binding Endocrinology, April 1, 2006; 147(4): 1884 - 1894. [Abstract] [Full Text] [PDF]
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 K. Herrick-Davis, E. Grinde, T. J. Harrigan, and J. E. Mazurkiewicz Inhibition of Serotonin 5-Hydroxytryptamine2C Receptor Function through Heterodimerization: RECEPTOR DIMERS BIND TWO MOLECULES OF LIGAND AND ONE G-PROTEIN J. Biol. Chem., December 2, 2005; 280(48): 40144 - 40151. [Abstract] [Full Text] [PDF]
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 J. Vendelin, V. Pulkkinen, M. Rehn, A. Pirskanen, A. Raisanen-Sokolowski, A. Laitinen, L. A. Laitinen, J. Kere, and T. Laitinen Characterization of GPRA, a Novel G Protein-Coupled Receptor Related to Asthma Am. J. Respir. Cell Mol. Biol., September 1, 2005; 33(3): 262 - 270. [Abstract] [Full Text] [PDF]
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S. Shacham, M. N. Cheifetz, M. Fridkin, A. J. Pawson, R. P. Millar, and Z. Naor Identification of Ser153 in ICL2 of the Gonadotropin-releasing Hormone (GnRH) Receptor as a Phosphorylation-independent Site for Inhibition of Gq Coupling J. Biol. Chem., August 12, 2005; 280(32): 28981 - 28988. [Abstract] [Full Text] [PDF]
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A. J. Pawson, S. Maudsley, K. Morgan, L. Davidson, Z. Naor, and R. P. Millar Inhibition of Human Type I Gonadotropin-Releasing Hormone Receptor (GnRHR) Function by Expression of a Human Type II GnRHR Gene Fragment Endocrinology, June 1, 2005; 146(6): 2639 - 2649. [Abstract] [Full Text] [PDF]
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 C. K. Cheng and P. C. K. Leung Molecular Biology of Gonadotropin-Releasing Hormone (GnRH)-I, GnRH-II, and Their Receptors in Humans Endocr. Rev., April 1, 2005; 26(2): 283 - 306. [Abstract] [Full Text] [PDF]
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A. Perron, P. Sarret, L. Gendron, T. Stroh, and A. Beaudet Identification and Functional Characterization of a 5-Transmembrane Domain Variant Isoform of the NTS2 Neurotensin Receptor in Rat Central Nervous System J. Biol. Chem., March 18, 2005; 280(11): 10219 - 10227. [Abstract] [Full Text] [PDF]
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D. S. Kang, C. Gustafsson, M. Morgelin, and L. M. F. Leeb-Lundberg B1 Bradykinin Receptor Homo-Oligomers in Receptor Cell Surface Expression and Signaling: Effects of Receptor Fragments Mol. Pharmacol., January 1, 2005; 67(1): 309 - 318. [Abstract] [Full Text] [PDF]
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D. Mesnier and J.-L. Baneres Cooperative Conformational Changes in a G-protein-coupled Receptor Dimer, the Leukotriene B4 Receptor BLT1 J. Biol. Chem., November 26, 2004; 279(48): 49664 - 49670. [Abstract] [Full Text] [PDF]
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A. U. Meysing, H. Kanasaki, G. Y. Bedecarrats, J. S. Acierno Jr., P. M. Conn, K. A. Martin, S. B. Seminara, J. E. Hall, W. F. Crowley Jr., and U. B. Kaiser GNRHR Mutations in a Woman with Idiopathic Hypogonadotropic Hypogonadism Highlight the Differential Sensitivity of Luteinizing Hormone and Follicle-Stimulating Hormone to Gonadotropin-Releasing Hormone J. Clin. Endocrinol. Metab., July 1, 2004; 89(7): 3189 - 3198. [Abstract] [Full Text] [PDF]
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K. Nakamura, S. Yamashita, Y. Omori, and T. Minegishi A Splice Variant of the Human Luteinizing Hormone (LH) Receptor Modulates the Expression of Wild-Type Human LH Receptor Mol. Endocrinol., June 1, 2004; 18(6): 1461 - 1470. [Abstract] [Full Text] [PDF]
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P. M. Gault, K. Morgan, A. J. Pawson, R. P. Millar, and G. A. Lincoln Sheep Exhibit Novel Variations in the Organization of the Mammalian Type II Gonadotropin-Releasing Hormone Receptor Gene Endocrinology, May 1, 2004; 145(5): 2362 - 2374. [Abstract] [Full Text] [PDF]
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 A. Ulloa-Aguirre, J. A. Janovick, A. Leanos-Miranda, and P. M. Conn Misrouted cell surface GnRH receptors as a disease aetiology for congenital isolated hypogonadotrophic hypogonadism Hum. Reprod. Update, March 1, 2004; 10(2): 177 - 192. [Abstract] [Full Text] [PDF]
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M. C. Overton, S. L. Chinault, and K. J. Blumer Oligomerization, Biogenesis, and Signaling Is Promoted by a Glycophorin A-like Dimerization Motif in Transmembrane Domain 1 of a Yeast G Protein-coupled Receptor J. Biol. Chem., December 5, 2003; 278(49): 49369 - 49377. [Abstract] [Full Text] [PDF]
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J. J. Carrillo, J. Pediani, and G. Milligan Dimers of Class A G Protein-coupled Receptors Function via Agonist-mediated Trans-activation of Associated G Proteins J. Biol. Chem., October 24, 2003; 278(43): 42578 - 42587. [Abstract] [Full Text] [PDF]
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A. Leanos-Miranda, A. Ulloa-Aguirre, T. H. Ji, J. A. Janovick, and P. M. Conn Dominant-Negative Action of Disease-Causing Gonadotropin-Releasing Hormone Receptor (GnRHR) Mutants: A Trait That Potentially Coevolved with Decreased Plasma Membrane Expression of GnRHR in Humans J. Clin. Endocrinol. Metab., July 1, 2003; 88(7): 3360 - 3367. [Abstract] [Full Text] [PDF]
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 T.A. Bramley, K. Campbell, and G.S. Menzies Human placental GnRH-like factors: II. Inhibition of enzymatic degradation of GnRH-II and [D-Trp6]GnRH ethylamide tracers by human term placental cytosol fractions reveals the presence of GnRH-binding protein(s) Mol. Hum. Reprod., May 1, 2003; 9(5): 291 - 300. [Abstract] [Full Text] [PDF]
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G. Y. Bedecarrats, K. D. Linher, and U. B. Kaiser Two Common Naturally Occurring Mutations in the Human Gonadotropin-Releasing Hormone (GnRH) Receptor Have Differential Effects on Gonadotropin Gene Expression and on GnRH-Mediated Signal Transduction J. Clin. Endocrinol. Metab., February 1, 2003; 88(2): 834 - 843. [Abstract] [Full Text] [PDF]
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K. Morgan, D. Conklin, A. J. Pawson, R. Sellar, T. R. Ott, and R. P. Millar A Transcriptionally Active Human Type II Gonadotropin-Releasing Hormone Receptor Gene Homolog Overlaps Two Genes in the Antisense Orientation on Chromosome 1q.12 Endocrinology, February 1, 2003; 144(2): 423 - 436. [Abstract] [Full Text] [PDF]
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R. Latif, P. Graves, and T. F. Davies Ligand-dependent Inhibition of Oligomerization at the Human Thyrotropin Receptor J. Biol. Chem., November 15, 2002; 277(47): 45059 - 45067. [Abstract] [Full Text] [PDF]
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M. C. Overton and K. J. Blumer The Extracellular N-terminal Domain and Transmembrane Domains 1 and 2 Mediate Oligomerization of a Yeast G Protein-coupled Receptor J. Biol. Chem., October 25, 2002; 277(44): 41463 - 41472. [Abstract] [Full Text] [PDF]
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L. F. G. Silveira, P. M. Stewart, M. Thomas, D. A. Clark, P. M. G. Bouloux, and G. S. MacColl Novel Homozygous Splice Acceptor Site GnRH Receptor (GnRHR) Mutation: Human GnRHR "Knockout" J. Clin. Endocrinol. Metab., June 1, 2002; 87(6): 2973 - 2977. [Abstract] [Full Text] [PDF]
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 A. Christopoulos and T. Kenakin G Protein-Coupled Receptor Allosterism and Complexing Pharmacol. Rev., June 1, 2002; 54(2): 323 - 374. [Abstract] [Full Text] [PDF]
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L. Wang, D. Y. Oh, J. Bogerd, H. S. Choi, R. S. Ahn, J. Y. Seong, and H. B. Kwon Inhibitory Activity of Alternative Splice Variants of the Bullfrog GnRH Receptor-3 on Wild-Type Receptor Signaling Endocrinology, September 1, 2001; 142(9): 4015 - 4025. [Abstract] [Full Text] [PDF]
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M. Beranova, L. M. B. Oliveira, G. Y. BÉdÉcarrats, E. Schipani, M. Vallejo, A. C. Ammini, J. B. Quintos, J. E. Hall, K. A. Martin, F. J. Hayes, et al. Prevalence, Phenotypic Spectrum, and Modes of Inheritance of Gonadotropin-Releasing Hormone Receptor Mutations in Idiopathic Hypogonadotropic Hypogonadism J. Clin. Endocrinol. Metab., April 1, 2001; 86(4): 1580 - 1588. [Abstract] [Full Text]
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 S. Rama and A.J. Rao Embryo implantation and GnRH antagonists: The search for the human placental GnRH receptor Hum. Reprod., February 1, 2001; 16(2): 201 - 205. [Abstract] [Full Text] [PDF]
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 G Milligan Oligomerisation of G-protein-coupled receptors J. Cell Sci., January 4, 2001; 114(7): 1265 - 1271. [Abstract] [PDF]
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S. Wolfahrt, B. Kleine, H. Jarry, and W. G. Rossmanith Endogenous regulation of the GnRH receptor by GnRH in the human placenta Mol. Hum. Reprod., January 1, 2001; 7(1): 89 - 95. [Abstract] [Full Text] [PDF]
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T. Madigou, E. Mañanos-Sanchez, S. Hulshof, I. Anglade, S. Zanuy, and O. Kah Cloning, Tissue Distribution, and Central Expression of the Gonadotropin-Releasing Hormone Receptor in the Rainbow Trout (Oncorhynchus mykiss) Biol Reprod, December 1, 2000; 63(6): 1857 - 1866. [Abstract] [Full Text]
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K. D. Karpa, R. Lin, N. Kabbani, and R. Levenson The Dopamine D3 Receptor Interacts with Itself and the Truncated D3 Splice Variant D3nf: D3-D3nf Interaction Causes Mislocalization of D3 Receptors Mol. Pharmacol., October 1, 2000; 58(4): 677 - 683. [Abstract] [Full Text]
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M.-L. Kottler, S. Chauvin, N. Lahlou, C. E. Harris, C. J. Johnston, J.-P. Lagarde, P. Bouchard, N. R. Farid, and R. Counis A New Compound Heterozygous Mutation of the Gonadotropin-Releasing Hormone Receptor (L314X, Q106R) in a Woman with Complete Hypogonadotropic Hypogonadism: Chronic Estrogen Administration Amplifies the Gonadotropin Defect J. Clin. Endocrinol. Metab., September 1, 2000; 85(9): 3002 - 3008. [Abstract] [Full Text]
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R. Grosse, S. Roelle, A. Herrlich, J. Hohn, and T. Gudermann Epidermal Growth Factor Receptor Tyrosine Kinase Mediates Ras Activation by Gonadotropin-releasing Hormone J. Biol. Chem., April 14, 2000; 275(16): 12251 - 12260. [Abstract] [Full Text] [PDF]
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R. Grosse, A. Schmid, T. Schoneberg, A. Herrlich, P. Muhn, G. Schultz, and T. Gudermann Gonadotropin-releasing Hormone Receptor Initiates Multiple Signaling Pathways by Exclusively Coupling to Gq/11 Proteins J. Biol. Chem., March 24, 2000; 275(13): 9193 - 9200. [Abstract] [Full Text] [PDF]
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H.-O. Chung, Q. Yang, K. J. Catt, and K. K. Arora Expression and Function of the Gonadotropin-releasing Hormone Receptor Are Dependent on a Conserved Apolar Amino Acid in the Third Intracellular Loop J. Biol. Chem., December 10, 1999; 274(50): 35756 - 35762. [Abstract] [Full Text] [PDF]
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F.-Y. Zeng, A. Hopp, A. Soldner, and J. Wess Use of a Disulfide Cross-linking Strategy to Study Muscarinic Receptor Structure and Mechanisms of Activation J. Biol. Chem., June 4, 1999; 274(23): 16629 - 16640. [Abstract] [Full Text] [PDF]
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C. A. Saura, T. Tomita, F. Davenport, C. L. Harris, T. Iwatsubo, and G. Thinakaran Evidence That Intramolecular Associations between Presenilin Domains Are Obligatory for Endoproteolytic Processing J. Biol. Chem., May 14, 1999; 274(20): 13818 - 13823. [Abstract] [Full Text] [PDF]
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C. Le Gouill, J.-L. Parent, C.-A. Caron, R. Gaudreau, L. Volkov, M. Rola-Pleszczynski, and J. Stankova Selective Modulation of Wild Type Receptor Functions by Mutants of G-Protein-coupled Receptors J. Biol. Chem., April 30, 1999; 274(18): 12548 - 12554. [Abstract] [Full Text] [PDF]
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 D. C. Danila, A. V. Schally, A. Nagy, and J. M. Alexander Selective induction of apoptosis by the cytotoxic analog AN-207 in cells expressing recombinant receptor for luteinizing hormone-releasing hormone PNAS, January 19, 1999; 96(2): 669 - 673. [Abstract] [Full Text] [PDF]
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J. Jakubik and J. Wess Use of a Sandwich Enzyme-linked Immunosorbent Assay Strategy to Study Mechanisms of G Protein-coupled Receptor Assembly J. Biol. Chem., January 15, 1999; 274(3): 1349 - 1358. [Abstract] [Full Text] [PDF]
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 M. Dosil, L. Giot, C. Davis, and J. B. Konopka Dominant-Negative Mutations in the G-Protein-Coupled alpha -Factor Receptor Map to the Extracellular Ends of the Transmembrane Segments Mol. Cell. Biol., October 1, 1998; 18(10): 5981 - 5991. [Abstract] [Full Text]
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A. Ulloa-Aguirre, D. Stanislaus, V. Arora, J. Vaananen, S. Brothers, J. A. Janovick, and P. M. Conn The Third Intracellular Loop of the Rat Gonadotropin-Releasing Hormone Receptor Couples the Receptor to Gs- and Gq/11-Mediated Signal Transduction Pathways: Evidence from Loop Fragment Transfection in GGH3 Cells Endocrinology, May 1, 1998; 139(5): 2472 - 2478. [Abstract] [Full Text] [PDF]
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K. M. Kroeger, A. C. Hanyaloglu, R. M. Seeber, L. E. C. Miles, and K. A. Eidne Constitutive and Agonist-dependent Homo-oligomerization of the Thyrotropin-releasing Hormone Receptor. DETECTION IN LIVING CELLS USING BIOLUMINESCENCE RESONANCE ENERGY TRANSFER J. Biol. Chem., April 13, 2001; 276(16): 12736 - 12743. [Abstract] [Full Text] [PDF]
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) or of splice variant and GnRH-R-tail cDNA (
) were
cotransfected into COS-7 cells, and GnRH-induced IP accumulation was
determined. One representative experiment of three is shown. Data
represent means ± SEM.