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Hormone Research Center and Department of Biology (L.W., D.Y.O., H.S.C., R.S.A., J.Y.S., H.B.K.), Chonnam National University, Kwangju, 500–757, Republic of Korea; and the Research Group for Comparative Endocrinology (J.B.), Department of Experimental Zoology, Utrecht University, Utrecht 3584, The Netherlands
Address all correspondence and requests for reprints to: Hyuk Bang Kwon, Hormone Research Center, Chonnam National University, Kwangju 500-757, Korea. E-mail: kwonhb{at}chonnam.chonnam.ac.kr
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Introduction |
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Recently, we have cloned three different GnRHR cDNAs from the bullfrog, designated bfGnRHR-1, bfGnRHR-2, and bfGnRHR-3, respectively (11). These receptors share about approximately 40–45% amino acid identity and exhibit different tissue distributions and ligand selectivity. The bfGnRHR-1 was predominantly expressed in the pituitary, indicating that bfGnRHR-1 may play a classical role in the neuroendocrine control of gonadotropin secretion. However, bfGnRHR-2 and -3 were mainly expressed in various brain regions, suggesting that they may play a role in other brain functions. Although the function of bfGnRHR-2 and -3 in the brain remains to be elucidated, they probably interact with cGnRH-II (and/or sGnRH if sGnRH exists in the bullfrog) whose cell bodies are localized in the midbrain and projecting to the spinal cord and posterior hypothalamic area (12). All three bfGnRHRs exhibited the typical characteristics of nonmammalian GnRHRs (10, 13, 14). All bfGnRHRs contain the cytoplasmic carboxyl tail that is absent in mammalian GnRHRs (15, 16). The Asn2.50 residue in transmembrane domain 2 (TM2) that is important for interaction with Asp7.49 residue in TM7 of the mammalian GnRHR is changed to Asp in bfGnRHRs (17). All bfGnRHRs responded better to cGnRH-II than mGnRH, whereas the mammalian GnRHR was more sensitive to mGnRH than cGnRH-II (18). Interestingly, the bfGnRHR-3 was most sensitive to sGnRH among the natural GnRHs examined (11). Although the sGnRH gene has not yet been identified in other vertebrates than in fishes, a recent study revealed the presence of a sGnRH-like peptide in mammalian brain (6), suggesting a possible interaction of the bfGnRHR-3 with sGnRH.
Northern blot and RT-PCR analysis revealed that each bfGnRHR gene produces multiple transcripts by alternative RNA processing and/or alternative use of transcriptional initiation sites as found in the mammalian GnRHR genes (19, 20, 21). Northern blot analysis indicated the presence of at least four different bfGnRHR-1 mRNA species (11). RT-PCR analysis revealed that there is one additional bfGnRHR-2 splice variant (sv), which is presumed to be generated by partial deletion of exon 1 (unpublished data). In this study we will describe the existence of four bfGnRHR-3 sv’s produced by alternative splicing procedures including exon skipping, partial retention of intronic sequences, and/or transcriptional slippage. The relative abundance of bfGnRHR-3 sv’s in the brain and pituitary, and the production of different proteins derived from the sv’s, suggest that these sv’s may play a role in modulating the biological effect of GnRH. To elucidate the biological relevance of these sv’s, we analyzed cellular expression and responsiveness of the bfGnRHR-3 sv’s to GnRH, and examined the effect of the sv’s on wild-type (wt) bfGnRHR-3 signaling in a transient expression system. In addition, the expression levels of the bfGnRHR-3 wt and sv transcripts were examined in hibernation and the prebreeding season.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Animals and tissue preparation
Bullfrogs were purchased from the local market in Kwangju,
Korea, housed in flow-through tanks under simulated natural conditions.
Frogs were killed by decapitation. The tissues of interest were quickly
dissected, immediately frozen in liquid nitrogen, and stored at -80 C
until use.
Isolation of bfGnRHR-3 sv cDNA
Total RNA was extracted using Tri-reagent (Sigma)
according to the manufacturer’s instructions.
Poly-(A)+ RNA was purified from total RNA using
QIAGEN Oligotex mRNA kit (QIAGEN, Chatsworth,
CA). The wt bfGnRHR-3 cDNA containing the entire open reading frame was
characterized as described previously (11). The bfGnRHR-3
sv cDNAs were identified by RT-PCR amplification using bullfrog cDNAs
from various brain regions and pituitary with primers flanking the
coding region: bf3-start,
5'-CGCGAATTCGCCACCATGAACGCTAGTGACCAACCTATGG-3'
[containing an EcoRI site (underlined)], a
Kozak translation initiation site (italics), and the
N-terminal coding region of bfGnRHR-3 cDNA (bold), and
bf3-stop,
5'-CGCCTCTAGATTCACATAAAGCTCTCTACGATTTGAC-3'
[containing an XbaI site (underlined), and the
C-terminal coding region of the bfGnRHR-3 cDNA (bold)],
respectively. The PCR products were inserted into the EcoRI
and XbaI sites of pcDNA3, yielding the pc-sv1, pc-sv2,
pc-sv3, and pc-sv4 constructs.
Determination of exon-intron boundaries of the bfGnRHR-3
gene
Genomic DNA was isolated from liver using a genomic DNA
isolation kit (QIAGEN). To determine the genomic
organization of the bfGnRHR-3 gene, primers flanking the
presumptive splice sites were designed: bf3-intron A-F,
5'-ATCTTGAACCCGTTGGGCATAG-3'; bf3-intron A-R,
5'-GTTGTACAGGGTCTCCAACCAGTG-3'; bf3-intron B-F,
5'-CCGCTCCTCATCATGGTCTT-3'; bf3-intron B-R, 5'-CATCCGAGCCCGCGGGATG-3'.
PCR products of approximately 0.8 kb (containing intron A) and
approximately 2.5 kb (containing intron B) were obtained, subcloned,
and sequenced using the Dye Terminator Cycle Sequencing Ready Reaction
Kit (Perkin-Elmer Corp.) and an automated ABI prism 377
DNA sequencer (Perkin-Elmer Corp.).
Epitope tagged bfGnRHR-3 cDNA constructs and Western blot
analysis
A stretch of nucleotides coding for a nine amino acid epitope
(YPYDVPDYA) derived from the influenza virus hemagglutinin protein
(HA-tag) was inserted just after the initiating methionine residue in
the extracellular N-terminal part of the bfGnRHR-3 wt and sv cDNAs by
PCR with VentR DNA polymerase using the HA-start
primer,
5'-CGCAAGCTTGCCACCATGTACC-CATACGACGTGCCAGACTACGCGAACGCTAGTGACCAACCTATGG-3'
[containing a HindIII site (underlined), a Kozak
translation initiation site, an HA-tag coding sequence
(italics), the N-terminal coding part of bfGnRHR-3 cDNA
(bold)], in combination with the bf3-stop primer (see
above). PCR products were digested with HindIII and
XbaI and inserted into the HindIII and
XbaI sites of pcDNA3, yielding the HA-wt, HA-sv1, HA-sv2,
HA-sv3, and HA-sv4 constructs. All constructs were sequenced.
HeLa cells were maintained at 37 C in DMEM with 10% heat-inactivated FBS, 1 mM glutamate, 100 U of penicillin, and 100 µg/ml streptomycin. HeLa cells were plated in 100-mm dishes and transfected with HA-wt and HA-sv constructs using the SuperFect transfection kit (QIAGEN). Forty-eight hours after transfection the cells were washed twice with chilled PBS, solubilized in 0.5 ml of lysis buffer with protease inhibitors (50 mM Tris-HCl, pH 8.0; 150 mM NaCl; 0.5% Nonidet P-40; 10 mM NaF; 1 mM sodium orthovanadate; 1 mM dithiothreitol; 10 µg/ml leupeptin; 10 µg/ml aprotinin; 10 µg/ml trypsin/chymotrypin inhibitor; 5 µg/ml pepstatin A; and 1 mM phenylmethylsulfonyl fluoride) and frozen at -80 C. The lysates were cleared by centrifugation. The supernatants were immunoprecipitated with anti-HA antibody overnight at 4 C and then incubated with Protein A-Sepharose beads (Amersham Pharmacia Biotech, Buckinghamshire, UK) to absorb the immunoprecipitates for 4 h. The beads were washed six times with 1 ml of lysis buffer. Bound proteins were eluted with an equal amount of SDS-loading buffer (0.125 M Tris-HCl; 4% SDS; 20% (vol/vol) glycerol; 0.2 M dithiothreitol; and 0.02% Bromophenol Blue, pH 6.8). The immunoprecipitates were separated by 12% SDS-PAGE and transferred onto nitrocellulose membrane. The membranes were blocked in PBS with 0.1% Tween 20 and 5% dried milk overnight, and then probed with anti-HA antibody (1:1000 dilution) (Roche Molecular Biochemicals, Mannheim, Germany). Immunoreactive proteins were detected using horseradish-peroxidase-conjugated secondary antibody and visualized using the enhanced chemiluminescence detection system (Amersham Pharmacia Biotech).
In vitro translation
To examine whether sv proteins are translated in
vitro, pc-wt, pc-sv1, pc-sv2, pc-sv3, and pc-sv4 were in
vitro transcribed with T7 polymerase and translated using the
rabbit reticulocyte lysate system according to the manufacturer’s
instruction (Promega Corp.). In vitro
translated proteins were separated using 12% SDS-polyacrylamide
gel and exposed to an x-ray film (Kodak).
Ligand binding assay
[His5,D-Tyr6]GnRH
(Peptron, Taejeon, Korea) was radioiodiated using the chloramine-T
method. Cell membranes were prepared 48 h after transfection.
Membranes resuspended in protein-free binding buffer (40 mM
Tris, pH 7.4; 2 mM MgCl2). The
membrane fraction (20 µg protein per each tube) was incubated with
I125-[His5,D-Tyr6]GnRH
with a range of concentration (0.1–2 nM) and 0.1% BSA.
Assays were incubated on ice overnight. Nonspecific binding was
observed in the presence of 100 µM cold
[His5,D-Tyr6]GnRH.
For displacement experiments, membranes were incubated on ice overnight
with
I125-[His5,D-Tyr6]GnRH
(100,000 cpm), 0.1% BSA and varying concentrations of mGnRH, cGnRH-II
and
[His5,D-Tyr6]GnRH.
The reaction was terminated by filtration through GF/C filter (Brandel,
Inc., Gaithersburg, MD) that was presoaked with binding buffer
containing 1% BSA, and washed twice with binding buffer.
Luciferase assay
HeLa cells were cultured in 24-well plates, and transfection was
performed using the SuperFect transfection kit (QIAGEN).
For each transfection, 50 ng of pc-wt, pc-sv1, pc-sv2, pc-sv3, or
pc-sv4, 200 ng of pCREluc along with 200 ng of internal control plasmid
pCMVßGal were used. The empty vector pcDNA3 was used to adjust the
total amount of DNA transfected to 0.7 µg. To demonstrate the
dose-dependent effect of pc-sv’s on pc-wt, 200 ng of pCREluc construct
and 50 ng of pc-wt construct were cotransfected with various amounts of
the pc-sv’s (50 ng, 150 ng, and 250 ng, respectively). Forty-two hours
post transfection, cells were treated with either mGnRH or cGnRH-II.
Because we found, in a preliminary time-course experiment, that GnRHs
exhibited highest activity in inducing luciferase reporter gene
expression after 6 h treatment, we selected this period for GnRH
treatment. After 6 h, cells were harvested and the luciferase
activity in cell extracts was determined using a luciferase assay
system according to standard methods in a Lumat LB9501 (EG & G,
Berthold, Germany). The luciferase values were normalized by the
ß-galactosidase values used as an internal control for transfection
efficiency. Transfection experiments were performed in triplicate and
repeated at least three times.
Green fluorescent protein (GFP)-fusion constructs of bfGnRHR-3
cDNAs and confocal laser-scanning microscopy
The enhanced GFP (EGFP) coding region was fused in-frame to the
C-terminal end of the bfGnRHR-3 wt and sv cDNAs by removing the stop
codon using PCR. The following primers were used: GFP-start,
5'-CGCAAGCTTGCCACCATGAACGCTAGTGACCAACCTATGG-3'
[containing an HindIII site
(underlined), a Kozak translation initiation site
(italics), and the N-terminal coding part of bfGnRHR-3 cDNA
(bold)], and GFPwt-stop,
5'-CGCCTCACGGATCCCTCTCTACGATTTGACGCTTACA-3',
or GFPsv1-stop,
5'-CGCCTCATGGATCCCCGCGGGATGTTGTTGTAGGAG-3', or
GFPsv2–4-stop,
5'-CGCCTCACGGATCCAGATGCCGCTTCCCCTCACCTTCAG-3',
in which BamHI restriction sites (underlined)
just in front of the stop codons (bold) were introduced. The
PCR products were digested with HindIII and BamHI
and ligated to HindIII and BamHI digested
pEGFP-N1 plasmid, generating wtGFP and sv1–4GFP constructs. HeLa cells
were transfected with pEGFP-N1 alone, or wtGFP, or the sv1–4GFP
constructs. Cells were transferred onto sterile glass coverslips in
6-well plates 48 h after transfection. After growing for another
12 h, cells were washed with PBS and fixed in 4% paraformaldehyde
at room temperature for 30 min. Fixed cells were washed with PBS and
mounted on microscope slides with 50% (vol/vol) glycerol in PBS. Image
analysis of GFP fluorescence in cells, transfected with pEGFP-N1
plasmid alone or with the various GFP-constructs, were performed under
a Leica Corp. TCS-NT laser-scanning confocal microscope
(Leica Corp., Heidelberg, Germany) equipped with an
argon/krypton laser using a 488 nm excitation filter and 515–540 nm
band pass emission filters. The images of individual cells were
manipulated on a Macintosh computer using the TCS-NT Image program
(Leica Corp.).
RT-PCR analysis of bfGnRHR-3 sv mRNAs
Five micrograms of total RNA prepared from the pituitary and
several brain tissues (olfactory lobe, telencephalon, hypothalamus,
cerebellum, and spinal cord) of a pool of six frogs were
reverse-transcribed and subjected to PCR. To examine the expression
patterns of the bfGnRHR-3 sv mRNAs, PCR was performed using
bf3-interF (5'-GCGAAAAAGAAGAACAAGGCCATG-3') and bf3-interR
(5'-CTCATCCGAGCCCGCGGGATG-3') primers. PCR products were separated by
agarose-gel electrophoresis, transferred to nylon membrane, and
hybridized with radiolabeled bfGnRHR-3 cDNA. As an internal
control, bullfrog GAPDH-specific primers, Rc-GAPDH-F
(5'-CAATCCAATGGGGAGCTTCTG-3') and Rc-GAPDH-R
(5'-CAATCCAATGGGGAGCTTCTG-3') were used, generating a PCR product of
450 bp. The PCR cycling parameters were denaturation at 95 C for 30
sec, followed by 30 cycles at 94 C for 15 sec, at 50 C for 10 sec, and
at 72 C for 30 sec. Although the results of these RT-PCR experiments
can be considered only as qualitative, the intensity of each
PCR-amplified band was analyzed to get an indication of quantitative
differences in expression using image analysis software (TINA,
Fuji Photo Film Co., Ltd.) after being photographed with a
chemoluminescence camera. The data shown are band densities in
arbitrary optical density units.
Data analysis
Binding assay and transfection experiments were performed in
triplicate and repeated at least three times. Statistical analysis was
performed by either one- or two-way ANOVA or t test.
Statistical significance was set at P < 0.05.
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Results |
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). The 1.3-kb PCR product most likely
corresponded to the previously identified bfGnRHR-3 cDNA
(11), although the diffuse nature of the band suggested a
mixture of multiple products with small differences in size, possibly
derived from alternative splicing. The 1.1-kb product seemed to
represent an unidentified sv form of bfGnRHR-3 mRNA. Sequence analysis
revealed that the 1.1-kb product (designated sv1) lacked 204 bp between
nucleotides 576 and 782 of the bfGnRHR-3 wt cDNA sequence. This
truncation of the sv1 mRNA is most likely due to exon skipping during
the pre-mRNA splicing (Fig. 1B). This exon skipping resulted in a frame
shift and introduced a premature stop codon, generating a truncated
protein of 211 amino acids. The 1.3-kb band contained at least four
different cDNAs: bfGnRHR-3 (wt) and three additional bfGnRHR-3 cDNA
sequences (designated sv2, sv3, and sv4) in which 47, 94, and 100
additional nucleotides were inserted between exons 2 and 3,
respectively. Hence, these sv2, sv3, and sv4 cDNAs were produced by
partial intron retention (Fig. 1B). In addition, the sv4 cDNA contains
an additional sequence (5'-AGCCAA-3') that was not found in intron B of
the bfGnRHR-3 gene. The retained intron sequence introduced
two premature stop codons. Because the sv2, sv3, and sv4 share the same
stop codon in retained intron B, the truncated bfGnRHR-3 proteins
derived from sv 2–4 are identical in amino acid sequence.
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0.6 kb), located in transmembrane domain (TM) IV, and intron B
(2.4 kb), located in intracellular loop 3, respectively (Fig. 2A). However, we cannot exclude the
possibility that additional introns are present in the 5' or 3'
untranslated region of the bfGnRHR-3. The first intron (intron A) in
the bfGnRHR-3 gene is much smaller than the intron
found at the similar position in mammalian GnRHR genes
(4- to 12-kb). The second intron is about 2.4 kb in size, which is
very similar to the mammalian GnRH gene (3 kb).
Exon-intron junction analyses indicated that the 5' splice site of
intron B loosely conformed to the consensus sequence, and that two
additional putative 5' splice sites are present within intron B.
Comparison of the sv sequences with the partial bfGnRHR-3
gene sequences indicated that sv1 is generated by skipping of exon 2
(by using the normal 3'-splice site of intron A and the 3'-splice site
of intron B), whereas sv2, sv3, and sv4 are generated by partial
retention of intron B sequences during the splicing process (Fig. 2B).
Interestingly, alternative splicing of the GnRHR primary transcript,
producing a mature mRNA with the complete deletion of exon 2, was also
observed in mouse (19) and human (22). This
suggests that a common mechanism of alternative splicing is used in
amphibians and mammals for their GnRHR genes. The
alternative splicing events within intron B of the bfGnRHR-3 pre-mRNA
further extend the observations found in mammalian GnRHR pre-mRNA
splicing, and probably result from competition among three cryptic 5'
splice sites present in this intron for the common 3' splice site of
intron B (Fig. 2B). These 5' splice sites loosely conformed to the
metazoan consensus sequence, GTRAGT, in which the initial GT
dinucleotide is almost invariant (Fig. 2B) (24, 25). The
3' splice site of intron B is in accordance with the conserved
consensus sequence YnXCAG (25). In
addition, transcriptional slippage (26) appears to
contribute to the generation of sv4, because 6 additional nucleotides
found in this cDNA are absent in the genomic DNA (Fig. 2B).
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, lane 1), of 27 kDa in cells
expressing the HA-sv1 protein (Fig. 3A, lane 2), and of 34 kDa in cells
expressing the HA-sv2, HA-sv3, and HA-sv4 proteins (Fig. 3A, lanes
3–5). It is notable that there was no significant difference in
expression levels among the wt and sv proteins. To examine the
translation of sv’s in vitro, the corresponding bfGnRHR-3
proteins were synthesized using an in vitro translation
rabbit reticulocyte system. This result showed that bfGnRHR-3 proteins
had similar molecular masses as found in transfected HeLa cells (Fig. 3B) The small difference in size between in vitro translated
receptors and HeLa cell-expressed receptors is most likely due to use
of different plasmids (HA-containing plasmids in HeLa cell-expressed
receptor) or a posttranslational modification such as glycosylation in
HeLa cells. The relative small protein size observed in sv proteins is
due to early introduction of stop codon by frame shift (sv1) or intron
B retention (sv2–4) (Fig. 3C).
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). When 20,000 cpm of
I125-[His5,D-Tyr6]GnRH
was incubated with 20 µg of membrane fraction, 16% bound per free
value was obtained from wt receptor containing membrane fraction. In
contrast, the bfGnRHR-3 sv’s showed a very low binding affinity
(maximum 3% and 0.7% binding for sv1 and sv2–4, respectively) to
I125-[His5,D-Tyr6]GnRH
(Fig. 4B). chicken GnRH-II, mGnRH, and
[His5,D-Tyr6]GnRH
inhibited
I125-[His5,D-Tyr6]GnRH
binding in a dose-dependent manner (data not shown). These results
indicate that these sv receptors could marginally bind to ligand,
although they are expressed at the same level as the wt receptor as
shown in Fig. 3A
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, reporter gene expression was
stimulated in a dose-dependent manner in response to mGnRH (Fig. 3A) or
cGnRH-II (Fig. 5B) in cells transfected with the pc-wt construct only.
None of the four sv’s alone was able to induce luciferase activity in
response to mGnRH or cGnRH-II (Fig. 5, A and B). The luciferase
expression was increased 20-fold in the presence of 100 nM
mGnRH (Fig. 5C) or 1 nM of cGnRH-II (Fig. 5D) in cells
expressing the wt bfGnRHR-3 protein. Cotransfection with increasing
amounts of pc-sv1 did not show any effect on the GnRH-induced
luciferase gene expression in cells expressing pc-wt (Fig. 5, C and D).
However, cotransfection of increasing amounts of pc-sv2, pc-sv3, or
pc-sv4 (from 50–250 ng) constructs with constant amounts of the pc-wt
(50 ng) construct, yielded a dose-dependent decrease of mGnRH- or
cGnRH-II-induced signaling (Fig. 5, C and D, respectively). Although
the inhibitory effect of pc-sv4 on pc-wt was relatively less than that
of pc-sv2 and pc-sv3; statistically there was no significant difference
in the repressive activity among these three isoforms. Similar results
were obtained in HepG2 cells (data not shown).
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. A highly localized, ring-like
distribution of fluorescence was detected in cells expressing the wtGFP
fusion protein (Fig. 6B), indicating plasma membrane-associated
localization of the wt receptor. The observed plasma
membrane-associated fluorescence was generally even in terms of the
intensity, although some cells showed a more punctuate fluorescence. In
contrast, the distribution of the fluorescence in cells transfected
with the GFP plasmid alone was diffuse, and present
throughout the cytosol and nucleus (Fig. 6A), consistent with GFP
itself not being localized to any particular intracellular compartment,
whereas no fluorescence was detected in untransfected cells (data not
shown). The fluorescence corresponding to the sv1GFP was noticeably
concentrated in the cytoplasmic compartment (Fig. 6C). In cells,
expressing the sv2–4GFP fusion proteins, a decreased
membrane-associated fluorescence was visualized, accompanied by a
larger amount of fluorescence in the endocytic vesicles (Fig. 6D). It
should be noted that because the sv2, 3, and 4 proteins are identical
in amino acid sequence, the fusion of these sv2, 3, and 4 with GFP
produces the identical protein.
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). The
overall expression levels of wt (yielding a 345 bp PCR product) were
significantly higher in the prebreeding season than in hibernation and
appeared more abundant in midbrain and hindbrain regions. Because the
differences in size of the sv2 (392 bp), sv3 (439 bp) and sv4 (445 bp)
PCR products are too small to be clearly separated on agarose gel (Fig. 7), we were unable to conclude whether there is a brain region-specific
expression for each of these variant transcripts. In terms of overall
expression level for these three transcripts (i.e. the upper
band representing the mixture of sv2, sv3, and sv4 transcripts), it is
apparent that the relative expression levels of sv2–4 were
dramatically decreased from hibernation to the prebreeding season. We
have compared the intensity of each EtBr-stained PCR band and
calculated their absolute OD value (Fig. 7B). In each brain region, the
ratio of the band intensity of the wt to sv2–4 is 0.47 in pituitary
(P), 0.95 in olfactory lobe (OL), 0.48 in telencephalon (T), 1.33 in
hypothalamus (H), 0.76 in cerebellum (C), and 0.89 in spinal cord (S),
respectively, in hibernation season, but increased to 3.76, 19.56, 4.0,
4.9, 3.2, and 5.1, respectively, in the same order in prebreeding
season. Therefore, the total fold increase of the relative expression
level of the wt to sv2–4 from hibernation to breeding season was 8,
20.6, 8.3, 3.7, 4.2, and 5.7, respectively. In addition, the primers
specific for GAPDH mRNA amplified a PCR product of 450 bp with a
similar intensity in all the tissues examined, verifying the quality
and integrity of the cDNA samples (Fig. 7A). Moreover, Southern blot
analysis was performed, confirming the RT-PCR amplified products being
bfGnRHR-3 specific (data not shown).
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Alternative RNA splicing is a commonly used strategy for creating a functionally diverse pool of gene products derived from a single gene, which is recognized as an important mechanism for the regulation of the wt receptor function (30, 31). In mammalian species, a single GnRHR gene generates multiple transcripts by alternative splicing and/or different use of transcriptional initiation sites (19, 20, 22). However, the functional relevance of these transcripts is poorly understood. The present study indicates that the bfGnRHR-3 gene produces at least five different RNA species by exon skipping and intron retention. Interestingly, exon 2 deletion was also found in the mammalian GnRHR gene (19, 22), indicating that the splicing pattern of GnRHR transcripts may be evolutionary conserved. Exon 2 deletion and intron B retention introduced premature stop codons, producing truncated protein. Immunoblot analysis and in vitro translation assay revealed that these truncated proteins are expressed with no significant differences in expression levels compared with that of the wt protein. However, these sv proteins exhibited a very low binding affinity to GnRH and were not able to induce signal transduction. The failure to induce signal transduction is most likely due to either low ligand binding affinity or lack of a key motif in the receptor that is involved in signal transduction. The low binding affinity of sv proteins may result from the truncated nature even though they have putative ligand binding sites (Asp98, Asn102, and Lys121 at the homologous position of human GnRHR) (11, 32, 33). The early introduction of stop codon generated proteins that lack TM5/6/7 or TM6/7 in sv1 and sv2–4, respectively. TM7 is known as a crucial counter part of interaction with TM2, which can stabilize receptor structure and provide signaling ability (17, 34, 35). Alternatively, a very low affinity of sv proteins to ligand may be due to an impaired translocation of sv proteins to cellular membrane. Confocal microscopy studies revealed that the wt receptor protein was membrane-associated, whereas sv’s were located somewhat differently. The sv1 protein was present only in the cytosol, indicating that the deletion of exon 2 seriously impairs the proper translocation of the truncated sv1 receptor into the cell membrane. The other sv’s (sv2–4) were expressed in both the cell membrane and cytosol. Therefore, it is assumed that such truncations affect the efficient translocation of these proteins to the cell membrane, which may provide little chances to bind with ligand. The other reason for the lack of signaling ability may be the absence of NPX2–3Y motif in TM7 that is involved in signal transduction in rhodopsin-like G protein-coupled receptors (GPCRs) (36). Because this motif is completely absent in these sv-derived proteins (for bfGnRHR-3: such as Ala278, Asp338 and DPITY at the homologous position), it is evident that they would lose the ability to induce signal transduction.
Although these sv proteins showed very low binding affinities to ligand
and absence of signaling ability via the cAMP signal transduction
pathway in transfected cells, they were able to inhibit wt receptor
signaling when cotransfected. Similar finding was observed in the human
GnRHR system (27). A sv of the human GnRHR exhibited a
specific and dose-dependent inhibition of GnRH-induced wt receptor
signal transduction (27). It is also notable that the sv
cDNAs of the bullfrog were more sensitive in inhibiting GnRH-induced wt
receptor signaling than that of human GnRHR. In case of human GnRHR, an
approximately 8- to 15-fold excess sv was required for inhibition of wt
signaling (27), whereas a 3-fold excess of bfGnRHR sv’s
was enough to inhibit wt signaling. Considering a high expression of
sv’s RNA transcripts in hibernation season, bfGnRHR sv proteins may
regulate the wt receptor function in this season. However, it is not
easy to explain how these sv proteins interfere with wt signaling. A
competition between truncated receptor and the wt receptor for ligands
may not be the case because the sv proteins have extremely low binding
affinities. One possible explanation is that coexpression of sv’s with
the wt receptor may decrease membrane expression of the wt receptor as
demonstrated by a recent study (27). This interpretation
is based on the possible defective intracellular transport due to the
formation of misfolded complexes between wt receptor and truncated
protein in the endoplasmic reticulum. This possibility is supported by
two facts: the GnRHR appears to have two receptor folding units (TM1–5
and TM6–7) (27) and 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 interaction (37, 38). Thus, it is possible
that TM1–5 of sv proteins may physically interact with TM6–7 of the
wt receptor, rustling in a reduced membrane expression. However, the
failure of sv1 to inhibit wt receptor signaling could not be explained
by the above interpretation. The alternative interpretation, therefore,
is that sv proteins (sv2–4 but not sv1) expressed in the cell membrane
could physically interact with the agonist-occupied wt receptor,
subsequently resulting in a reduced signaling. Currently, there are
increasing lines of evidence indicating that G protein-coupled
receptors could function as dimers (homo- or hetero- or multimers)
(39). A recent study using fluorescence resonance energy
transfer analysis clearly demonstrated that the GnRHR can form dimers
or multimers after agonist but not antagonist binding
(40). Because the receptor microaggregation is an early
event (<1 min) of agonist occupancy of the receptor, such a
microaggregation appears a component of signal transduction, leading to
the agonist activation of the receptor. Therefore, it is possible that
an interaction of the agonist-occupied wt receptor with truncated
receptor may interfere with wt receptor signaling. Considering the fact
that sv2–4 (that could be expressed in the cell membrane) but not sv1
(expressed only in the cytoplasm) could inhibit the wt receptor
signaling, the membrane expression of truncated protein appears a
prerequisite for inhibition of wt signaling. However, elucidation of
the detailed molecular mechanism under which sv inhibits wt receptor
signaling will require further studies such as cellular distribution of
wt bfGnRHR-3-GFP in the presence of sv proteins, coimmunoprecipitation
of wt receptor and sv proteins, and fluorescence resonance energy
transfer analysis(40) or bioluminescence resonance energy
transfer analysis (41) for examining direct physical
interaction wt receptor and sv’s.
A particularly interesting finding was the differential expression of
the bfGnRHR-3 wt and sv mRNAs under different physiological conditions.
The ratios of wt/sv mRNAs were much higher in the prebreeding season
than those observed during hibernation (Fig. 7). Thus, it appears that
the inhibitory effect of sv’s on wt functioning can vary in different
seasons, such that it may be more effective in inhibition of wt
receptor signaling in hibernation than in the prebreeding season.
Recently, it has been reported that there is a variable expression of
differently sized GnRHR transcripts in cyclic ewes (42),
although the significance of this finding is yet unclear. Differential
expression levels of sv’s and wt indicate that the mechanism
generating the sv’s may play an important role in the regulation of
the wt receptor signaling under different physiological conditions.
In addition, it should be also noted that the wt bfGnRHR-3 expression was markedly augmented in the pituitary gland in the prebreeding season. In our previous study (11), we clearly demonstrated by Northern blot analysis the predominant expression of bfGnRHR-1 in the pituitary, whereas bfGnRHR-3 was localized in the brain regions. The expression of the wt bfGnRHR-3 receptor in the pituitary in prebreeding season, as revealed by the more sensitive RT-PCR approach in this study, raises the possibility that bfGnRHR-3 may also participate in the neuroendocrine function as does the bfGnRHR-1, allowing reproductive activity of the frog. However, regarding the much higher expression level of bfGnRHR-1 than bfGnRHR-3 in the pituitary, it is reasonable to assume that bfGnRHR-1 plays the major role in regulating reproductive function in the bullfrog.
In summary, we have identified five different transcripts of the bfGnRHR-3, generated by alternative splicing. The observations that the wt receptor signaling is inhibited by sv-derived proteins and that the seasonal changes in expression levels of the alternatively spliced transcripts occur, suggest that posttranscriptional regulation of the bfGnRHR-3 gene is an important mechanism in regulating the wt receptor function under different physiological conditions.
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Acknowledgments |
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Footnotes |
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Abbreviations: cGnRH II, Chicken GnRH-II; EGFP, enhanced GFP; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; GnRHR, GnRH receptor; HA, hemagglutinin; mGnRH, mammalian GnRH; sGnRH, salmon GnRH; sv, splice variant (sv1–4 of bfGnRHR-3); TM, transmembrane domain; wt, wild-type (bullfrog GnRHR-3 wild-type).
Received March 2, 2001.
Accepted for publication May 15, 2001.
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