Mol. Hum. Reprod. Advance Access originally published online on July 28, 2005
Molecular Human Reproduction 2005 11(8):591-600; doi:10.1093/molehr/gah205
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Splice variants of the relaxin and INSL3 receptors reveal unanticipated molecular complexity
1Serono Research Institute, One Technology Place, Rockland, MA, USA, 2Howard Florey Institute, University of Melbourne, Victoria, Australia and 3Department of Animal and Dairy Sciences, Mississippi State University, MS, USA
4 To whom correspondence should be addressed at: Serono Research Institute, One Technology Place, Rockland, MA 02370, USA. E-mail: marco.muda{at}serono.com
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Abstract |
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LGR7 and LGR8 are G protein-coupled receptors that belong to the leucine-rich repeat-containing G-protein coupled receptor (LGR) family, including the thyroid-stimulating hormone (TSH), LH and FSH receptors. LGR7 and LGR8 stimulate cAMP production upon binding of the cognate ligands, relaxin and insulin-like peptide 3 (INSL3), respectively. We cloned several novel splice variants of both LGR7 and LGR8 and analysed the function of four variants. LGR7.1 is a truncated receptor, including only the N-terminal region of the receptor and two leucine rich repeats. In contrast, LGR7.2, LGR7.10 and LGR 8.1 all contain an intact seven transmembrane domain and most of the extracellular region, lacking only one or two exons in the ectodomain. Our analysis demonstrates that although LGR7.10 and LGR8.1 are expressed at the cell surface, LGR7.2 is predominantly retained within cells and LGR7.1 is partially secreted. mRNA expression analysis revealed that several variants are co-expressed in various tissues. None of these variants were able to stimulate cAMP production following relaxin or INSL3 treatment. Unexpectedly, we did not detect any direct specific relaxin or INSL3 binding on any of the splice variants. The large number of receptor splice variants identified suggests an unforeseen complexity in the physiology of this novel hormone-receptor system.
Key words: GPCR/INSL3/LGR7/LGR8/relaxin/splice variants
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Introduction |
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The leucine-rich repeat-containing G-protein coupled receptor (LGR) family comprises eight members in the human. The LGR family can be divided into three groups based on sequence similarity (Hsu et al., 2000
). One group consists of the glycoprotein hormone receptors, FSH receptor (FSHR), LH receptor (LHR), and thyroid-stimulating hormone receptor (TSHR). The orphan receptors LGR4, LGR5 and LGR6 form the second group whereas the third group includes LGR7 and LGR8, the recently identified relaxin and insulin-like peptide 3 (INSL3) receptors, respectively (Hsu et al., 2002; Kumagai et al., 2002).
Relaxin and INSL3 are key factors in the regulation of a variety of developmental processes related to reproduction. INSL3 is essential for the development of the gubernaculum during testis descent (Ivell and Bathgate, 2002) and recently was also demonstrated to be involved in germ cell maturation in males and females (Kawamura et al., 2004). Relaxin is essential for the development of the reproductive tract and nipple during pregnancy and is also a key regulator of collagen turnover in reproductive and nonreproductive tissues (Bathgate et al., 2003). Seven genes encoding relaxin family members are present in the human genome. The peptides encoded by these genes are relaxin-1, relaxin-2, INSL3 [also known as Leydig insulin-like peptide (LEY IL) or relaxin like factor (RLF)], the recently discovered relaxin-3 (Bathgate et al., 2002), INSL4 (also known as placentin or early placenta insulin-like (EPIL) peptide; INSL4/Placentin/EPIL) (Chassin et al., 1995; Koman et al., 1996), INSL5 (Conklin et al., 1999) and INSL6 (Hsu, 1999; Lok et al., 2000). Relaxin-3 is predominantly expressed in the brain, is unlikely to be a circulating hormone and has no actions associated with the other relaxin genes (Bathgate et al., 2003). Relaxin-1 and INSL4 orthologues are not present in non-primates (Bathgate et al., 2002), hence throughout this article relaxin refers to the single relaxin in non-primates, whereas the two human relaxin peptides will be referred to as H1 and H2 relaxin. Recently, in addition to relaxin, LGR7 has been described as a specific receptor for relaxin-3 (Sudo et al., 2003), although relaxin-3 will also interact with two receptors unrelated to LGRs, GPCR135 (Liu et al., 2003a) and GPCR142 (Liu et al., 2003b). Furthermore, INSL3 has been shown to be the selective ligand of the LGR8 receptor (Kumagai et al., 2002) and consistently mice deficient in either INSL3 or LGR8 share a similar phenotype. Indeed, studies from INSL3 and LGR8 null mice demonstrated the critical role of this receptor ligand pair in the regulation of gubernacular differentiation (Gorlov et al., 2002; Ferlin et al., 2003). No other relaxin family peptide has been shown to interact with LGR7 or LGR8 (Hsu et al., 2002; Ivell and Bathgate, 2002; Kumagai et al., 2002; Bathgate et al., 2003; Sudo et al., 2003; Lin et al., 2004). Although INSL5 has recently been demonstrated to be a specific ligand for GPCR142 (Liu et al., 2005), the receptors for INSL4 and INSL6 are unknown. Relaxin and other relaxin family peptides have been implicated in a variety of biological functions not restricted to mammalian reproduction, suggesting a more widespread role for these hormones in the regulation of diverse functions in various organs such as lung, brain, kidney and heart.
LGR7 and LGR8, like other LGR family members, are unique GPCRs because of the presence of a very large extracellular domain (ectodomain). As has been previously shown for the glycoprotein hormone receptors, relaxin binds to the ectodomain of the LGR7 receptor. Hence, studies with an ectodomain only soluble LGR7 protein (7BP) have demonstrated it will bind relaxin (Hsu et al., 2002). Furthermore, studies using chimeric LGR7/LGR8 receptors show that primary ligand binding is in the ectodomain and there is a secondary binding site in the transmembrane domains (Sudo et al., 2003). While undertaking molecular cloning of LGR family members in our laboratory, it became clear that alternative splicing is a common occurrence within the LGR family. Previous reports from other laboratories, describing LGR splice variants, support this observation. Indeed splice variants were previously reported for all the glycoprotein hormone receptors, FSHR (Kraaij et al., 1998; Tena-Sempere et al., 1999), TSHR (Graves et al., 1992), LHR (Loosfelt et al., 1989; Misrahi et al., 1996; Nakamura et al., 2004), LGR4 and LGR6 (M.Muda and D.Taylor, unpublished data). Following the finding that one LHR receptor splice variant was able to modulate the functional property of the wild-type LHR receptor (Nakamura et al., 2004), the identification of LGR7 and LGR8 splice variants was of particular interest. We report here the identification of 29 splice variants for LGR7 and three variants for LGR8. The variants can be grouped into two main classes: those that contain the transmembrane region, and those that encode only the truncated extracellular region. We have started analysing the function of some of the variants, namely LGR7.1, LGR7.2, LGR7.10 and LGR8.1.
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Materials and methods |
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Reagents and antibodies
Anti-FLAG M2 monoclonal antibody, biotinylated and peroxidase conjugated versions, was purchased from Sigma (St. Louis, MO, USA). [125I]-Labelled streptavidin and peroxidase conjugated streptavidin were purchased from Amersham Biosciences (Piscataway, NJ, USA). Ez-Link Sulfo-NHS-LC-Biotin was purchased from Pierce (Rockford, IL, USA). Quantitation of cAMP was performed using Tropix chemiluminescent immunoassay system for 96-well microplates (Applied Biosystems, Bedford, MA, USA). Porcine relaxin was purified from pregnant sow ovaries as previously described (Sherwood and O’Byrne, 1974
). Recombinant H2 relaxin was a gift from Bas Medical (San Mateo, CA, USA). Human INSL3 was synthesized as previously described (Fu et al., 2004). Porcine relaxin and recombinant H2 relaxin for some of the studies were purchased from the National Hormone and Peptide Program (Torrance, CA, USA).
Identification and cloning of full-length LGR7.1
The NCBI database was searched using the human LGR7 nucleotide sequence (accession number NM021634) as query. One EST clone was identified encoding a partial sequence that corresponded to the published human LGR7 nucleotide sequence (accession number BG611610
[GenBank]
). The EST clone was purchased from Invitrogen (Carlsbad, CA, USA), and the complete DNA sequence determined using specific sequencing primers and the BigDye version 3.1 (Applied Biosystems, Foster City, CA, USA). DNA sequence determination was done on an ABI 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). The full LGR7.1 coding sequence was obtained as a PCR product of 562 bp using the following primers: sense primer 5'-ATGACATCTGGTTCTGTCTT-3', antisense primer 5'-CTTCATTTTTCAGAGCCATC-3'. Human placenta Marathon-ready cDNA (BD Biosciences Clontech, Palo Alto, CA, USA) was used as the template. PCR was performed in a final volume of 100 µl containing 1x Expand High Fidelity buffer, 200 µM dNTPs, 250 pmoles each of cloning primers, 3.5 units of Expand High Fidelity enzyme mix (Roche, Indianapolis, IN, USA) and 2 µl of cDNA using a 9600 GeneAmp PCR System (Perkin Elmer, Torrance, CA, USA). Amplified product was cloned into dT-treated pBSK (Stratagene, La Jolla, CA, USA).
Cloning of LGR7, LGR8 and splice variants LGR7.2, LGR7.10 and LG8.1
To identify novel splice variants, we utilized a full-length nested PCR approach using cDNA prepared from various tissues. We decided to focus on the following tissue cDNAs: adrenal, ovary, testis, pituitary, placenta and prostate (Marathon-Ready cDNA, BD Biosciences Clontech). Nested sets of primer were designed corresponding to the 5'- and 3'-untranslated sequences of LGR7 based on the genomic sequence: sense primer 5'-ACCAAGAC CAAATTTTGCTC-3', nested sense primer 5'-TGCTCACTTTCATTAAT CAG-3', antisense primer 5'-TCCTCATGTCCTTGTAAAAT-3' and nested antisense primer 5'-CACAGTATTCTCTGCGAAGA-3'. Nested primers for LGR8 PCR were sense primer 5'-GCCTCAGATTGATTACAATGTTCTT TCTAC-3', nested sense primer 5'-GATTACAATGTTCTTTCTACTTCATT TCAT-3', antisense primer 5'-AGATGTCATCTTCCAAAAGCTGTCC-3' and nested antisense primer 5'-CTGTTTTAGGTAGTCCACTGAAAGTC-3'. Two sequential PCR were performed using Expand High Fidelity polymerase as suggested by the manufacturer in a final volume of 100 µl containing 1x Expand High Fidelity buffer, 200 µl dNTPs, 250 pmoles each of 5' forward and 3' reverse primers, 3.5 units of Expand High Fidelity enzyme mix and 5 µl of cDNA. To obtain specific full-length product, we performed a second PCR using identical conditions with the nested primers. Amplified products were visualized using ethidium bromide on 1% agarose gels in 1x Tris/borate/EDTA buffer (Invitrogen). PCR products were gel purified and cloned into pCR4Blunt-Topo (Invitrogen). The restriction endonuclease PstI was used to screen LGR7 clones, and SpeI and HindIII were used for screening the LGR8 clones. Following restriction digest analysis of up to 60 independent clones per tissue, DNA sequence analysis was performed to confirm novel sequences.
mRNA analysis and tissue distribution
To confirm mRNA tissue expression of LGR7 and LGR8 splice variants, we designed internal sets of primers that allowed the amplification of specific regions within the LGR7 and LGR8 coding sequence. One set of nested primers was used for the simultaneous detection of LGR8 and LGR8.1 mRNA expression. Sense and antisense primers were used for a first PCR reaction followed by a second PCR using nested sense and antisense primers, listed in Table I. Similarly, for LGR7 and its variants three nested sets of primer were used to allow detection of the regions spanning the novel exons splicing found in LGR7.1, LGR7.2 and LGR7.10. The sets of nested primers used allowed the simultaneous detection of LGR7 canonical sequence as well as the alternative splice variants, primers are listed in Table I. PCR conditions were as described above, and following the first round 1 µl of the first PCR reaction was used for the nested PCR and were run simultaneously using identical conditions. Following amplification, products were separated using 4% NuSieve 3:1 agarose gels (Cambrex Bio Science, Rockland, ME, USA) and detected using ethidium bromide.
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Expression plasmids, cell culture, transfection and peptide treatment
For cellular expression, LGR7, LGR8 and their variants were subcloned into the pMT expression vector (Muda et al., 1996). LGR7, LGR8 and variants were tagged via an N-terminal FLAG epitope tag as previously described (Hsu et al., 2002). All receptor constructs were sequence verified.
HEK 293T cells were grown under 5% CO2 in Dulbecco’s modified Eagles medium (DMEM) 10% FBS supplemented with 1x penicillin/streptomycin (Invitrogen). The day before transfection cells were seeded at 2 x 106 cells per 100 mm Petri dishes with 10 ml of growth media. The next day, cells were transfected with plasmid DNA using Fugene (Roche) according to the manufacturer’s instructions. In all transfections constant amounts of plasmid DNA were used by complementing when needed to 10 µg with pBSK. After 48–72 h cells were lifted using Dulbecco’s phosphate buffered saline (PBS) 2.7 mM KCl, 1.4 mM KH2PO4, 4.3 mM NaH2PO4, 137 mM NaCl, containing 5 mM EDTA and 5 mM EGTA. Cells were then collected by centrifugation, at 1000 rpm for 4 min, resuspended, counted and plated into 96-well plates at 3 x 104 cells per well in 50 µl assay media (DMEM no phenol red, 0.5 mM 3-isobutyl-1-methyl xanthine (IBMX, Sigma) 0.1% bovine serum albumin (BSA, Fraction V, Sigma), 1x penicillin/streptomycin (Invitrogen) and incubated at 37°C for 1 h. Dilutions of porcine relaxin, H2 relaxin or human INSL3 were then added to each well and incubated for 12–16 h at 37 C. Following stimulation cells were lysed, and total cAMP was measured.
Receptor surface expression FLAG detection
Twenty-four hours after Fugene transfection cells were lifted, as described above, and re-suspended in DMEM 10% FBS media at 1 x 106 cells per ml. Two millilitres of cell suspensions were transferred into six-well poly-l-lysine-coated plates (Biocoat, Becton Dickinson, Palo Alto, CA, USA). Plates were then incubated at 37°C overnight to allow cells to attach. Cells were washed twice with 1 ml PBS before incubating with 1 ml of biotinylated M2 antibody at 5 µg/ml in PBS 5% BSA at room temperature for 30 min with agitation. Cell monolayers were washed three times with 2 ml PBS, and 400,000 counts per minute (cpm) of [125I]-labelled streptavidin were added in PBS 5% BSA. After 30 min of incubation with agitation at room temperature, plates were washed three times with PBS. Antibody complexes were recovered using 1 M NaOH, and samples were counted with an auto-gamma counter Cobra II (Packard, Meriden, CT, USA). Triplicate wells for each splice variant were tested in parallel to LGR7 and/or LGR8 and empty vector in each assay and tested at least three times.
Biotinylation of cell surface proteins
Biotinylation of cell-surface forms of LGRs was performed using the protocol described previously (Bai et al., 1998). 293 T cells were transfected as described above, 48 h after transfection cells were rinsed with PBS and incubated with Sulfo-NHS-LC-Biotin (Pierce) 1 mM in PBS. After quenching with 1 ml Tris, 0.5 M, pH 7.5 at room temperature, cells were lysed at 4°C in 1 ml lysis buffer [10 mM Tris pH 7.4, 150 mM NaCl, 2 mM EDTA, 1 mM EGTA, 1% Triton X-100, 0.5% NP40, Protease Inhibitors Mix (Roche), Iodoacetamide 100 mM]. Insoluble material was removed by centrifuging at 15,000 r.p.m. for 15 min at 4°C. FLAG-tagged receptors were immunoprecipitated from solubilized lysates using 60 µl of M2 agarose beads (50% suspension equilibrated in lysis buffer). FLAG-tagged receptors were eluted from the beads in 100 µl of 2x sodium dodecyl sulphate (SDS) sample buffer with 100 mM dithiothreitol (DTT) by incubating at 60°C for 30 min.
Eluted samples were separated on 7 or 12% (LGR7.1) SDS-polyacrylamide gels and transferred to PVDF membranes. Blotted membranes were blocked with 5% non fat milk in 1x Tris-buffered saline (TBS) 0.1% Tween-20 and then incubated with streptavidin horse-radish peroxidase (HRP) conjugated for 1 h at room temperature (1:5000) (5% milk in 1x TBS 0.1% Tween-20) to identify the biotinylated form of the receptors. In parallel, identical blots were incubated with anti-FLAG M2 HRP conjugated (diluted 1:1000) for 1 h at room temperature with 5% milk in 1x TBS/0.1% Tween-20 to reveal total immunoprecipitated FLAG-tagged receptors. Following five washes using 1x TBS/0.1% Tween-20, membranes were developed using an enhanced chemiluminescence (ECL) detection system following the manufacturer’s instructions (Amersham Biosciences).
Binding assays
pMT constructs encoding LGR7, LGR8 and LGR7/LGR8 splice variants were transfected into HEK 293T cells in 75 cm flasks as previously described (Sudo et al., 2003). After 24 h the cells were lifted in EDTA phosphate solution and then plated in 24- or 6-well poly-L-lysine coated plates for relaxin and INSL3 binding or cell surface binding assays, respectively. Assays were performed in parallel 24 h later. Relaxin binding studies were performed as previously described (Sudo et al., 2003) using two concentrations (100 pM and 500 pM) of [33P]-labelled H2 relaxin. [125I]-Labelled human INSL3 was obtained from Perkin-Elmer (Boston, MA, USA), and binding studies were performed using a concentration of 100 pM of radioligand. Specific binding was determined in the presence and absence of an excess of H2 relaxin or human INSL3 (1 µM). Cell surface binding was tested in parallel for each transfected construct in six-well plates as described above. Splice variants were tested in parallel to LGR7 and/or LGR8 and empty vector transfected cells in triplicate wells within each assay. Each variant was tested in at least three separate assays. Specific binding data (total binding minus nonspecific binding) have been pooled from the individual experiments and plotted as mean ± SEM cpm.
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Results |
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Using human LGR7 and LGR8 nucleotide sequences (accession number NM021634 and accession number NM130806) as queries, we searched the NCBI database and identified one EST clone encoding a partial nucleotide sequence with some identity to the LGR7 sequence. We obtained the corresponding EST clone (accession number BG611610 [GenBank] ) and completed its sequence. This analysis revealed that this partial cDNA sequence represents a bona fide novel alternative spliced mRNA of LGR7. Indeed, this cDNA clone encodes two novel exons and a characteristic poly-A tail preceded by a canonical upstream poly-A signal sequence, AAUAAA. As the coding sequence in the EST clone lacked the start codon, we completed its sequence using PCR and cDNAs from human placenta. Assembly of the LGR7.1 coding region with the 3' sequence from the EST clone sequence generated a cDNA sequence of 1775 bp. Using the software program SIM4 (Florea et al., 1998
), we aligned and compared the LGR7 and LGR7.1 sequences with the human genomic sequence, Celera Release 27. Of the 18 exons encompassing the coding sequence of LGR7 (Figure 1A), exon 1 encodes most of the putative signal sequence, exon 2 encodes the low-density lipoprotein class A (LDLa) module, and a short connecting sequence (exon 3) precedes the cysteine rich region flanking the leucine rich repeats (LRRs). The 10 LRRs, present in the extracellular domain, span exons 5–14 and the seven transmembrane domain is encoded by the last four exons, 15–18 (Figure 1A). In contrast, LGR7.1 is encoded by 17 exons, which are identical from exon 1 to exon 6 to LGR7 then a novel exon, named 6.A, is used. This exon introduces a short amino acid sequence, SRAVKDGSEK, and a stop codon, suggesting that LGR7.1 encodes a protein of 190 amino acids characterized by a N-terminal portion of the extracellular domain of LGR7 (Figure 1A and B). After exon 6.A, LGR7.1 sequence is identical to LGR7 from exons 7–15 (Figure 1A), then a second novel exon, named 15.A encoding an alternative poly-A signal sequence is used (Figure 1B). Following addition of a poly-A tail LGR7.1 is predicted to be expressed as a mRNA of approximately 1.9 Kb. Importantly, and consistent with this, previously reported northern blot analysis of LGR7 mRNA expression revealed two main bands, one of which is consistent with the molecular size predicted for LGR7.1 mRNA (Hsu et al., 2000).
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We then decided to verify if other variants were expressed in human tissues. Using commercially available human cDNAs as templates, we have identified and cloned 29 alternative LGR7 variants and three alternative LGR8 variants (Tables II and III). Remarkably, in our analysis, splice variants of LGR7 and LGR8 were more represented than the canonical LGR7 and LGR8 sequences.
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Splice variants were named by a numerical suffix to the original gene in the order of isolation. Similar to LGR7.1 analysis, each of the novel identified sequence variants was analysed using the SIM4 program by comparing it with the human genomic sequence. Two LGR7 splice variants and one LGR8 splice variant, named LGR7.2, LGR7.10 and LGR8.1, were chosen for further studies.
LGR7.2 was cloned twice from ovary and prostate cDNAs and its sequence is identical to LGR7 except that it lacks two LRRs corresponding to exons 12 and 13 close to the seven transmembrane domain which starts at exon 15 (Figure 2A). In addition to this deletion, because of the novel exon junction, LGR7.2 contains a single amino acid substitution, phenylalanine in place of leucine (Figure 2B). Subsequent analysis revealed that LGR7.2 mRNA lacking exons 12 and 13 might be broadly expressed (see below). Consistent with this, the commercially available clone for LGR7, from a full-length cDNA library collection (clone number AB3603-B02, Origene, Rockville, MD, USA), when fully sequenced was found to correspond to LGR7.2.
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LGR7.10 was also cloned from ovary cDNA, but in contrast to LGR7.2 lacks only one exon, exon 3 (Figure 2A). Although LGR7.10 has been described previously (Hsu et al., 2000
), no characterization other than its inability to increase cAMP production in response to relaxin was reported (Hsu et al., 2002).
We cloned LGR8.1 from uterine cDNA, and remarkably 38 of 60 clones isolated from this tissue contained LGR8.1 rather than LGR8. No LGR8.1 was found in 58 clones obtained from adrenal gland (Table III). Consistent with this, LGR8.1 mRNA expression was detected in the uterus but not in seven other tissues examined (see below). The LGR8.1 form is identical in sequence to LGR8 with the exception of one missing LRR encoded by exon 11 (Figure 2C). In contrast to LGR7.2 and LGR7.10, LGR8.1 has no amino acid substitution because of the alternative splicing event (Figure 2D).
To further confirm and validate the expression of the different splice forms, we used a second PCR-based analysis using primers that were designed to simultaneously amplify the variant form and the canonical sequences. Using this method, we confirmed the expression of all the alternative exon structures previously identified by full-length cloning (Figure 3). In our analysis, using primer sets that span three separate regions of the coding sequence, we detected a band corresponding to canonical LGR7 sequence in seven of eight tissues. LGR7.1 and LGR7.2 mRNA were broadly expressed and detected in seven of eight tissues examined, whereas LGR7.10 was detected only in four of the tissues analysed (Figure 3). Finally, in contrast to LGR7 and its variants, mRNA expression of LGR8 and LGR8.1 were more restricted and detected in only two tissues (Figure 3). In conclusion, our analysis demonstrated the expression of distinct splice variants and that several splice forms are typically expressed in the same tissue.
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Previous studies on the LGR7 signalling mechanism revealed that a single amino acid substitution mimicked the naturally occurring gain-of-function mutation of the LH receptor which leads to constitutive cAMP production (Hsu et al., 2000). Subsequent studies showed that cells expressing LGR7 increase cAMP production in response to relaxin or H3 relaxin stimulation (Sudo et al., 2003). Similarly, LGR8 was recently shown to mediate cAMP production following relaxin or INSL3 stimulation (Kumagai et al., 2002). Therefore, the interaction of relaxin peptides with LGR7 or LGR8 induces a conformational change that results in the activation of cAMP synthesis, most likely through the Gs type of heterotrimeric G-proteins (Hsu et al., 2002). In early studies, a gain-of-function mutant of LGR7.10 displayed constitutive activity (Hsu et al., 2000), in contrast to LGR7 wild type. Later work, consistent with our results found that LGR7.10 did not respond to relaxin stimulation (Hsu et al., 2002). To better understand these apparently contradictory results, we reanalysed the responses to relaxin and INSL3 of LGR7.10 and the other splice variants as well as their membrane localization. In our hands, consistent with previous results, porcine or recombinant H2 relaxin were both active on LGR7 and LGR8 receptors (Figure 4a), whereas they did not activate LGR7.10. Surprisingly, but similar to LGR7.10 none of the variants identified was able to increase either basal cAMP synthesis or induce cAMP production following relaxin stimulation (Figure 4a). Furthermore, although the LGR8 transfected cells responded to INSL3 stimulation, none of the splice variants responded to INSL3 (Figure 5a).
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We then monitored the binding of [33P]-labelled H2 relaxin and [125I]-labelled INSL3 to LGR7, LGR8 and splice variants using transfected 293 T cells. Consistent with previous reports, our analysis revealed that both LGR7 and LGR8 bound [33P]-labelled H2 relaxin with affinity in the low nanomolar range, Kd 
0.1 nM and 1 nM, respectively. We then tested the splice variants for specific binding using two concentrations of [33P]-labelled H2 relaxin. Unexpectedly, our analysis demonstrated that none of the splice variants tested showed specific binding for [33P]-labelled H2 relaxin, at both concentrations tested (Figure 4b).
We next tested INSL3, the proposed physiological ligand of LGR8. Consistent with previous results (Kumagai et al., 2002; Sudo et al., 2003), [125I]-labelled INSL3 bound LGR8 with high affinity (Kd 0.1 nM) whereas it did not bind to LGR7 (Figure 5b). Similar to relaxin, neither LGR8.1 nor any of the other splice variants, demonstrated any specific [125I]-labelled INSL3 binding (Figure 5b). As both results, lack of cAMP response and lack of binding, could have been explained by receptors that do not have proper cell surface expression, we analysed their cell surface localization.
We decided to monitor the molecular composition and localization of the various variants using two methods: first, intact cells were incubated with FLAG biotinylated-antibody followed by [125I]-labelled streptavidin, and second cellular localization was determined by cell-surface protein biotinylation reaction, followed by immuno purification and streptavidin detection. Using anti-FLAG antibodies on intact cells (see Materials and methods) followed by [125I]-labelled secondary antibody, we did not measure notable surface expression of either LGR7.1 or LGR7.2. In contrast, LGR7, LGR8 and also LGR7.10 and LGR8.1 splice variants were detected at the cell surface although with distinct levels of expression (Figure 6).
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Analysis of cell surface expression using a biotinylation assay demonstrated similar results. Cell surface localized LGR7 and LGR7.10 were detected as biotinylated proteins migrating between 85 and 95 kDa, whereas LGR8 and LGR8.1 were detected in the region of 80–90 kDa (Figure 7A and C). Consistent with the lack of detection on the cell surface using antibodies, LGR7.2 did not highlight as a specific band in the biotinylation assay, indicating that this variant receptor form is mostly retained within the cell (Figure 7A). Finally, LGR7.1 was not biotinylated at the cell surface but a protein of approximately 25 kDa, most likely an immature form, was detected as an intracellular protein (Figure 7B). Somewhat consistent with this hypothesis, cell culture supernatant from cells expressing FLAG-tagged LGR7.1 displayed low levels of a soluble secreted protein around 25–30 kDa, but only when using a very strong promoter (data not shown). Owing to the low level of secretion, the significance of the soluble secreted form is still unclear. On the other hand, LGR7.10 and LGR8.1 were both detected as biotinylated bands, indicating that these variants were present on the cell surface. In conclusion, both methods indicate that although some variants, for example LGR7.2, are mostly retained within the cell, other splice variants, like LGR7.10 or LGR8.1 are partially or efficiently expressed at the surface.
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We started dissecting the function of the first cloned variants LGR7.1, LGR7.2 and LGR8.1 and included LGR7.10 to confirm and complete previous results (Hsu et al., 2002
). The analysis of the exon–intron structure of LGR7.1 revealed 17 exons, two of which were novel. LGR7.1 mRNA size is consistent with a band of approximately 1.9 Kb previously detected by Northern analysis (Hsu et al., 2000). The LGR7.2 sequence is identical to LGR7 except that it lacks two LRRs corresponding to exons 12 and 13, whereas LGR7.10 lacks only exon 3. LGR8.1 lacks one LRR, exon 11, and appears to be more represented than LGR8 in uterine mRNA. In contrast to LGR7 and LGR8, intact cells expressing the splice variants described in this report did not display binding of relaxin and INSL3 or activate the cAMP signalling pathway in response to both peptides. Using two distinct methods, we demonstrated that two variants, LGR7.2 and LGR7.1, are largely retained inside the cell. In contrast, LGR7.10 was detected at the cell membrane, but at a low level, and consistent with our results it was previously shown to be unresponsive to relaxin (Hsu et al., 2002). In spite of its membrane localization, LGR7.10 did not bind relaxin, suggesting a dual role for exon 3, efficient cell-surface membrane localization and relaxin binding. The primary relaxin binding sites in LGR7 have recently been demonstrated to be within the LRR repeats 4, 5, 6 and 8; hence, it is unlikely that the region coded by exon 3 is necessary for direct binding. However, deletion of exon 3 might remove an important structural element essential for the overall ectodomain structure. Consistent with this, the low cell surface expression observed in our experiments may be explained by inefficient receptor processing because of a partial loss of native structure. Further studies are necessary to refine the role of the region encoded by exon 3 within LGR7 and LGR7.10.
Similar to the LGR7 variants, LGR8.1 was unable to bind relaxin or INSL3 and consequently stimulate cAMP production in response to both peptides. However, loss of exon 11 in LGR8.1, in contrast to the LGR7 variants, does not alter cell surface expression. This suggests that no major structural change takes place in the ectodomain of the LGR8.1 receptor. This observation, together with lack of binding, points to an essential role for the LRR encoded by exon 11 for the interaction with relaxin and INSL3. However, our results are preliminary, and it is still a matter of some speculation whether residues within the exon 11 are responsible for the direct interaction with relaxin and INSL3. Exon 11 encodes LRR7, and this region was not shown to be necessary for relaxin binding (Bullesbach and Schwabe, 2005). It is possible that loss of exon 11 compromises the complex interaction between three binding residues in relaxin and LRR 4, 5, 6 and 8. In contrast to LGR7.10 and LGR8.1, lack of relaxin and INSL3 binding on cells expressing LGR7.1 and LGR7.2 was explained by the lack of cell surface expression of these two variants. Interestingly, a recent report (Nakamura et al., 2004) acknowledged that a natural LH receptor splice variant lacking exon 9 is retained inside the cell and exerts its function by modulating the cell surface functional expression of the full-length LH receptor. It will be interesting to test whether in a similar manner LGR7.2 affects the processing of the full-length LGR7 form. Finally, defining the role of LGR7.1 was difficult in part due to the renowned problematic expression in heterologous systems of the truncated (ectodomain only) versions of LGRs. Indeed, in our hands, only a small fraction of total LGR7.1 protein expressed was secreted in the media, and detection of LGR7.1 by western analysis in conditioned media was dependent on the use of a very strong promoter element (data not shown). In spite of a lack of strong experimental evidence, we still think that LGR7.1 may represent a bona fide secreted version of LGR7, and future analysis using different expression systems might be able to tackle this issue.
In conclusion, our work established that several splice variants of LGR7 and LGR8 are expressed in human tissues. It is tempting to speculate that some of the splice variants identified might affect LGR7, LGR8 as well as each other’s function via direct intramolecular interactions as was reported for LHR (Nakamura et al., 2004). Moreover, the LH receptor itself was shown to self-associate (Tao et al., 2004) and, given the structural similarity among LGRs receptors, we put forward the hypothesis that some of the variants identified may have similar functions. The large number of variants identified drastically underscores the importance of alternative splicing in the physiology of the LGR family of G-protein coupled receptors.
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The authors thank Yieh Ping Wan (Perkin-Elmer life and analytical sciences) for provision of 125I-labelled INSL3, Dr John Wade (HFI, Australia) for provision of human INSL3, BAS Medical for provision of H2 relaxin used at HFI, Dr Xuliang Jiang for comments and helpful discussion, Dr Sean McKenna (SRI, USA) and Prof. Geoffrey Tregear (HFI, Australia) for support and encouragement. The sequence data have been submitted to the GenBank under accession numbers AY899848 [GenBank] , AY899849 [GenBank] , AY899850 [GenBank] , AY899851 [GenBank] . Research at the Howard Florey Institute was supported by a National Health and Medical Research (NHMRC) Grant to R.A.D.B., Application ID 300012.
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