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Department of Obstetrics and Gynecology (K.N., S.Y., Y.O., T.M.), School of Medicine, Gunma University, and Core Research for Evolutional Science and Technology (CREST) (K.N., T.M.), Japan Science and Technology, Gunma 371-8511, Japan
Address all correspondence and requests for reprints to: Kazuto Nakamura, Department of Obstetrics and Gynecology, School of Medicine, Gunma University, Gunma 371-8511, Japan. E-mail: nkazuto{at}med.gunma-u.ac.jp.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Over the past several years, many groups have documented that GPCRs can form dimers and oligomers, including the m3 muscarinic (6), ß2-adrenergic (7), V2 vasopressin (8), metabotropic glutamate (9),
-opioid (10), and D3 dopamine receptors (11). Those findings suggest that dimerization may be a universal aspect of GPCR biology. Moreover, it has been reported that some splice variants of GPCRs (e.g., GnRH receptor and D3 dopamine receptor) modulate the function of the wild-type receptor by forming a receptor dimer (11, 12). Furthermore, the coexpression of defective LHR fragments partially reconstitutes ligand-induced signal transduction (13). These results prompted us to investigate whether the interaction of hLHR(exon 9) with hLHR alters the function of the wild-type receptor.
To date, there is no direct evidence to indicate whether or not hLHR dimers actually exist. In this study, we used immunoprecipitation of the tagged hLHR and hLHR(exon 9), using c-myc for hLHR and flag for hLHR(exon 9), to examine whether these receptors can form receptor complexes. We also examined the effect of the receptor complexes of hLHR and hLHR(exon 9) on agonist-induced cAMP accumulation and internalization of hLHR. Furthermore, we explored the impact of the formation of receptor complexes on the trafficking of hLHR. We found that hLHR and hLHR(exon 9) can form receptor complexes. The reductions in the protein levels for both hLHR and hLHR(exon 9) are followed by the formation of receptor complexes between hLHR and hLHR(exon 9).
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). We sequenced both PCR products that represented the small one (615 bp) lacking exon 9 of hLHR cDNA. This result suggested hLHR(exon 9) in the human ovary although we were unable to prove that hLHR(exon 9) protein is expressed in the human ovary. To determine whether hLHR and hLHR(exon 9) form receptor complexes, we used 293 cells, transiently expressing Myc-hLHR 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) (14, 15, 16), and for hLHR(exon 9), 60 kDa (lane 5) (Fig. 2). 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). This finding was specific: this did not occur among cells individually expressing the receptors (data not shown). To confirm this further, we cotransfected 293 cells with Myc-hLHR and FLAG-hLHR. As shown in Fig. 2, Myc-hLHR was not coimmunoprecipitated with FLAG-hLHR, indicating that this phenomenon is specific to FLAG-hLHR(exon 9) (lane 3).
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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. 3C), correspondingly increasing the expression of FLAG-hLHR(exon 9) (Fig. 3B). These results combined suggest the receptor complexes of hLHR reduce the expression of Myc-hLHR.
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, in the 293 cells expressing FLAG-hLHR(exon 9), no detectable binding of [125I]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 from 122,000 ± 6,000 to 53,700 ± 5,100, without changing the affinity for [125I]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 1 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 7-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. We used different receptor numbers at the cell surface to confirm this and analyzed cAMP responsiveness by the transfection with different amounts of hLHR plasmid. From the results shown in Table 2, the mechanism underlying the increase of EC50 and the decrease in the maximal response was due to the reduction of receptor number, suggesting the coexpression of FLAG-hLHR(exon 9) negatively affect the signaling condition of wild-type hLHR.
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, 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. (17), 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 60-kDa 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.
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). Together with the results shown in Table 1
, and Figs. 4 and 5, we can safely conclude that FLAG-hLHR(exon 9) is trapped in the endoplasmic reticulum, as with the immature form of the wild-type receptor.
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, increasing the amount of Myc-hLHR plasmid for transfection led to the gradual reduction of FLAG-hLHR(exon 9). On the other hand, Myc-hLHR expression was not attenuated by the increase in the amount of FLAG-hLHR plasmid for transfection (Fig. 6B). This suggests that, as proposed above, the receptor complex formation of hLHR reduces FLAG-hLHR(exon 9) as it does Myc-hLHR.
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, it was clearly demonstrated that the immature receptor of Myc-hLHR associated with FLAG-hLHR(exon 9), consistent with Fig. 2. Next, we used ß-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. Consistent with the results of Kishi and Ascoli (20), the gradient chosen could clearly separate the lysosomal compartment from endosomes and plasma membrane. This gradient was unable to separate endosomes from the plasma membrane, which was not a potential problem for this study, because the purpose of this gradient fractionation was to obtain the lysosomal compartment.
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, 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. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Many mutations in gonadotropin receptors have been reported (21). 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 normal regular menstrual cycle (1), although the functional meaning of LHR(exon 9) was yet unknown. In previous findings from this laboratory, three transcripts (5.4, 3.6, 2.4 kb) of hLHR mRNA were detected by Northern blot of the human ovary (5), indicating that one may encode LHR(exon 9). Laue et al. (4) showed that naturally occurring mutant hLHR lacking exon 8 caused Leydig cell hypoplasia. This mutant hLHR is also incapable of binding to hCG. Although we do not know the exact mechanism, we think that a deletion around this region of hLHR induces a conformational change, which results in the loss of binding ability to hCG.
A recent report (11) demonstrated that dopamine type 3 receptor splice variant, D3nf, which does not 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 FLAG-hLHR(exon 9) reduced the expression level of Myc-hLHR (Fig. 3) as well as FLAG-hLHR(exon 9) receptor protein (Fig. 6) 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. 7 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 protein-coupled 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 (22). 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 (8, 12, 23). In the case of hLHR, this sequence is likely, if 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 (24). A recent report lends further credence to this hypothesis (23) by showing that a naturally occurring truncated mutant of human chemokine receptor 5 exerts a dominant negative effect on wild-type chemokine receptor 5. Although we did not attempt to establish the intracellular location of the FLAG-hLHR(exon 9), 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. 5, 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. 4). Several studies have suggested that some mutant GPCRs have a negative effect on the signaling of their receptors (8, 12). To test our hypothesis that FLAG-hLHR(exon 9) also has a negative effect on Myc-hLHR function, we first applied the cAMP assay. As shown in Table 1, it was shown that the coexpression of Myc-hLHR and FLAG-hLHR(exon 9) suppressed signaling ability of Myc-hLHR, induced by shifting the EC50 toward a significantly higher agonist concentration. One possible explanation for the reduction of maximal cAMP accumulation by coexpression may involve competition between wild-type and FLAG-hLHR(exon 9) for Gs-proteins, resulting in the reduction of the agonist-stimulable G protein pool. However, we do not think that this is the case for hLHR, because FLAG-hLHR(exon 9) remains in the endoplasmic reticulum, where hLHR is supposed to be incapable of binding to G protein. Whaley et al. (25) carefully explored the effect of the varying levels of ß2-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; c.f. Table 1). Our data in Table 2 support this idea to interpret the mechanism underlying the attenuation of signal conduction of Myc-hLHR induced by hCG. We assumed that FLAG-hLHR(exon 9) cannot change the conformation of Myc-hLHR, causing an inappropriate interaction with the G protein at the plasma membrane. 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.
In conclusion, the findings presented herein show that hLHR is associated with hLHR(exon 9) and that the receptor complexes between wild-type hLHR and hLHR(exon 9) can negatively control the function of wild-type hLHR. However, the clinical significance of the receptor complexes of hLHR 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 hLHR and hLHR(exon 9) contribute to the regulation of functions or various pathological conditions in the ovary.
Our findings contribute to the understanding of the role of receptor complexes of hLHR, the function of hLHR, and the functions of the family of GPCRs.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Construction of hLHR and hLHR(exon 9) Expression Vectors
cDNAs encoding hLHR and hLHR(exon 9) were inserted separately into the mammalian expression vector pcDNA3.1 (Invitrogen, San Diego, CA). The receptors were tagged with c-myc for wild-type human LH receptor (Myc-hLHR) or flag epitopes for wild-type LH receptor (FLAG-hLHR) and hLHR(exon 9)[FLAG-hLHR(exon 9)]. 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.
Expression in Mammalian Cells
293 Cells were maintained as monolayer cultures at 37 C in MEM (DMEM) supplemented with 10% newborn calf serum and antibiotics. 293 Cells were transiently transfected with pcDNA3.1 vectors using the Ca2+ phosphate method (26). 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 d after transfection. To establish cell lines stably, 293 cells were plated into 100-mm dishes and transfected with either Myc-hLHR or FLAG-hLHR(exon 9) or with both constructs. Cells were selected in media containing 700 µg/ml G418. Stable cell lines were maintained in the media containing G418.
RT-PCR
Human ovaries were removed from patients who had undergone salpingo-oophorectomy 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. Total RNA (5 µg) was extracted from ovaries to generate first-strand cDNA with a cDNA synthesis kit (Life Technologies, Inc., Gaithersburg, MD). The entire 2-µl cDNA synthesis reaction volume was combined in a 50-µl final reaction volume for PCR amplification containing 0.25 µM of each oligonucleotide primer and 1.5 IU Taq DNA polymerase. The primer sequences were: 5'-TTCGGATCCTACATCTGGAGAAGATGCACAATG and 3'-TCGAGAATTCAGGTGAATAGCATAGGTGATGGTG for hLHR, and 5'-CCAAGGTCATCCATGACAACT and 3'-CACCCTGTTGCTGTAGCCAAA for glyceraldehyde-3-phosphate dehydrogenase.
Thirty cycles of PCR amplification were performed. Each cycle consisted of 90 sec denaturation at 95 C, 150 sec annealing at 62 C, and 150 sec at 70 C for enzymatic extension. After PCR, LH receptor PCR-product DNA was then electrophoresed on 2% agarose gels and visualized after ethidium bromide staining by UV fluorescence.
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 x 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). The lysates (500 µg) were immunoprecipitated with the agarose-conjugated anti-c-myc antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or the agarose-conjugated anti-flag M2 antibody (Sigma Chemical Co., St. Louis, MO) 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 (27). 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).
Endo H and PNGase F Treatment of Myc-hLHR and FLAG-hLHR(exon 9)
293 Cells expressing either Myc-hLHR or FLAG-hLHR(exon9) were solubilized as described for Western blotting. Detergent-soluble extracts (500 µg) of cells expressing either Myc-hLHR or FLAG-hLHR(exon9) were incubated in the presence or absence of Endo H (Roche Clinical Laboratories, Indianapolis, IN) or PNGase F (Roche). Endo H treatment before immunoprecipitation was performed by incubation of the detergent-solubilized cell extracts in 750 µl 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 µl 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.
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) in PBS. The cells were lysed and immunoprecipitated for Western blotting as described in SDS-PAGE, Western Blotting, and Immunoprecipitation except that HRP-conjugated streptavidin (Pierce) was used to detect biotinylated receptors.
Hormone Binding Experiments
Equilibrium binding parameters for hCG in Table 1were measured during an overnight incubation (4 C) of intact cells with increasing amounts of [125I]hCG (specific activity, 85.7 µCi/µg) as described elsewhere (28). Binding of [125I]hCG to intact cells was performed during an overnight incubation with 100 ng/ml [125I]hCG at 4 C in ice-cold buffer (Waymouth MB 752/1 medium containing 50 µg/ml gentamicin, 20 mM HEPES, and 1 mg/ml BSA), and detergent extracts used to measure [125I]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 µl of detergent solution/1 million cells as described elsewhere (29). The detergent concentration was diluted to 0.1%, and triplicate aliquots of the extracts were incubated with 100 ng/ml [125I]hCG at 4 C for overnight. The third aliquots also received 5 µg/ml of crude hCG (Sigma) to correct for nonspecific binding. The free and bound hormones were separated as described elsewhere (28).
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 µg/ml gentamicin, 20 mM HEPES, and 1 mg/ml BSA with the presence of a phosphodiesterase inhibitor, 0.5 mM 3-isobutyl-1-methylxanthine (Sigma). The different parameters that describe the concentration-response curves were calculated as described elsewhere (30).
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 x 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 x g for 20 min at 4 C. The contents of the gradients were then collected from the top (500 µl/fraction), and aliquots of each of the four fractions were combined to assay ß-hexosaminidase activity used as a marker for lysosomes (31) and perform immunoprecipitations for Western blotting to detect Myc-hLHR and FLAG-hLHR(exon 9) as described in SDS-PAGE, Western Blotting, and Immunoprecipitation.
Other Methods
Statistical analysis (t test with two-sided P values) was performed using StatFlex (Artech Inc., Osaka, Japan).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: ECL, Enhanced chemiluminescence; Endo H, endoglycosidase H; GPCR, G protein-coupled receptor; hCG, human chorionic gonadotropin; hLHR, human LH receptor; HRP, horseradish peroxidase; PNGaseF, N-glycosidase F; PVDF, polyvinylidene difluoride.
Received for publication December 19, 2003. Accepted for publication March 9, 2004.
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A. Kern, D. Hubbard, A. Amano, and G. D. Bryant-Greenwood Cloning, Expression, and Functional Characterization of Relaxin Receptor (Leucine-Rich Repeat-Containing G Protein-Coupled Receptor 7) Splice Variants from Human Fetal Membranes Endocrinology, March 1, 2008; 149(3): 1277 - 1294. [Abstract] [Full Text] [PDF]
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-phage DNA with BamHI and HindIII. The picture is representative of two independent experiments. GAPDH, Glyceraldehyde-3-phosphate dehydrogenase.
