The effect of splice variant of the human luteinizing hormone (LH) receptor on the expression of gonadotropin receptor

Molecular and Cellular Endocrinology 260–262 (2007) 117–125

The effect of splice variant of the human luteinizing hormone (LH) receptor on the expression of gonadotropin receptor Takashi Minegishi a,b,∗ , Kazuto Nakamura a,b , Soichi Yamashita a , Yuki Omori a a

Department of Obstetrics and Gynecology, Graduate School of Medicine, Gunma University, Maebashi, Gunma, Japan b Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology, Tokyo, Japan Received 8 August 2005; accepted 27 November 2005

Abstract A splice variant of human lutropin/choriogonadotropin-receptor [hLHR (exon 9)] that lacks exon 9 was previously cloned in the corpus luteum of a woman with a regular menstrual-cycle. Supported by detergent soluble binding assay and receptor biotinylation experiment, the receptor binding assay shows hLHR (exon 9) is neither expressed at the cell surface nor have the capability of binding to hCG. Interactions between hLHR (exon 9) with the immature bands of gonadotropin receptors not with the mature bands were seen. This phenomenon is specific among gonadotropin receptors since human thyrotropin-receptor (hTSHR) failed to be coimmunoprecipitated. Furthermore, this receptor complex attenuated the receptor protein level within the cells. To elucidate the mechanism underlying the decrease in receptor protein by this receptor complex, we performed a Percoll-fractionation experiment, which indicated the receptor complex drove hLHR to the lysosome instead of the plasma-membrane. Moreover, the expression of hLHR (exon 9) mRNA was seen at all phases of the menstrual cycle and relatively increased as the luteal phase progressed. These results reveal a novel mechanism of regulation for gonadotropin receptor expression. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Human ovary; LH receptor; FSH receptor; Splice variant

1. Introduction All GPCRs share a common serpentine structure consisting of seven transmembrane helices linked by three alternating intracellular and extracellular loops. The extracellular regions are involved in ligand binding, while the intracellular regions are primarily involved in signaling. The lutropin/choriogonadotropinreceptor (LHR), along with the receptors for follitropin-receptor (FSHR) and thyrotropin-receptor (TSHR), form a subfamily of the superfamily of GPCRs (Segaloff and Ascoli, 1993; Dufau, 1998), distinguished by their large extracellular N-terminal domain encoding almost half size of the receptor. When we cloned hFSHR (Minegishi et al., 1991) and hLHR (Minegishi et al., 1990), we also cloned a splice variant of hLHR, from the corpus luteum of a woman with a regular menstrual cycle, lacking exon 9 coding 62 amino acids [hLHR (exon 9)]. The

∗ Corresponding author at: Department of Obstetrics and Gynecology, Graduate School of Medicine, Gunma University, 3-39-22 Showa-machi, Maebashi, Gunma 371-8511, Japan. Tel.: +81 27 220 8420; fax: +81 27 220 8443. E-mail address: [email protected] (T. Minegishi).

0303-7207/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2005.11.051

physiological function of hLHR (exon 9) in the human ovary is yet unknown. In addition, two additional deletion mutants within the N-terminal extracellular domain of hLHR (i.e., exons 8 and 10) were found (Laue et al., 1996; Gromoll et al., 2000). In both cases, these naturally occurring mutations cause Leydig cell hypoplasia, resulting in complete or partial feminization of the external genitalia. Moreover, we detected three transcripts (5.4, 3.6, and 2.4 kb) for hLHR mRNA in the human ovary by Northern blot analysis, showing both wild-type hLHR and hLHR (exon 9) are generated by alternative splicing (Minegishi et al., 1997a). These data suggest that hLHR (exon 9) may have a specific function in the ovary. Since reports demonstrate that many GPCRs exist as dimers or oligomers, dimerization or oligomerization of GPCRs is important for receptor function, including agonist affinity, potency, and efficacy and G protein specificity (Bai, 2004). The recent report describing that FSH and FSHR complexes form dimers in the crystal graphy (Fan and Hendrickson, 2005), expanding the possibility of receptor dimers being an important factor for gonadotropin receptors. Furthermore, the discovery of heterodimerization of GPCRs complicates the understanding of the general mechanism of GPCR modulation and function.

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For instance, the heterodimeric ␬-␦ opioid receptor synergistically bind receptor selective ligands with higher binding affinity (Jordan and Devi, 1999). In the case of ␤-adrenergic receptor (␤AR), the heterodimerization of ␤1 AR and ␤2 AR inhibits ␤2 AR-dependent activation of the ERK1/2 MAPK signalling pathway (Lavoie et al., 2002). In addition, heterodimerization of type 1 somatostatin receptor (SSTR1) and SSTR5 reduces the internalization of SSTR5 (Rocheville et al., 2000). We examined whether gonadotropin receptors, hLHR and hFSHR, form an association with hLHR (exon 9) by coimmunoprecipitation. Our study clearly highlights the importance of hLHR (exon 9) expression in regulating gonadotropin receptor function in the human ovary. 2. Materials and methods 2.1. Hormones and reagents Purified hCG (CR-129) was kindly supplied by Dr. A. Parlow and the National Hormone and Pituitary Program (National Institute of Diabetes and Digestive and Kidney Disease, National Institutes of Health). Anti-cAMP serum was donated by Dr. Takashi Matozaki (Biosignal Research Center Institute for Molecular and Cellular Regulation, Gunma University, Japan). [125 I]Sodium was purchased from Amersham Pharmacia Biotech (Arlington Heights, IL, USA).

2.2. Construction of hLHR, hLHR (exon 9) and hFSHR expression vectors cDNAs encoding hLHR and hLHR (exon 9) were inserted separately into the mammalian expression vector pcDNA3.1, hFSHR and hTSHR (Invitrogen, San Diego, CA, USA). The receptors were tagged with c-myc for wild-type human FSH receptor (Myc-hFSHR) and wild-type human LH receptor (MychLHR) or flag epitopes for wild-type LH receptor (FLAG-hLHR), hLHR (exon 9) [FLAG-hLHR (exon 9)] and hTSHR (FLAG-hTSHR). The receptor cDNAs were modified using PCR to insert after signal peptide, a 10-residue c-myc epitope (EQKLISEEDL) for the Myc-hLHR, and an eight-residue flag epitope (DYKDDDDK) for the FLAG-hLHR and FLAG-hLHR (exon 9). Their identity was verified by sequencing on both strands.

2.3. Expression in mammalian cells Human embryonic kidney (293) cells were obtained from American Type Culture Collection (Manassas, VA, USA) and maintained as monolayer cultures at 37 ◦ C in MEM (DMEM) supplemented with 10% newborn calf serum and antibiotics. Two hundred and ninety-three cells were transiently transfected with pcDNA3.1 vectors using the Ca2+ phosphate method (Chen and Okayama, 1987). An equal amount of pcDNA3.1 vector was cotransfected with each receptor construct so that the total amount of DNA used was consistent with studies involving transfections with two constructs. We checked the transfection efficiency with these receptor constructs, which was varied between 40 and approximately 50%. For each experiment, the transfected 293 cells were used 2 days after transfection. To establish cell lines stably, 293 cells were plated into 100-mm dishes and transfected with either Myc-hLHR or FLAGhLHR (exon 9) or with both constructs. Cells were selected in media containing 700 ␮g/ml G418. Stable cell lines were maintained in the media containing G418.

2.4. RNA extraction, RT-PCR and quantitative RT-PCR Human ovaries were removed from patients who had undergone salpingooophorectomy for gynecological diseases. Informed consent was obtained from all human subjects, and this study was approved by the Gunma University School Institutional Review Board. The menstrual history and basal body temperature

record were used to determine the date of menstrual cycle. After whole ovaries were removed, leading follicles (10 and 14 days follicle) and corpora lutea (21 and 27 days corpus luteum) were isolated and stored in liquid nitrogen until use. For PCR amplification, 5 ␮g of RNA was used to generate first strand cDNA using a cDNA synthesis kit (SuperScript First-Strand Synthesis System for RT-PCR, Invitrogen) following the manufacturer’s instructions. The entire 2 ␮l cDNA synthesis reaction volume was combined in a 100 ␮l final reaction volume for PCR amplification containing 0.25 ␮M of each oligonucleotide primer and 1.5 IU Taq DNA polymerase (SIGMA). The primer sequences were: 5 -TTCGGATCC-TACATCTGGAGAAGATGCACAATG (LHR-S1); 3 -TCGAGAATTC-AGGTGAATAGCATAGGTGATGGTG (LHR-AS2) for hLHR; 5 -CTCACCAAGCTTCGAGTCATCCAA (hFSHR-exon 1-F); 3 -TCAAATCCTCTGCTGTAGCTGGAC (hFSHR-exon 10-R) for hFSHR; 5 -CCAAGGTCATCCATGACAACT (GAPDH-F); 3 -CACCCTGTTGCTGTAGCCAAA (GAPDH-R) for GAPDH. In all, 30 cycles of PCR amplification were performed using a DNA thermal cycler. Each cycle consisted of 90 s denaturation at 95 ◦ C, 150 s annealing at 62 ◦ C, and 150 s at 70 ◦ C for enzymatic extension. After DNA amplification the PCR mixture, amplified DNA fragments were recovered from agarose gels by QIAquick Gel Extraction Kit (Qiagen, Valencia, CA, USA). After precipitated with ethanol, DNA fragments were subcloned into pGEM-T Easy Vector Systems (Promega Corp., Madison, WI, USA). Real-time quantitative PCR was carried out using the ABI Sequence Detection System (Applied Biosystems, Foster City, CA, USA). QuantiTect SYBR Green PCR Master Mix (QIAGEN) was used for all reactions in the ABI Sequence Detection System and conditions were optimized for the real-time PCR as recommended by the manufacturer. The primers sequences were: 5 -ACACTTTATTCTTCCATGCTTGCTGAG (hLHR-exons 10–11F); 3 -ATTAAAAGCATCTGGTTCAGGAGCACA (hLHR-exons 10–11R) to amplify the region between exons 10 and 11 of hLHR; 5 -TGCTGTGCTTTTAGAAACTTGCCAACA (hLHR-exons 9–10F); 3 -CCTTACTGTGCTTTCACATTGTTTGGA (hLHR-exons 9–10R) to amplify the region between exons 9 and of hLHR, and 5 -CTCACCAAGCTTCGAGTCATCCAA (hFSHR-exon 1-F); 3 -AAGGTTGGAGAACACTCTGCCTCT (hFSHR-exon 3-R) to amplify the region between exons 1 and 3 of hFSHR. The assay analysis was carried out using the ABI PRISM 7000 SDS-software, and was expressed as copy numbers determined from standard curves run in each assay. The expression at exons 10 and 11 region of hLHR indicates the sum expression of both full-length hLHR and hLHR (exon 9). On the other hand, the expression at exons 9 and 10 region of hLHR indicates the expression of the full-length hLHR. Therefore, the expression of hLHR (exon 9) is expressed as the difference between exons 10 and 11 region and exons 9 and 10 region of hLHR.

2.5. SDS-PAGE, Western blotting, and immunoprecipitation Cells were lysed in a solution (lysis buffer) containing 0.5% Nonidet P-40, 200 mM NaCl, 20 mM HEPES, 1 mM EDTA, pH 7.4, during a 30-min incubation at 4 ◦ C. The lysates were clarified by centrifugation (100,000 × g for 30 min), and the amount of protein present in the supernatants was measured using the DC protein assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The lysates (500 ␮g) were immunoprecipitated with the agarose-conjugated antic-myc antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) or the agarose-conjugated anti-flag M2 antibody (Sigma Chemical Co., St. Louis, MO, USA) overnight. After extensive washing, the immunoprecipitation complex was eluted by vigorous mixing of the agarose in sodium dodecyl sulfate sample buffer for 15 min at room temperature. The eluant was then resolved on sodium dodecyl sulfate gels and electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes as described elsewhere (Quintana et al., 1993). After blocking, expression of the different proteins was determined with the HRP-conjugated anti-myc antibody (Santa Cruz Biotechnology) or the HRP-conjugated anti-flag M2 antibody (Sigma), and the proteins were finally visualized using enhanced chemiluminescence (ECL Plus) (Amersham Pharmacia Biotech).

T. Minegishi et al. / Molecular and Cellular Endocrinology 260–262 (2007) 117–125 Table 1 hFSHR, hLHR, and hLHR (exon 9) mRNA levels during the menstrual cycle Menstural cycle

hFSHR hLHR hLHR (exon 9)

10 days

14 days

21 days

27 days

6000 ± 480 5500 ± 510 1900 ± 400

8000 ± 530 8400 ± 790 5000 ± 460

3000 ± 320 21,000 ± 1400 22,000 ± 1600

520 ± 62 6300 ± 570 8600 ± 780

Amounts of hFSHR, hLHR, and hLHR (exon 9) mRNA were determined by quantitative real-time RT-PCR as described in Section 2. Each number represents the means ± S.E.M of three independent experiments. Each mRNA level is shown in copies per 100 ng of RNA.

2.6. Endo H and PNGase F treatment of Myc-hLHR and FLAG-hLHR (exon 9) Two hundred and ninety-three cells expressing either Myc-hLHR or FLAGhLHR (exon 9) were solubilized as described for Western blotting. Detergentsoluble extracts (500 ␮g) of cells expressing either Myc-hLHR or FLAG-hLHR (exon 9) were incubated in the presence or absence of Endo H (Roche Clinical Laboratories, Indianapolis, IN, USA) or PNGase F (Roche). Endo H treatment before immunoprecipitation was performed by incubation of the detergentsolubilized cell extracts in 750 ␮l of lysis buffer for 15 h at 37 ◦ C with 300 mU/ml Endo H. Detergent-solubilized cell extracts digested with PNGase F in 750 ␮l of lysis buffer before immunoprecipitation were incubated for 15 h at 37 C with 32 U/ml PNGase F. After digestion, detergent-solubilized cell extracts were further treated, followed by immunoprecipitation, SDS-PAGE, and Western blotting as described in SDS-PAGE, Western Blotting, and Immunoprecipitation.

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3-isobutyl-1-methylxanthine (Sigma). The different parameters that describe the concentration–response curves were calculated as described elsewhere (Hipkin et al., 1995).

2.10. Percoll gradient method Transfected cells were washed twice with cold homogenization buffer (0.25 M sucrose, 10 mM HEPES, 1 mM EDTA, pH 7.4), scraped into a small volume of the same buffer, and collected by centrifugation at 4 ◦ C. The cells were then resuspended in cold homogenization buffer, and they were lysed by forcing them through a 21-gauge needle 10 times. Postnuclear supernatants were prepared by centrifuging the homogenates at 800 × g for 10 min at 4 ◦ C. The supernatants were saved, and the pellets were rehomogenized and centrifuged again. The two supernatants were combined, and a 2-ml aliquot was thoroughly mixed with 8 ml of a Percoll solution (with a density of 1.047 g/ml) prepared in homogenization buffer. These mixtures were then centrifuged at 33,000 × g for 20 min at 4 ◦ C. The contents of the gradients were then collected from the top (500 ␮l/fraction), and aliquots of each of the four fractions were combined to assay ␤-hexosaminidase activity used as a marker for lysosomes (Hall et al., 1978) and perform immunoprecipitations for Western blotting to detect MychLHR and FLAG-hLHR (exon 9) as described in SDS-PAGE, Western Blotting, and Immunoprecipitation.

2.11. Other methods Statistical analysis (t test with two-sided P values) was performed using StatFlex (Artech Inc., Osaka, Japan).

3. Results 2.7. Biotinylation of receptor Transfected cells were washed four times with ice-cold PBS (10 mM sodium phosphate; 150 mM NaCl, pH 8) and then biotinylated during a 30-min incubation with freshly prepared 0.5 mg/ml solutions of sulfosuccinimidyl-6(biotinamid) hexanoate (Pierce Chemical Co., Rockford, IL, USA) in PBS. The cells were lysed and immunoprecipitated for Western blotting as described in SDS-PAGE, Western Blotting, and Immunoprecipitation except that HRPconjugated streptavidin (Pierce) was used to detect biotinylated receptors.

2.8. Hormone binding experiments Equilibrium binding parameters for hCG in Table 1 were measured during an overnight incubation (4 ◦ C) of intact cells with increasing amounts of [125 I]hCG (specific activity, 85.7 ␮Ci/␮g) as described elsewhere (Fabritz et al., 1998). Binding of [125 I]hCG to intact cells was performed during an overnight incubation with 100 ng/ml [125 I]hCG at 4 ◦ C in ice-cold buffer (Waymouth MB 752/1 medium containing 50 ␮g/ml gentamicin, 20 mM HEPES, and 1 mg/ml BSA), and detergent extracts used to measure [125 I]hCG binding were obtained by solubilizing the cells in 0.5% Nonidet P-40, 20 mM HEPES, 100 mM NaCl, 20% glycerol, and 1 mM EDTA, pH 7.4, using a constant ratio of 100 ␮l of detergent solution/1 million cells as described elsewhere (Nakamura and Ascoli, 1999). The detergent concentration was diluted to 0.1%, and triplicate aliquots of the extracts were incubated with 100 ng/ml [125 I]hCG at 4 ◦ C for overnight. The third aliquots also received 5 ␮g/ml of crude hCG (Sigma) to correct for nonspecific binding. The free and bound hormones were separated as described elsewhere (Fabritz et al., 1998).

2.9. cAMP accumulation To obtain concentration-response curves for the hCG-induced increases in cAMP, the double antibody RIA method was used to measure intracellular cAMP levels in cells (plated in 35-mm wells) that had been incubated with seven different concentrations of hCG for 30 min at 37 ◦ C in 1 ml of Waymouth MB 752/1 medium containing 50 ␮g/ml gentamicin, 20 mM HEPES, and 1 mg/ml BSA with the presence of a phosphodiesterase inhibitor, 0.5 mM

3.1. The association of Myc-hLHR, Myc-hFSHR and FLAG-hLHR (exon 9) To determine whether hLHR and hLHR (exon 9) form receptor complexes, we used 293 cells, transiently expressing MychLHR and/or FLAG-hLHR (exon 9). The 293 cells expressing Myc-hLHR or FLAG-hLHR (exon 9) were subjected to electrophoresis, and the following receptor species were revealed: 85 kDa (mature receptor), and 68 kDa (immature receptor) for hLHR, consistent with previous reports (lane 1) (Davis et al., 1997; Hipkin et al., 1992; VuHai-LuuThi et al., 1992), and for hLHR (exon 9), 60 kDa (lane 5) (Fig. 1). We did not detect any lower or higher molecular bands for either hLH or hLHR (exon 9). Among the cells coexpressing both receptors, using differential immunoprecipitation, immature forms of Myc-hLHR (68 kDa) were coprecipitated using the anti-flag antibody, which indicated that FLAG-hLHR (exon 9) can interact only with the immature form of Myc-hLHR (lane 2). To confirm this further, we cotransfected 293 cells with Myc-hLHR and FLAG-hLHR. As shown in Fig. 1, Myc-hLHR was not coimmunoprecipitated with FLAG-hLHR, indicating that this phenomenon is specific to FLAG-hLHR (exon 9) (lane 3). To examine whether Myc-hFSHR and FLAG-hLHR (exon 9) form complexes, we transiently expressed Myc-hFSHR and/or FLAG-hLHR (exon 9). Western blots of extracts of cells that had been transiently transfected with Myc-hFSHR display two bands: 87 kDa and 81 kDa (Fig. 2 lane 1). In the 293 cells co-expressing both Myc-hFSHR and FLAG-hLHR (exon 9), immature forms of Myc-hFSHR (81 kDa) were co-precipitated by the anti-FLAG antibody (Fig. 2 lane 2), using differential immunoprecipitation.

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and 4). We also examined whether hTSHR and FLAG-hLHR (exon 9) form complexes, and hTSHR was not coprecipitated by the same method (data not shown). 3.2. hLHR (exon 9) expresses in the human ovary of menstrual cycle

Fig. 1. Detection of hLHR association by immnoblotting and coimmunoprecipitation. Two hundred and ninety-three cells expressing Myc-hLHR (lane 1), FLAG-hLHR (lane 4), and FLAG-hLHR (exon 9) (lane 5), and coexpressing both Myc-hLHR and FLAG-hLHR (exon 9) (lane 2) or both Myc-hLHR and FLAG-hLHR (lane 3) were solubilized as described in Section 2, immunoprecipitated (IP) with anti-c-myc antibody or anti-flag antibody, resolved by 7.5% reducing SDS-PAGE, transferred to a PVDF membrane, and probed with an indicated antibody (IB). The blot is representative of three independent experiments.

Fig. 2. Detection of Myc-hFSHR association by immunoblotting and coimmunoprecipitation. Two hundred and ninety-three cells co-expressing both MychFSHR and FLAG-hLHR (exon 9) (lanes 1 and 2) or both Myc-hFSHR and FLAG-hLHR (lane 3), or both Myc-hFSHR and FLAG-hTSHR (lane 4) were solubilized as described in Section 2, immunoprecipitated (IP) with anti-c-Mycantibody (lane 1) or anti-FLAG-antibody (lanes 2–4), resolved by 7.5% reducing SDS-PAGE. After transferred to a PVDF membrane, proved with anti-c-Mycantibody (IB). The blot is representative of three independent experiments.

On the other hand, in the 293 cells expressing both Myc-hFSHR and FLAG-hLHR, or both Myc-hFSHR and FLAG-hTSHR which is categorized to a subfamily of the superfamily of GPCRs as hFSHR and hLHR, no bands were detected (Fig. 2, lanes 3

We examined whether hLHR (exon 9) is expressed in the ovary of the menstrual cycle. We previously showed that the amounts of hLHR mRNA increase from preovulatory follicles to the corpus luteum of midluteal phase and subsequently decrease at the late luteal phase. As shown in Fig. 3B, corresponding to our previous data (Minegishi et al., 1997a), the expression of both wild-type hLHR (801 bp) and hLHR (exon 9) (615 bp) changed across the luteal phase and hLHR (exon 9) were seen at every phase of the cycle. To confirm this, a quantitative realtime PCR was performed. As shown in Table 1, hLHR (exon 9) was expressed at every phase of the cycle, indicating that hLHR (exon 9) is naturally expressed at all phases of the menstrual cycle. Moreover, the expression of hLHR (exon 9) mRNA relative to hLHR was increased as the luteal phase progressed. Since we detected a single band (817 bp) by RT-PCR with primers to amplify exon 1 to exon 10 (Fig. 3A) for hFSHR, we designed one pair of primers for a quantitative RT- PCR. In contrast to hLHR and hLHR (exon 9), FSHR mRNA levels clearly declined from 8000 ± 530 to 3000 ± 320 in copy numbers after ovulation, supporting our previous data (Minegishi et al., 1997a). 3.3. Co-expression of FLAG-hLHR (exon 9) with the wild-type Myc-hLHR decreases expression of the wild-type hLHR We cotransfected 293 cells with Myc-hLHR, by increasing the amount of FLAG-hLHR (exon 9) plasmid, to examine the interaction between Myc-hLHR and FLAG-hLHR (exon 9). As in the 293 cells with increasing FLAG-hLHR (exon 9) expression, Fig. 4A shows that both the 85-kDa and 68-kDa bands of Myc-hLHR were diminished. On the other hand, coimmunoprecipitation of immature forms of Myc-hLHR (68 kDa) increased (Fig. 4C), correspondingly increasing the expression of FLAG-hLHR (exon 9) (Fig. 4B). These results combined suggest the receptor complexes of hLHR reduce the expression

Fig. 3. RT-PCR of hFSHR and hLHR transcripts in human ovaries. Total RNA samples were prepared from the ovary. RT-PCR was performed for hFSHR (follicle from 14 day of menstrual cycle) (A) or hLHR (lane 1; follicle from 10 days of menstrual cycle lane 2; follicle from 14 days of menstrual cycle lane 3; corpus luteum from 21 days of menstrual cycle lane 4; corpus luteum from 27 days of menstrual cycle) (B) as described in Section 2. DNA size markers (MW) were provided by digesting λ-phage DNA with BamHI and HindIII. The picture is representative of the three independent experiments.

T. Minegishi et al. / Molecular and Cellular Endocrinology 260–262 (2007) 117–125

Fig. 4. Coimmunoprecipitation of Myc-hLHR with FLAGhLHR (exon 9). Two hundred and ninety-three cells transiently transfected with Myc-hLHR and increasing amounts of FLAG-hLHR (exon 9). The total amount of DNA used for each transfection condition was kept constant by the addition of an appropriate amount of pcDNA3.1 vector. Two hundred and ninety-three cells were solubilized as described in Section 2, immunoprecipitated (IP) with anti-c-myc antibody or anti-flag antibody, resolved by 7.5% reducing SDSPAGE, transferred to a PVDF membrane, and probed with an indicated antibody (IB). (A) Two hundred and ninety-three cells cotransfected with MychLHR and FLAG-hLHR (exon 9) were immunoprecipitated by anti-c-myc antibody, and immunoblotted by anti-c-myc antibody to detect the expression of Myc-hLHR. (B) Two hundred and ninety-three cells cotransfected with Myc-hLHR and FLAG-hLHR (exon 9) were immunoprecipitated by M2 anti-flag antibody and immunoblotted by M2 anti-flag antibody to detect the expression of FLAG-hLHR (exon 9). (C) Two hundred and ninety-three cells cotransfected with MychLHR and FLAG-hLHR (exon 9) were immunoprecipitated by M2 anti-flag antibody and immunoblotted by anti-c-myc antibody to detect the Myc-hLHR coimmnoprecipitated with FLAG-hLHR (exon 9). The blot is representative of three independent experiments. Each blot was exposed for a different time to detect the signal.

of Myc-hLHR. We then examined whether the formation of receptor complexes of hLHR affected the binding affinity for hCG and signaling condition. As shown in Table 2, in the 293 cells expressing FLAG-hLHR (exon 9), no detectable binding of [125 I]hCG was found, indicating that FLAG-hLHR (exon 9)

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is not expressed on the cell surface. The coexpression of FLAGhLHR (exon 9) with Myc-hLHR reduced receptor number from 122,000 ± 6000 to 53,700 ± 5100, without changing the affinity for [125 I]hCG. These results lead us to consider the negative control of receptor function thorough the formation of receptor complexes, including receptor number and signal transduction. Thus, the ability to transduce a hCG signal was measured by quantitating cAMP accumulation in cells incubated with increasing concentrations of hCG. Table 2 shows the basal levels of cAMP are quite similar to cells expressing both Myc-hLHR and FLAG-hLHR (exon 9). In contrast, EC50 for a hCG-induced cAMP response is about seven-fold higher in cells expressing Myc-hLHR and FLAG-hLHR (exon 9) than in cells expressing Myc-hLHR alone, and the maximal response in cells expressing both receptors was significantly reduced when compared with cells expressing Myc-hLHR alone. The above Western blot and the binding assay data demonstrated that the receptor complexes formation of FLAG-hLHR (exon 9) with Myc-hLHR caused the decrease in the level of Myc-hLHR in the 293 cells. 3.4. FLAG-hLHR (exon 9) is retained within the cells rather than being expressed only at the cell surface The following experiments were designed to ascertain whether FLAG-hLHR (exon 9) is expressed at the cell surface. This was established by the biotinylation of the cell surface proteins followed by Western blotting with horseradish peroxidase (HRP)-conjugated streptavidin. As shown in Fig. 5, streptavidin blots of anti-myc immunoprecipitates of lysates obtained from biotinylated cells transfected with Myc-hLHR resulted in the visualization of a prominent (85 kDa) band, which coincides with the data presented by Min et al. (Min and Ascoli, 2000), whereas the anti-myc blot resulted in both the 85-kDa and 68-kDa bands. On the other hand, in FLAG-hLHR (exon 9) expressed alone, streptavidin blots failed to detect the 60kDa band of FLAG-hLHR (exon 9). These findings indicate that FLAG-hLHR (exon 9) is retained within the cells rather than being expressed only at the cell surface. Although the receptor biotinylation experiment suggested that FLAG-hLHR (exon

Table 2 Effect of receptor complex formation on 125 I-hCG binding and cAMP responsiveness of transiently transfected 293 cells Receptor

Myc-hLHR FLAG-hLHR (exon 9) Myc-hLHR + FLAG-hLHR (exon 9)

hCG bindinga

hCG-stimulated cAMP accumulationb

Kd (nM)

Receptors/cell

Basal cAMP (pmol/105 cells)

EC50 (pM)

Maximal response (pmol/105 cells)

0.72 ± 0.17 ND 0.34 ± 0.02

122000 ± 6000 ND 53700 ± 5100c

2.6 ± 0.7 ND 2.1 ± 0.3

5.6 ± 2.3 ND 37.9 ± 10.9*

36.4 ± 2.2 ND 19.9 ± 3.4*

a Two hundred and ninety-three cells were transfected with either Myc-hLHR alone or Myc-hLHR and FLAG-hLHR (exon 9), and cells were used to measure equilibrium binding parameters for 125 I-hCG during an overnight incubation at 4 ◦ C. Each number represents the mean ± S.E.M. of three independent experiments. ND: not detectable. The receptor number was significantly lower when Myc-hLHR was co-expressed with FLAG-hLHR (exon 9) than when expressed alone (* p < 0.05). b Two hundred and ninety-three cells were incubated in the presence of 0.5 mM MIX with increasing concentrations of hCG for 15 min at 37 ◦ C. The methods used to measure cAMP and to calculate the EC50 and maximal response for hCG stimulation are described in Section 2. Each value represents the mean ± S.E.M. of four independent experiments. Duplicate dishes were used in each experiments. The EC50 was significantly higher and the maximal response was significantly lower when Myc-hLHR was co-expressed with FLAG-hLHR (exon 9) than when expressed alone (* p < 0.05).

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3.5. Co-transfection of FLAG-LHR (exon 9) drives Myc-hLHR to the lysosome

Fig. 5. Biotinylation of Myc-hLHR and FLAG-hLHR (exon 9). Two hundred and ninety-three cells were transiently transfected with Myc-hLHR or FLAGhLHR (exon 9). The cell surface proteins were covalently modified with biotin, and lysates were prepared, immunoprecipitated with anti-c-myc antibody or M2 anti-flag antibody, and resolved by SDS-PAGE. After electrophoretic blotting, the biotinylated proteins were visualized using horseradish peroxidaselabeled streptavidin and ECL Plus as described in Section 2. Myc-hLHR or FLAGhLHR (exon 9) that was not biotinylated was visualized as described in Fig. 1. The blot is representative of three independent experiments.

9) was retained intracellularly, further experiments with Endo H (endoglycosidase H), cleaving high-mannose form of carbohydrates from glycoproteins, supported this, based on the fact that the removal of these mannose residues from glycoproteins normally occurs within the Golgi apparatus during posttranslational modification. Therefore, glycoproteins that are sensitive to Endo H can indicate whether the glycoproteins still reside within the endoplasmic reticulum (Kornfeld and Kornfeld, 1985). As with previous data (Rozzell et al., 1995), the 68-kDa band of Myc-hLHR, sensitive to Endo H, underwent a shift to 58 kDa, whereas treatment with PNGaseF (N-glycosidase F) caused the migration of both the 85- and 68-kDa bands to 58-kDa band. FLAG-hLHR (exon 9) exhibited a shift in molecular mass in response to Endo H or PNGaseF, similar to the immature form of the wild receptor (Fig. 6).

To examine the mechanism of Myc-hLHR reduction through cotransfection of FLAG-hLHR (exon 9), Percoll gradient was used to fractionate the postnuclear supernatant from cells stably expressing either or both Myc-hLHR and FLAG-LHR (exon 9). The failure of Myc-hLHR to correctly traffic to the plasma membrane, when coexpressed in the same cell as FLAG-hLHR (exon 9), may represent an artifact of the transient expression assay system. At first, we tried to ascertain whether the receptor association between Myc-hLHR and FLAG-hLHR (exon 9) occurred in stable cell lines. As shown in Fig. 7A, it was clearly demonstrated that the immature receptor of Myc-hLHR associated with FLAG-hLHR (exon 9), consistent with Fig. 1. Next, we used ␤-hexosaminidase activity, known as the biochemical marker for lysosomes, to collect the lysosomes fraction. As shown in Fig. 7B, fraction number 4 had a hexosaminidase activity 10 times higher compared with other fractions, suggesting that the majority of lysomes are separated into this fraction. The Western blot data in Fig. 7C show that the 85-kDa band of the mature Myc-hLHR was prominent in fractions 1 and 2, whereas, in lane 4, very faint bands were observed, suggesting that Myc-hLHR is hardly located in the lysosomal compartment. On the other hand, FLAG-hLHR (exon 9) is separated in nos. 2 and 3: especially in the no. 3 fraction, suggesting that these fractions contain the endoplasmic reticulum. In 293 cells expressing Myc-hLHR, both the mature receptor (85 kDa) and the immature receptor (68 kDa) could be seen in lanes 2 and 3. As shown in Fig. 7D, the migration of both Myc-hLHR and FLAG-hLHR (exon 9) in fraction 4 was enhanced in 293 cells expressing both Myc-hLHR and FLAG-hLHR (exon 9), as compared with the 293 cells expressing Myc-hLHR alone. These results led us to consider that the expression of FLAG-hLHR (exon 9) drives Myc-hLHR to the lysosome, where the receptors are eventually degraded. 4. Discussion

Fig. 6. Glycosylation condition of Myc-hLHR and FLAG-hLHR (exon 9). Two hundred and ninety-three cells expressing either Myc-hLHR or FLAG-hLHR (exon 9) were solubilized in detergent as described in Section 2 and were incubated in the absence (−) or presence of either Endo H or PNGase F. After the incubation, each receptor was immunoprecipitated, resolved by 7.5% reducing SDS-PAGE, transferred to a PVDF membrane, probed with either anti-c-myc or M2 anti-flag antibody, and visualized using ECL plus. The blot is representative of three independent experiments.

Many mutations in gonadotropin receptors have been reported (Themmen and Huhtaniemi, 2000). Activating and inactivating mutations with very different phenotypic effects have been identified. LHR (exon 9), one of the splice variants among hLHR, was cloned in the corpus luteum of a patient with a regular menstrual cycle (Minegishi et al., 1990), although the functional meaning of LHR (exon 9) was yet unknown. In previous findings from this laboratory, three transcripts (5.4, 3.6 and 2.4 kb) of hLHR mRNA were detected by Northern blot of the human ovary (Minegishi et al., 1997a), indicating that one may encode LHR (exon 9). Laue et al. (Laue et al., 1996) showed that naturally occurring mutant hLHR lacking exon 8 caused Leydig cell hypoplasia due to the loss of binding ability to hCG. Although we do not know the exact mechanism, a deletion around this region of hLHR might induce a conformational change, which results in the loss of binding ability to hCG. A recent report (Elmhurst et al., 2000) demonstrated that dopamine type 3 receptor splice variant, D3nf, which does not

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Fig. 7. Distribution of Myc-hLHR and FLAG-hLHR (exon 9) in lysosomes of 293 cells stably expressing either or both Myc-hLHR and FLAG-hLHR (exon 9). Cells stably express indicated constructs. Receptor numbers are 139,000 ± 6000 for Myc-hLHR and 64,500 ± 5000 for Myc-hLHR + FLAG-hLHR (exon 9), respectively. We did not measure the receptor number of FLAG-hLHR (exon 9) because the receptors are not expressed at the cell surface. The receptor expression level was evaluated by the signal intensity of Western blot. (A) Two hundred and ninety-three cells stably expressing both Myc-hLHR and FLAG-hLHR (exon 9) were solubilized as described in Section 2, immunoprecipitated (IP) with anti-c-myc antibody or anti-flag antibody, resolved by 7.5% reducing SDS-PAGE, transferred to a PVDF membrane, and probed with the indicated antibody (IB). The blot is representative of three independent experiments. (B) Postnuclear supernatants of 293 cells were prepared and fractionated on Percoll gradients as described in Section 2. The contents of the gradients were then collected from the top (500 ␮l/fraction), and aliquots of each of the four fractions were combined to assay ␤-hexosaminidase activity. The result shows the distribution of endogenous ␤-hexosamindase activity present in 293 cells. Data represent the mean increase relative to the combined fraction no. 1 (±S.E.M.; n = 5). The absence of an error bar indicates that the S.E.M. was too small to be observed graphically. (C) Two hundred and ninety-three cells stably expressing either Myc-hLHR or FLAG-hLHR (exon 9) were prepared and fractionated on Percoll gradients as described in Section 2. The contents of the gradients were then collected from the top (500 ␮l/fraction), and aliquots of each of the four fractions were combined, immunoprecipitated (IP) with anti-c-myc antibody or anti-flag antibody, resolved by 7.5% reducing SDS-PAGE, transferred to a PVDF membrane, and probed with the indicated antibody (IB). The blot is representative of three independent experiments. (D) Two hundred and ninety-three cells stably expressing either or both Myc-hLHR and FLAG-hLHR (exon 9) were prepared and fractionated on Percoll gradients as described in Section 2. At this time, the contents of the gradients were then collected from four fractions at the bottom, which were combined, immunoprecipitated (IP) with anti-c-myc antibody or anti-flag antibody, resolved by 7.5% reducing SDS-PAGE, transferred to a PVDF membrane, and probed with the indicated antibody (IB). The blot is representative of three independent experiments.

bind ligands and is incapable of signal transduction, could form a heterooligomer, and the coexpression of D3nf with D3 dopamine receptor attenuated the ligand binding to the D3 dopamine receptor. In the case of the dopamine receptor, D3nf behaves as a natural antagonist at the cell surface, but D3nf did not significantly alter the amount of D3 dopamine receptor expressed. In contrast, the receptor complexes with Myc-hLHR and FLAGhLHR (exon 9) reduced the expression level of Myc-hLHR (Fig. 4) in 293 cells, resulting in the attenuation of the expression of Myc-hLHR at the plasma membrane. The results of Western blots using Percoll gradient fractionation in Fig. 6 indicated that hLHR formed complexes with hLHR (exon 9), which are transferred to the lysosome, where they are eventually degraded, instead of being translocated from the endoplasmic reticulum to the transducing organelle. The mutant rhodopsin, G proteincoupled photoreceptor, retained in the endoplasmic reticulum, trapped wild-type rhodopsin, demonstrating that the mutant misfolded rhodopsin molecules might interfere with the maturation of wild-type rhodopsin in the endoplasmic reticulum, allowing it to be eventually degraded (Colley et al., 1995). A number of

truncated receptor variants have recently been described, resulting in the reduction of the full-length receptor expressions by coexpression of truncated and wild receptors (Zhu and Wess, 1998; Grosse et al., 1997; Benkirane et al., 1997). In the case of hLHR, we assume that the receptor complex in the endoplasmic reticulum prevents wild-type hLHR from the association with molecules such as chaperone, previously demonstrated to be involved in the maturation of gonadotropin receptors (Rozell et al., 1998). A recent report lends further credence to this hypothesis (Benkirane et al., 1997) by showing that a naturally occurring truncated mutant of human chemokine receptor 5 exerts a dominant negative effect on wild-type chemokine receptor 5. Based on the comparison between the effects of Endo H and PNGase F digestion of Myc-hLHR and FLAG-hLHR (exon 9), as shown in Fig. 6, FLAG-hLHR (exon 9) is retained in the endoplasmic reticulum, which coincides with the biotinylation experiment where FLAG-hLHR (exon 9) was not biotinylated (Fig. 5). We then examined whether the formation of receptor complexes of hLHR affected the binding affinity for hCG and signaling condition. As shown in Table 2, in the 293 cells expressing

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FLAG-hLHR (exon 9), no detectable binding of [125 I]hCG was found, indicating that FLAG-hLHR (exon 9) is not expressed on the cell surface. The coexpression of FLAG-hLHR (exon 9) with Myc-hLHR reduced receptor number, without changing the affinity for [125 I]hCG. These results lead us to consider the negative control of receptor function thorough the formation of receptor complexes, including receptor number and signal transduction. Thus, the ability to transduce a hCG signal was measured by quantitating cAMP accumulation in cells incubated with increasing concentrations of hCG. Table 2 shows the basal levels of cAMP are quite similar to cells expressing both Myc-hLHR and FLAG-hLHR (exon 9). In contrast, EC50 for a hCG-induced cAMP response is about seven-fold higher in cells expressing Myc-hLHR and FLAG-hLHR (exon 9) than in cells expressing Myc-hLHR alone, and the maximal response in cells expressing both receptors was significantly reduced when compared with cells expressing Myc-hLHR alone. These results showed that the FLAG-hLHR (exon 9) caused the reduction of expression of functional receptor number and affected the signaling condition of wild-type hLHR. Whaley et al. (Whaley et al., 1994) carefully explored the effect of the varying levels of ␤2 -adrenergic receptor on the activation of adenylyl cyclase, suggesting that the receptor expression levels could inversely affect the functional properties of the receptor (i.e. an increase in EC50 and a decrease in the maximal response; cf. Table 2). Our data in Table 2 support this idea to interpret the mechanism underlying the attenuation of signal conduction of Myc-hLHR induced by hCG. Based on the data that FLAG-hLHR (exon 9) stays inside the endoplasmic reticulum, we suspect the decrease of hCG responsiveness in cells expressing both Myc-hLHR and FLAG-hLHR (exon 9) is due to the reduction of Myc-hLHR expression at the plasma membrane. Taken together, we hypothesize that exon 9 of hLHR involves in both receptor insertion to the plasma membrane and hormone binding, based on the fact that exon 9 codes leucine-rich repeats 8–9 and N-terminal part to hinge region. In this study, we found that the 68-kDa immature receptor of Myc-hLHR and also 81 kDa immature receptor of hFSHR forms receptor complexes with FLAG-hLHR (exon 9). The decrease in Myc-hLHR and Myc-hFSH expression at the cell surface was due to the reduction of immunoreactive protein in the 293 cells, as confirmed by Western blot. The coimmunoprecipitation study with Myc-hLHR, Myc-hFSHR and FLAG-hLHR (exon 9) revealed that these receptors physically form receptor complexes. We propose the misfolded mutant hLHR form a receptor association with wild type gonadotropin receptors. This phenomenon cannot occur with TSHR, categorized in the same subclass of GPCR with the large glycosylated extracellular domain. In the RT-PCR experiment (Fig. 3), we detected hLHR (exon 9) mRNA from the late follicular phase to the late luteal phase. Therefore, both hLHR and hLHR (exon 9) are expressed during the entire luteal phase. To quantify the mRNA levels of hFSHR, hLHR, and hLHR (exon 9), we performed the RT-PCR (Table 1). As the luteal phase progressed, hLHR (exon 9) increased relative to hLHR, demonstrating that hLHR (exon 9) expressed more than hLHR in the late luteal phase. On the other hand, hFSHR mRNA levels were markedly

decreased after ovulation, consistent with the previous results from this laboratory (Minegishi et al., 1997b). This results suggested that hLHR (exon 9) dominantly existed against hFSHR from the post-ovulation to the early luteal phase. Given that hFSHR forms the receptor complex with hLHR (exon 9) in the physiological condition, this association could negatively regulate hFSHR expression as hLHR (exon 9) is increasingly expressed (Yamashita et al., 2005). In conclusion, the findings presented herein show that hFSHR and hLHR are associated with hLHR (exon 9) and that the receptor complexes between wild-type receptors and hLHR (exon 9) can negatively control the function of wild-type receptors. However, the clinical significance of the receptor complexes of gonadotropin receptors is yet unclear. It is well known that many substances, including LH, hCG, and cytokines, can regulate luteal function. Further study is required to verify whether the receptor complexes between gonadotropin receptors and hLHR (exon 9) contribute to the regulation of functions or various pathological conditions in the ovary. Acknowledgements We thank Dr. Yumiko Abe and Hiroko Matuda for expert technical assistance. We also wish to thank Dr. Mario Ascoli for suggestions with different technical aspects of this project; Rika Kuroda for the preparation of this manuscript; and Dr. Takashi Matozaki (Biological Research Center Institute for Molecular and Cellular Regulation, Gunma University, Japan) for anticAMP serum. References Bai, M., 2004. Dimerization of G-protein-coupled receptors: roles in signal transduction. Cell. Signal. 16, 175–186. Benkirane, M., Jin, D.Y., Chun, R.F., Koup, R.A., Jeang, K.T., 1997. Mechanism of transdominant inhibition of CCR5-mediated HIV-1 infection by ccr5delta32. J. Biol. Chem. 272, 30603–30606. Chen, C., Okayama, H., 1987. High-efficiency transformation of mammalian cells by plasmid DNA. Mol. Cell Biol. 7, 2745–2752. Colley, N.J., Cassill, J.A., Baker, E.K., Zuker, C.S., 1995. Defective intracellular transport is the molecular basis of rhodopsin-dependent dominant retinal degeneration. Proc. Natl. Acad. Sci. USA 92, 3070–3074. Davis, D.P., Rozell, T.G., Liu, X., Segaloff, D.L., 1997. The six N-linked carbohydrates of the lutropin/choriogonadotropin receptor are not absolutely required for correct folding, cell surface expression, hormone binding, or signal transduction. Mol. Endocrinol. 11, 550–562. Dufau, M.L., 1998. The luteinizing hormone receptor. Annu. Rev. Physiol. 60, 461–496. Elmhurst, J.L., Xie, Z., O’Dowd, B.F., George, S.R., 2000. The splice variant D3nf reduces ligand binding to the D3 dopamine receptor: evidence for heterooligomerization. Mol. Brain Res. 1, 63–74. Fabritz, J., Ryan, S., Ascoli, M., 1998. Transfected cells express mostly the intracellular precursor of the lutropin/choriogonadotropin receptor but this precursor binds choriogonadotropin with high affinity. Biochemistry 37, 664–672. Fan, Q.R., Hendrickson, W.A., 2005. Structure of human follicle-stimulating hormone in complex with its receptor. Nature 433, 269–277. Gromoll, J., Eiholzer, U., Nieschlag, E., Simoni, M., 2000. Male hypogonadism caused by homozygous deletion of exon 10 of the luteinizing hormone receptor: differential action of the luteinizing hormone (LH) and human chorionic gonadotropin (hCG). J. Clin. Endocrinol. Metab. 85, 2281–2286.

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