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Department of Physiology, University of Turku, Kiinamyllynkatu 10, FIN-20520 Turku, Finland; and the Medical Research Council Reproductive Biology Unit, Center for Reproductive Biology (H.M.F.), Edinburgh, United Kingdom EH3 9 EW
Address all correspondence and requests for reprints to: Dr. Ilpo Huhtaniemi, Department of Physiology, University of Turku, FIN-20520 Turku Finland. E-mail: ilpo.huhtaniemi{at}utu.fi
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Abstract |
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In conclusion, marmoset monkey LHR seems to lack the sequence corresponding to exon 10 of the LHR gene in other mammalian species. The truncation does not alter LHR function, as the monkey receptor protein bound hCG and evoked cAMP and inositol trisphosphate responses comparable to those of the human LHR containing the exon 10-encoded structure. As the sequence homologous to exon 10 is missing in the other two glycoprotein receptors, i.e. those of FSH and TSH, this extra exon is apparently inserted into the LHR messenger RNA of some species during evolution from intronic sequences by a change in alternative splicing.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Unlike many members of the 7-transmembrane domain receptor family, which are intronless (1), the 3 glycoprotein hormone receptor genes contain 9 (TSH and FSH receptors) or 10 (LHR) introns. LHR is composed of an extracellular N-terminal half and a plasma membrane-associated C-terminal half of the full-length protein (1). The extracellular domain is encoded by the first 10 and a part of the 11th exon, and it is capable of high affinity hormone binding. The rest of exon 11 encodes the transmembrane and intracellular parts of the receptor, which are capable of low affinity hormone binding and signal transduction (6, 7, 8, 9).
Recent studies on serial mutagenesis of the LHR complementary DNA
(cDNA) emphasize that the structural integrity of the receptor protein
is important for its functions. Several point mutations or deletion of
Lys583, located in the third extracellular loop, maintain
the affinity of the receptor to ligand, but greatly diminish the
ligand-mediated signaling (10, 11). Two point mutations of the LHR in
the ionizable amino acid residues, i.e. Glu332
Lys332 and Asp333Lys333,
displayed nearly the same binding affinity for hCG as wild-type
receptor, but they also failed to evoke increased cAMP production (12).
Deletion of amino acids 1–11 or the leucine-rich repeats 1, 2, 3, 4,
5, and 6 individually, by contrast, resulted in a loss of detectable
binding activity (13).
Numerous mutations of the LHR gene have also been found to be involved in pathophysiological conditions. The syndrome of familial male-limited precocious puberty is caused by mutations mainly located in the third intracellular loop or in the sixth transmembrane segment of the LHR, resulting in its constitutive action (14, 15, 16). Inactivating mutations of the human LHR lead to Leydig cell hypoplasia, a form of male pseudohermaphroditism resulting from the failure of fetal testicular Leydig cells to differentiate and produce testosterone due to defective LH/hCG signal transduction (17, 18, 19, 20).
To further understand the structure-function relationships of the LHR gene and to determine whether there are structural differences in the LHR between different species, we undertook the cloning of the complete nucleotide sequence as well as functional expression of marmoset monkey LHR cDNA. The intriguing finding was that in the monkey LHR cDNA, the DNA sequence corresponding to exon 10 of the LHR gene in all other species studied to date is missing in all splice variants. The truncated monkey LHR cDNA was successfully expressed in COS cells, allowing high hCG binding and hCG-mediated cAMP and inositol trisphosphate (IP3) production.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Reverse transcription-PCR (RT-PCR) and PCR analyses
For RT-PC and PCR reactions, several primers were designed based
on the conserved sequences of the human, porcine, rat, and mouse LHR
cDNAs. These primers were named human oligonucleotide (oh; Fig. 1
and Table 1). Subsequently, several
other primers specific for the monkey LHR cDNA were designed according
to the known partial monkey LHR cDNA sequences generated by RT-PCR;
these primers were named monkey oligonucleotide (om; Fig. 1 and Table 1).
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). The reverse transcribed, single strand cDNAs were
further amplified by PCR using the various primer pairs (see Fig. 1);
in each case, the antisense primer was the same as the primer used in
the RT reaction. Fifty microliters of the RT-PCR mixture contained 1
nM of each oligo primer, 200 mM deoxy-NTPs, 1.5
mM MgCl2, 20 U RNasin (Promega), 12.5 U avian
myeloblastosis virus reverse transcriptase, and 2.5 U Taq
DNA polymerase (Promega). The reaction was started at 50 C for 10 min
(at room temperature), followed by a period of 3 min at 97 C, and then
run for 35–40 PCR cycles (96 C, 1 min; 57 C, 1 min; 72 C, 2 min), and
final extension for 10 min at 72 C. The PCR reaction from monkey
genomic DNA was performed with the hot start method. The reaction was
started at 97 C for 5 min, followed by 52 C for 2 min, and 72 C for 3
min, and then run 30 PCR cycles (97 C, 1 min; 52 C, 1, 72 C, 2 min) and
final extension for 10 min at 72 C. The RT-PCR and PCR products were
resolved in 1–1.5% agarose gels, and the cDNA fragments with expected
sizes were cut from the gels and isolated using the QIAEX II gel
extraction kit (Qiagen, Chatsworth, CA). The DNA fragments were
subcloned into the T vector (Promega) according to the manufacturer’s
instruction for sequencing and construction of the full-length LHR cDNA
(see below).
Construction of the monkey and human LHR cDNAs for expression
studies
The overlapping fragments of the monkey LHR cDNA were digested
by restriction endonucleases (Promega) at appropriate sites (see Fig. 1) and ligated by the T4 DNA ligase (Promega) using standard molecular
biology methods. The complete monkey LHR cDNA was excised from the T
vector by digestion with SacII and SalI, rendered
blunt ended using the Klenow and T4 DNA polymerase enzymes (Promega),
and subcloned into the eukaryotic expression vector
pZeoSV2+ (Invitrogen, San Diego, CA), prepared by
digestion with EcoRV and dephosphorylation to yield
pZeoSV2+/monkey LHR (sense) and
pZeoSV2+/monkey LHR (antisense). As control,
human LHR cDNA was excised from pBS-hLHR by digestion with
EcoRV and BamHI and subcloned into
pZeoSV2+ at the HindIII and
BamHI sites to yield pZeoSV2+/human
LHR.
Sequencing
The partial and complete monkey LHR cDNAs inserted into the T
vector were sequenced from both strands using an automatic sequencing
machine (Perkin-Elmer, Foster City, CA). The nucleotide and amino acid
sequences were further analyzed by DNASTAR (DNASTAR, Madison, WI) and
BLAST Search (23) computer programs.
Northern hybridization analysis
Twenty micrograms of total RNA from monkey testis were resolved
on 1.2% formaldehyde denaturing agarose gel and transferred onto nylon
membrane (Hybond-N, Amersham, Aylesbury, UK). Prehybridization and
hybridization were performed as previously described (24). Briefly, the
filters were prehybridized for at least 4 h at 65 C in a solution
containing 50% formamide, 3 x SSC (1 x SSC = 150
mM NaCl and 15 mM sodium citrate, pH 7.0),
5 x Denhardt’ solution, 1% SDS, 0.1 g/liter heat-denatured calf
thymus DNA, and 100 mg/liter yeast transfer RNA. Hybridization was
carried out at 68 C overnight in the same solution after adding the
32P-labeled complementary RNA (cRNA) probe. After
hybridization, the membranes were washed twice with 2 x SSC and
0.1% SDS at room temperature for 30 min each time and twice with
0.1 x SSC and 0.1% SDS at 66 C for 2 h each time. After
treatment of the membrane with ribonuclease A (1 mg/liter in 2 x
SSC) at room temperature for 30 min, followed by washing with 2 x
SSC at 42 C for 30 min, the membranes were exposed to x-ray film (Kodak
XAR-5, Eastman Kodak, Rochester, NY) at -70 C for 3 days. The
molecular sizes of the messenger RNA (mRNA) species were determined by
comparison with RNA mol wt markers (Promega). The
32P-labeled cRNA was synthesized using a Riboprobe
synthesis II kit (Promega), [32P]UTP (Amersham), and
full-length monkey LHR cDNA that was subcloned into the T vector
(pGEM-5Z) as template.
Southern hybridization analysis of genomic DNA
Forty micrograms of monkey and human genomic DNAs were digested
with XhoI. The digested DNA fragments were resolved in a
0.7% agarose gel and transferred onto nylon membrane by the capillary
transfer method. The probe was generated by PCR amplification of exon
10 of the hLHR gene using as the primer pair oligos h9 and h10. The
amplified fragment was labeled with [32P]CTP using the
Multiprime DNA labeling system (Amersham), except that oligos h9 and
h10 were used instead of hexamer primers. Prehybridization and
hybridization were carried out as previously described (24).
Transfections
COS cells were maintained in DMEM-Ham’s F-12 medium (1:1; Life
Technologies, Grand Island, NY) supplemented with 10% FCS and 0.1
g/liter gentamicin (Biological Industries, Bet-HaEmek, Israel). Cells
(0.5 x 106) were plated on 9-cm diameter plates 1 day
before transfection and transfected using the calcium phosphate
precipitation method (25) with 10–15 µg of the plasmid under
investigation and 3 µg of the ß-galactosidase expression vector as
control for transfection efficiency. Precipitates were left on cells
for 12 h and subsequently washed with PBS and fed fresh medium.
Cells were used for analysis 2–3 days after transfection. In the case
of stable transfection, the cells were split into medium containing 200
mg/liter Zeocin (Invitrogen), and the selection was maintained for 3
weeks. After selection, the cells were pooled to create nonclonal
stable cell lines and maintained in medium containing 50–100 mg/liter
Zeocin.
[125I]hCG binding study
hCG (CR-127, NIDDK) was iodinated to a specific activity of
35,000 cpm/ng and 37% specific binding of radioactivity to an excess
of LHRs, as determined according to the method of Catt et
al. (26). For LHR binding measurements, the cells were washed
twice with cold PBS and scraped by rubber policeman into Dulbecco’s
PBS containing 0.1% BSA (D-PBS). The cells were pelleted by
centrifugation at 1,500 rpm and washed twice with D-PBS. For single
point binding measurements, triplicate aliquots of cells (0.2 x
106) were incubated with 150,000 cpm
[125I]iodo-hCG in the presence or absence of 50 IU
unlabeled hCG (Pregnyl, Organon, Oss, The Netherlands) in a total
volume of 250 µl. For Scatchard analysis, similar aliquots of cells
were incubated with increasing doses of [125I]iodo-hCG
(up to 500,000 cpm/tube). For competition studies, the same aliquots
were incubated with increasing amounts of unlabeled hCG (CR 127;
0.1–400 ng/tube) or recombinant human FSH (rhFSH; Org 32489, Organon;
0.1–400 mIU/tube) in the presence of 150,000 cpm
[125I]iodo-hCG. After overnight incubation at room
temperature, the cells were washed with 4 ml ice-cold D-PBS and
centrifuged, and the radioactivity in the cell pellets was counted in a
-spectrometer. Nonspecific binding was determined in the presence of
50 IU Pregnyl, and all data were corrected for nonspecific binding.
cAMP and IP3 assays
Transfected cells were plated at a density of 50,000 cells/well
(24-well plates) 24 h before stimulation. The cells were
stimulated for 3 h in a medium containing 0.2 mmol/liter
3-isobutyl-1-methylxanthine (Aldrich-Chemie, Steinheim, Germany) with
increasing doses of hCG (CR-127; 0–1000 µg/liter) or rhFSH (0–1000
IU/liter). All stimulations were performed in quadruplicate. At the end
of the incubation, the medium was collected for measurement of
extracellular cAMP using a standard RIA method (27).
For IP3 measurements, stably transfected cells were cultured in six-well plates at a concentration of 5 x 105 cells/well in inositol-free DMEM-Ham’s F-12 medium. After 24-h incubation, the cells were washed twice with HEPES-3 buffer (10 mM HEPES, 58 mM NaCl, 0.3 mM NaH2PO4, 3.4 mM sodium acetate, 5 mM KCl, 0.6 mM MgSO4, 1.5 mM CaCl2, 2 mM D-glucose, and 0.1% fatty acid-free BSA, pH 7.2) and stimulated without or with increasing doses (0–5000 µg/liter) of hCG for 20 min in the presence of LiCl. Accumulation of IP3 was terminated, and the substance was extracted by keeping the cells on ice with 10% ice-cold perchloric acid, followed immediately by neutralization with 1.5 M KOH in 60 mM HEPES, pH 7.2. Production of IP3 was determined by the inositol 1,4,5-trisphosphate [3H]RRA Kit (New England Nuclear Research Products, DuPont de Nemours Co., Boston, MA) according to the manufacturer’s instructions. The sensitivity of the system was approximately 0.1 pmol.
Presentation of data
The results are presented as the mean ± SEM
when appropriate.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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According to the partial monkey LHR cDNA sequences generated by RT-PCR, several other primers specific to the monkey sequence were synthesized. Using primer pairs oh1 and om1, oh2 and om2, om3 and om5, and om4 and oh8, four overlapping monkey LHR cDNA fragments were generated by RT-PCR. These fragments were subcloned into the T vector and sequenced.
The complete monkey LHR cDNA (Fig. 2) was constructed by
ligating cDNA fragments M1 and M2 at the AvaI site, followed
by ligation of H2 at BglII, M3 at ApaI, and M4 at
XbaI into the T vector (see Fig. 1). The complete monkey LHR
cDNA was excised from the T vector and subcloned into the eukaryotic
expression vector pZeoSV2+ to yield
pZeoSV2+/monkey LHR (sense) and
pZeoSV2+/monkey LHR (antisense).
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). These data strongly support the evidence that
exon 10 is absent in monkey LHR cDNA.
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),
indicating that the sequence corresponding to exon 10 is present in the
monkey gene, but spliced out in all of the LHR mRNA splice variants.
Furthermore, RT-PCR and PCR reactions were performed using primer pair
oligos h9 and h10, corresponding to exon 10 of the human LHR cDNA, from
testicular RNA and genomic DNA, and the 81-bp PCR products were
observed in the human and monkey genomic DNAs, but not in the monkey
testicular RNA (results not shown). Hence, exon 10 is present in monkey
genomic DNA, but is absent in LHR cDNA.
Comparison of monkey, human, porcine, and rat LHR cDNA and amino
acid sequences
Figures 2 and 4 show the nucleotide and deduced
amino acid sequences of the cloned monkey LHR cDNA as well as
comparisons between the cognate monkey, human, porcine, and rat
sequences. The translation initiation codon at position 1 of the
testicular LHR cDNA defines the start of a 2031-bp nucleotide open
reading frame (676 amino acids). Compared with the human, porcine, and
rat LHR cDNAs, the striking feature of the monkey LHR cDNA is the
absence of the sequence encoding the entire exon 10 (81-bp nucleotides,
27 amino acids) of the human, porcine, and rat cDNAs (Figs. 2 and 4).
The overall nucleotide sequence homology is 94% with human, 88% with
porcine, and 83% with rat, respectively. At the deduced amino acid
level, three potential N-linked glycosylation sites were
found in the extracellular domain that are conserved in the monkey,
human, porcine, and rat sequences; the other three potential
N-linked glycosylation sites in exon 10 were only conserved
in the human, porcine, and rat sequences (Fig. 4).
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, A and B).
No specific binding was observed in cells transfected with
pZeoSV2+/mLHR (antisense) and
pZeoSV2+ without insert (Fig. 5, A and B).
Scatchard analysis of the binding data demonstrated that the affinity
of [125I]iodo-hCG binding to the cells transfected with
pZeoSV2+/mLHR (Kd = 0.25
nM) was similar to that to cells transfected with
pZeoSV2+/hLHR (Kd = 0.20
nM; Fig. 5C). As shown in Fig. 5D, increasing doses of
human LH, but not rhFSH, were able to dose dependently displace the
[125I]iodo-hCG binding.
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). In contrast, rhFSH did not
stimulate cAMP production in either cell type (data not shown). The
EC50 values for cAMP production in monkey and human
LHR-expressing cells were 40 and 32 ng/ml, respectively. Unlike the
untransfected cells, which showed no elevation of IP3 (data
not shown), the cells expressing monkey and human LHR displayed similar
dose-dependent increases in IP3 production, which were
about 4.5- and 5.5-fold over the basal production rate (Fig. 6B). The
EC50 values for IP3 production in monkey and
human LHR-expressing cells were 90 and 37 ng/ml, respectively.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Alternative splicing is a widespread device for gene regulation and for generating isoform diversity (28). Previous studies demonstrated that in addition to mRNA of the LH holoreceptor, various sizes of splice variants of the LHR mRNA have been found in rat, mouse, porcine, ovine, and human species, consistent with deletions of complete or partial exons within the genomic structure (29, 30, 31, 32). The common method of alternative splicing, called cassette exon exclusion (whole exon deletion) (28), has been reported in numerous LHR splice variants in rat, pig, ovine, and human (1, 32, 33, 34). A truncated form of LHR without the entire exon 10 has been found in the ovine ovary (34), but not in other species studied to date, which indicates that alternative splicing of transcripts of a given gene is species specific. The extent of translation as well as the physiological significance of these mRNA forms are still unknown (1).
To confirm that the cloned monkey LHR cDNA encodes a functional LHR protein, we transfected the cDNA constructed in the expression plasmid pZeoSV2+ into COS cells. These cells as well as those transfected with the human LHR cDNA demonstrated specific high affinity binding of hCG. As a control, hCG binding was not observed in cells transfected with antisense monkey LHR cDNA or a plasmid without insert. The affinities (Kd) determined to the recombinant monkey LHR (Kd = 0.25 nM) and human LHR (Kd = 0.2 nM) were similar to those previously determined by others (human LHR, Kd = 0.12 nM; rat LHR, Kd = 0.17 nM; and porcine LHR, Kd = 0.18 nM) (12, 29, 35). Our data indicated that the cloned monkey LHR cDNA encodes a receptor protein that has binding characteristics to [125I]iodo-hCG similar to those previously reported using LHR from other species.
Davis et al. (36) reported that hCG stimulates phosphoinositide hydrolysis in isolated bovine corpus luteum cells. This raises the possibility that besides cAMP production, LH and hCG may also stimulate phospholipase C, generating inositol phosphates as second message. This was further confirmed by other studies, and it was proposed that all glycoprotein hormone receptors also stimulate inositol phosphate turnover (2, 37, 38, 39, 40). The exact nature of the G protein involved in LHR-mediated activation of phospholipase C is still not known. A recent study by Hirsch et al. (41) using chimeric LH and FSH receptors demonstrated that the C-terminal third of the human LHR is involved in phospholipase C activation. Our results show that, like in cells transfected with human LHR cDNA, transfected cells with monkey LHR cDNA displayed dose-dependent increases in IP3 accumulation in response to hCG, indicating that both receptor forms are efficiently coupled to phospholipase C activation. Compared with a previous report that the concentrations required to elicit half-maximal stimulation of IP3 production were about 20- to 30-fold higher than those given 50% stimulation of cAMP (2), our present data shown that the cells expressing monkey and human LHR responded to increasing doses of hCG with similar sensitivities of cAMP and IP3 production. This difference might be due to the different methodologies and cell types used. The cells expressing monkey LHR responded to increasing doses of hCG with cAMP responses similar to those of cells transfected with human LHR, but a response to rhFSH was absent in both cells. The EC50 values for cAMP responses in monkey and human LHR-expressing cells are 40 and 32 ng/ml, respectively, compatible with previous reports (2, 35). Hence, monkey and human LHR appeared to be functionally very similar. Taken together, the cloned truncated form of monkey LHR cDNA indeed encodes a functional LHR with specific hCG binding and hCG-stimulated cAMP and IP3 production.
The absence of exon 10 in the monkey LHR cDNA is interesting, and raises the question of whether the sequences present in exon 10 are important for LHR function in other species. The three glycoprotein hormone receptors, i.e. those of LH, FSH, and TSH, are all characterized by a large extracellular ligand-binding domain with a leucine repeat structure (1). Despite different number of exons and differences in overall size, the genes for the glycoprotein hormone receptors are structurally remarkably similar. With the exception of exon 10, the other exons of the LHR share structural homology in amino acid sequence with respective structures of the FSH and TSH receptors. Hence, exon 10 might be inserted into the LHR mRNA in some, but not all, species by alternative splicing, and it may not be essential for receptor function (1, 3, 4, 5).
All three glycoprotein hormone receptors have been shown to have N-linked carbohydrates (1). Recent studies using wild-type and mutant LHR expressed in COS cells demonstrated that the N-linked carbohydrate chains, specifically those attached to the two glycosylation sites Asn173 and Asn152, are critical for the assembly of a high affinity hormone-binding site within the extracellular domain in the rat LHR (42). In contrast, the putative glycosylation sites Asn269, Asn277, and Asn291 in exon 10 did not contribute to either hormone binding or membrane transport and insertion of the receptor protein (42). However, Rajaniemi et al. (43) indicated that the N-linked carbohydrate chains do not contribute to surface expression, hormone binding, and signal transduction once the receptor has been inserted into the membrane in a functional active form. Zhang et al. (44) analyzed the cysteine residues in rat LHR and demonstrated that the functional hormone-binding domain uses all cysteines N-terminal to exon 7, and the other cysteine residues, including Cys282 (exon 10) and Cys314 (exon11) are not essential for hormone-binding activity or plasma membrane insertion.
Analysis of compound heterozygous mutations of the LHR gene in Leydig cell hypoplasia patients demonstrated that an A872G transition, resulting in an Asn291Ser substitution and abolition of a potential N-glycosylation site in the extracellular domain (exon 10) appeared to have no effect on the activity of the human LHR (20). This is in accordance with the above finding that this site is not glycosylated or that glycosylation of this site is not critical for either ligand binding or signaling (20, 42). In contrast, the deletion of exon 8 in the extracellular domain of these patients greatly reduced ligand binding and ligand-induced cAMP production in transfected cells. Hence, the polypeptide encoded by exon 8, unlike that encoded by exon 10, plays an important role in LHR expression and signal transduction (20). Our present findings indicated that exon 10 is not crucial for LHR function.
The Northern hybridization analysis of monkey testicular RNA using a cRNA probe corresponding to the cloned monkey LHR cDNA revealed multiple transcripts with approximate molecular sizes of 5.5, 4.0, 2.7, 1.9, and 1.4 kDa. The predominant bands were 5.5 and 1.4 kb in size. This result is in agreement with those reported previously by us and others in other species (1, 22). The relative size and abundance of these transcripts differ from one species to another and also from tissue to tissue. These different transcripts derive from the alternate splicing and different transcription initiation site as well as polyadenylation signals (1, 45, 46, 47, 48, 49). A recent study indicated the existence of multiple sites of splicing and polyadenylation in the rat LHR gene, resulting in generation of different transcription sizes (46). Lu et al. and Hu et al. (47, 48) also demonstrated that in the rat, a long 3'-untranslated region containing two polyadenylation domains accounts for the size difference in LHR mRNAs. In the human, the 5'-noncoding region is unusually long, and the transcription initiation site is located 1085 bp upstream of the translation start site (49).
In conclusion, we have cloned LHR cDNA from marmoset monkey testis, and the recombinant cDNA was successfully expressed in COS cells, producing receptor protein with specific high affinity hCG binding and hCG-mediated cAMP and IP3 responses. The striking feature of the monkey LHR cDNA is the lack of an 81-bp nucleotide sequence encoding exon 10 in LHR cDNAs of other mammalian species. Our data provide strong evidence, of more general interest, that exon 10 (present in other species) is not essential for LHR function.
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Footnotes |
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Received December 23, 1996.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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H. Li, B. Degenhardt, D. Tobin, Z.-x. Yao, K. Tasken, and V. Papadopoulos Identification, Localization, and Function in Steroidogenesis of PAP7: A Peripheral-Type Benzodiazepine Receptor- and PKA (RI{alpha})-Associated Protein Mol. Endocrinol., December 1, 2001; 15(12): 2211 - 2228. [Abstract] [Full Text] [PDF]
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R. S. Kumar, S. Ijiri, and J. M. Trant Molecular Biology of the Channel Catfish Gonadotropin Receptors: 2. Complementary DNA Cloning, Functional Expression, and Seasonal Gene Expression of the Follicle-Stimulating Hormone Receptor Biol Reprod, September 1, 2001; 65(3): 710 - 717. [Abstract] [Full Text] [PDF]
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J. Bogerd, M. Blomenröhr, E. Andersson, H.H.A.G.M. van der Putten, C.P. Tensen, H.F. Vischer, J.C.M. Granneman, C. Janssen-Dommerholt, H.J.Th. Goos, and R.W. Schulz Discrepancy Between Molecular Structure and Ligand Selectivity of a Testicular Follicle-Stimulating Hormone Receptor of the African Catfish (Clarias gariepinus) Biol Reprod, June 1, 2001; 64(6): 1633 - 1643. [Abstract] [Full Text]
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R. Sampath Kumar, S. Ijiri, and J. M. Trant Molecular Biology of Channel Catfish Gonadotropin Receptors: 1. Cloning of a Functional Luteinizing Hormone Receptor and Preovulatory Induction of Gene Expression Biol Reprod, March 1, 2001; 64(3): 1010 - 1018. [Abstract] [Full Text]
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P. R. Manna, T. El-Hefnawy, J. Kero, and I. T. Huhtaniemi Biphasic Action of Prolactin in the Regulation of Murine Leydig Tumor Cell Functions Endocrinology, January 1, 2001; 142(1): 308 - 318. [Abstract] [Full Text] [PDF]
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P. R. Manna, J. Kero, M. Tena-Sempere, P. Pakarinen, D. M. Stocco, and I. T. Huhtaniemi Assessment of Mechanisms of Thyroid Hormone Action in Mouse Leydig Cells: Regulation of the Steroidogenic Acute Regulatory Protein, Steroidogenesis, and Luteinizing Hormone Receptor Function Endocrinology, January 1, 2001; 142(1): 319 - 331. [Abstract] [Full Text] [PDF]
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A. P. N. Themmen and I. T. Huhtaniemi Mutations of Gonadotropins and Gonadotropin Receptors: Elucidating the Physiology and Pathophysiology of Pituitary-Gonadal Function Endocr. Rev., October 1, 2000; 21(5): 551 - 583. [Abstract] [Full Text]
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J. Gromoll, U. Eiholzer, E. Nieschlag, and M. Simoni Male Hypogonadism Caused by Homozygous Deletion of Exon 10 of the Luteinizing Hormone (LH) Receptor: Differential Action of Human Chorionic Gonadotropin and LH J. Clin. Endocrinol. Metab., June 1, 2000; 85(6): 2281 - 2286. [Abstract] [Full Text]
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M. Tena-Sempere, P. R. Manna, and I. Huhtaniemi Molecular Cloning of the Mouse Follicle-Stimulating Hormone Receptor Complementary Deoxyribonucleic Acid: Functional Expression of Alternatively Spliced Variants and Receptor Inactivation by a C566T Transition in Exon 7 of the Coding Sequence Biol Reprod, June 1, 1999; 60(6): 1515 - 1527. [Abstract] [Full Text]
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P. R. Manna, P. Pakarinen, T. El-Hefnawy, and I. T. Huhtaniemi Functional Assessment of the Calcium Messenger System in Cultured Mouse Leydig Tumor Cells: Regulation of Human Chorionic Gonadotropin-Induced Expression of the Steroidogenic Acute Regulatory Protein Endocrinology, April 1, 1999; 140(4): 1739 - 1751. [Abstract] [Full Text]
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P. R. Manna, M. Tena-Sempere, and I. T. Huhtaniemi Molecular Mechanisms of Thyroid Hormone-stimulated Steroidogenesis in Mouse Leydig Tumor Cells. INVOLVEMENT OF THE STEROIDOGENIC ACUTE REGULATORY () PROTEIN J. Biol. Chem., February 26, 1999; 274(9): 5909 - 5918. [Abstract] [Full Text] [PDF]
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P. Rodien, F. Cetani, S. Costagliola, M. Tonacchera, L. Duprez, T. Minegishi, C. Govaerts, and G. Vassart Evidences for an Allelic Variant of the Human LC/CG Receptor rather than a Gene Duplication: Functional Comparison of Wild-Type and Variant Receptors J. Clin. Endocrinol. Metab., December 1, 1998; 83(12): 4431 - 4434. [Abstract] [Full Text]
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F.-P. Zhang, T. El-Hafnawy, and I. Huhtaniemi Regulation of Luteinizing Hormone Receptor Gene Expression by Insulin-Like Growth Factor-I in an Immortalized Murine Leydig Tumor Cell Line (BLT-1) Biol Reprod, November 1, 1998; 59(5): 1116 - 1123. [Abstract] [Full Text]
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) and human (
) LHR cDNAs. D,
Displacement of [125I]hCG binding from monkey LHR by hCG
(
) and rhFSH (
