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
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ABSTRACT
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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 4550 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.
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INTRODUCTION
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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.
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RESULTS
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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. 1
).
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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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.
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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
-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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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.
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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. 3
). 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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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.
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To ascertain the expression and subcellular localization of the GnRH
receptor isoforms, immunocytochemical studies were performed. Confocal
fluorescence microscopy on nonpermeabilized (Fig. 4
, 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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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.
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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. 5
) 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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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.
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Inhibition of GnRH Receptor Signaling by Coexpression of a
Truncated Splice Variant
When increasing amounts of splice variant cDNA (1.03.75 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. 6A
). Basal values remained
constant under these experimental conditions, whereas agonist-induced
phosphoinositide hydrolysis was reduced by 3050% 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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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.
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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. 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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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.
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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. 8
). 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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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) ( ) 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.
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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. 9
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 2025% 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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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 (0.254 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.
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DISCUSSION
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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
(4550 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
T31 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 receptors 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 gonadotropes
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
|
---|
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 117 of the GnRH receptor wild type cDNA plus
KpnI site) and primer 2 (nt positions 970987 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
424 (see Fig. 1
) 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 723753 including an endogenous
EcoN I-site (nt positions 723733) followed by a premature stop codon
(TGA) and an annealing region for pCMV-5 corresponding to nt positions
34213438 of pCMV-5. The antisense primer 1 contained a
SmaI site and covered nt positions 34653483 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 (12 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) 2024 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 6973, 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.
 |
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