Chorionic Gonadotropin Receptor Research Articles

Alternately spliced luteinizing hormone/human chorionic gonadotropin receptor mRNA in human breast epithelial cells

Luteinizing hormone (LH)/human chorionic gonadotropin (hCG) bind to a common transmembrane glycoprotein receptor, which is a member of the G protein-coupled receptor family. In human, the LH/hCG receptor gene is composed of 11 exons and 10 introns and its coding region is over 60 kb long. Human chorionic gonadotropin is a glycoprotein hormone containing a cystine-knot folding motif that is found in peptide growth factors known to activate the expression of homeogenes. In the present work we present evidence that hCG down-regulates all the three transcripts of HOXA1 at early stages of hCG treatment in the immortalized human breast epithelial cells (MCF-10F), whereas HOXA1-S1, the largest transcript, as well as HOXA1-S3, the smallest transcript, were up-regulated in the cancer cell lines MDA-MB-231 and MCF-7, respectively. This divergent reaction of hCG was associated with the pattern of LH/hCG receptors and splicing forms expression in human breast epithelial cells. MCF-10F cells expressed the full-length (1191 bp) compared with the cancer-derived cells MCF-7 and MDA-MB-231 that was weakly or not expressed. Isoform 1 (1117 bp) was silent in MCF-10F and expressed weakly in the cancer cells. The isoforms 2 (1006 bp) and 3 (932 bp) of LH/hCG gene receptor were silent in all the cell lines, whereas isoforms 4 (892 bp), 6 (626 bp) and 7 (441 bp) were silent in MCF-10F cells and expressed in the cancer cell lines. Instead isoform 5 (707 bp) showed in the three cell lines the strongest expression in MCF-10F cells. This difference in the expression of alternate splicing of LH/hCG receptor mRNA among the MCF-10F, MCF-7 and MDA-MB-231 cells, may explain the divergent response of these cells to HOXA1 activation by hCG.

Cell-free membrane desensitization assay for G protein-coupled receptors

Publisher Summary This chapter presents a cell-free membrane model to evaluate G protein-coupled receptor (GPCR) desensitization. When receptor desensitization requires proteins and enzymes present in compartments other than the plasma membrane, this assay model is unsuitable. Evidence from the older literature, however, demonstrates that this membrane model can be applied to receptors other than the luteinizing hormone (LH)/ chorionic gonadotropin (CG) receptor. Moreover, in preliminary studies, the existence of GTP-dependent desensitization of the LH/CG receptor was demonstrated in plasma membranes obtained from HEK 293 cells transfected stably with the LH/CG receptor. Demonstrating cell-free, GTP-dependent desensitization of the LH/CG receptor in a heterologous membrane suggests that the proteins required to mediate at least LH/CG receptor desensitization are also present in non-ovarian membranes and that these events may not be unique to ovarian follicular membranes and the LH/CG receptor. This preliminary result is consistent with the possibility that a GTP-dependent step is also obligatory for the desensitization of other G protein-coupled receptors.

Development of a Fluorescence Based High Throughput Assay for Antagonists of the Human Chorionic Gonadotropin Receptor Extracellular Domain: Analysis of Peptide Inhibitors

Development of a Fluorescence Based High Throughput Assay for Antagonists of the Human Chorionic Gonadotropin Receptor Extracellular Domain: Analysis of Peptide Inhibitors

Development of a Fluorescence Based High Throughput Assay for Antagonists of the Human Chorionic Gonadotropin Receptor Extracellular Domain: Analysis of Peptide Inhibitors

A simple method for prompt fluorescent detection of inhibitors of human chorionic gonadotropin (hCG) binding to the extracellular domain of the human luteinizing hormone/chorionic gonadotropin (hLH/CG) receptor was developed for high throughput screening (HTS). Construction and analysis of a recombinant phage that displays the extracellular binding domain of the hLH/CG receptor on its surface and specifically binds hCG was previously described. To facilitate the identification of molecules that disrupt the interaction of hCG with its receptor, a method for prompt fluorescent detection of these phage bound to hCG was developed. This technique is extremely sensitive and employs fluorescent labels (PBXL dyes) that are derived from red and blue-green algae. Antibodies labeled with PBXL dye were able to specifically detect phage that display the extracellular domain of the hLH/CG receptor when bound to hCG immobilized in 96-well microplates. Decreases in fluorescence correlate with the concentration of exogenous hCG or hCG antagonists in the assay. This prompt fluorescence detection assay was optimized in a 96-well format as a model system for HTS applications that target the receptors for the group of hormones known as the gonadotropins. Low-affinity molecules that disrupt binding of the phage-displayed receptor extracellular domain to hCG can be rapidly identified in this high throughput screen.

Palmitoylation of the luteinizing hormone/human chorionic gonadotropin receptor regulates receptor interaction with the arrestin-mediated internalization pathway.

The luteinizing hormone/human chorionic gonadotropin receptor (LH/hCGR) undergoes palmitoylation at cysteine residues 621 and 622 located in the carboxyl terminal tail of the receptor. This study examined the biological function of palmitoylation with respect to its effect on receptor internalization. Coexpression of wild-type (WT) or C621/622G mutant receptors with arrestin-2 increased receptor internalization in 293T cells. Furthermore, measurements of rate enhancement upon overexpression of arrestin indicate that the palmitoylation deficient mutant receptor is more prone to utilizing the arrestin mediated internalization pathway than the WT receptor. Coexpression of G-protein-coupled receptor kinase 4 (GRK4) with wild type receptor resulted in an increase in internalization, while coexpression with the mutant receptor did not result in further enhancement of internalization. Additionally, 293T cells expressing mutant receptor were responsive to hCG with respect to production of inositol phosphates. Taken together, these results suggest that the palmitoylation state of the receptor governs internalization by regulating the accessibility of the receptor to the arrestin-mediated internalization pathway.

Retention of Glucose on Oligosaccharide Chains Linked to the LH/hCG Receptor Prevents Cell Surface Expression

In the first step of asparagine-linked oligosaccharide chain maturation, terminal glucose residues are removed from the high mannose oligosaccharide core by glucosidases I and II. The role that glucose residues play in trafficking the luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor from the endoplasmic reticulum to the cell surface was investigated. Glucosidases I and II were inhibited by incubating 293 T cells transiently transfected with LH/hCG receptor cDNA with 5 mM 1-deoxynojirimycin (DNJ). DNJ treatment resulted in a marked reduction in cell surface [125I]hCG binding. Similar results were obtained from glucosidase I-deficient Lec 23 Chinese hamster ovarian (CHO) cells and wild-type CHO cells that were transiently transfected with LH/hCG receptor cDNA. Immunoprecipitation followed by Western blotting of transfected 293 T cells incubated in the presence or absence of 5 mM DNJ revealed that there is substantially less receptor in DNJ-treated cells than in control cells. These results show that the removal of glucose residues is necessary for trafficking the LH/hCG receptor to the cell surface.

Palmitoylation of the luteinizing hormone/human chorionic gonadotropin receptor regulates receptor interaction with the arrestin-mediated internalization pathway

Palmitoylation of the luteinizing hormone/human chorionic gonadotropin receptor regulates receptor interaction with the arrestin-mediated internalization pathway

An overview of the past, present, and future of nongonadal LH/hCG actions in reproductive biology and medicine.

Even though there were hints, it was not until 1986 that a number of laboratories worldwide began to demonstrate unequivocally the presence and functions of luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptors in various female and male nongonadal tissues. There was no species specificity but there was tissue specificity in the nongonadal LH/hCG receptor distribution. Nongonadal receptor levels were lower but they were regulated and processed and used signaling mechanisms similarly to gonadal receptors. Although still greater understanding is needed, gains made in the last decade demonstrate that nongonadal actions of LH/hCG are physiologically important and may have relevance to better understanding several diseases and their treatment. A recently developed LH receptor knockout model has begun to reaffirm and extend the importance of nongonadal LH signaling in the body.

Targeted disruption of luteinizing hormone/human chorionic gonadotropin receptor gene.

LH/hCG receptors were disrupted by gene targeting in embryonic stem cells. The disruption resulted in infertility in both sexes. The gonads contained no receptor mRNA or receptor protein. Serum LH levels were greatly elevated, and FSH levels were moderately elevated in both sexes; estradiol and progesterone levels decreased but were not totally suppressed in females; testosterone levels were dramatically decreased and estradiol levels moderately elevated in males. The external and internal genitalia were grossly underdeveloped in both sexes. Abnormalities included ambiguous vaginal opening, abdominal testes, micropenis, dramatically decreased weights of the gonads and reproductive tract, arrested follicular growth beyond antral stage, disarray of seminiferous tubules, diminished number and hypotrophy of Leydig cells, and spermatogenic arrest beyond the round spermatid stage. LH/hCG receptor gene disruption had no effect on FSH receptor mRNA levels in ovaries and testes, progesterone receptor (PR) levels in ovaries and androgen receptor (AR) levels in testes. However, it caused a dramatic decrease in StAR and estrogen receptor-alpha (ERalpha) mRNA levels and an increase in ERbeta mRNA levels in both ovaries and testes. Estradiol and progesterone replacement therapy in females and testosterone replacement in males, to determine whether phenotype and biochemical changes were a consequence of decreased gonadal steroid levels or due to a loss of LH signaling, revealed complete restoration of some and partial restoration of others. Nevertheless, the animals remained infertile. It is anticipated that the LH receptor knockout animals will increase our current understanding of gonadal and nongonadal actions of LH and hCG.

Targeted Disruption of Luteinizing Hormone/Human Chorionic Gonadotropin Receptor Gene

Targeted Disruption of Luteinizing Hormone/Human Chorionic Gonadotropin Receptor Gene

Treatment of preterm labor with human chorionic gonadotropin: a new modality

Background: Previous studies have demonstrated that human myometrium contains receptors for human chorionic gonadotropin (hCG) and that hCG can inhibit myometrial contractions in vitro.Objective: To use for the first time hCG as a tocolytic agent in the treatment of preterm labor.Methods: The study group included 100 women with preterm labor; 50 were assigned to receive HCC and 50 placebo. Assignment was made with stratification according to four categories of gestational age between 20–35 weeks. One half of the surviving infants were followed-up at 18 months. The protocol of the dosage consisted of one single dose of hCG 5,000 IV and 10,000 units of hCG in 500 dextrose as a drip of 20 drops per minute.Results: The mean length of time from randomization to delivery differs significantly for the two groups: 28.8 days for the hCG group and 15 days for the placebo treatment group (highly statistically significant, P <0.001). There was statistically significant difference (P <0.05) between the two groups regarding the induction of delivery before 37 weeks and the proportion of infants weighing less than 2,500 g. Human chorionic gonadotropin treatment was associated with an improvement score on the Bayley psychomotor development index.Conclusions: Human chorionic gonadotropin exhibits potent tocolysis with no fetal side effects. This preliminary study suggests that HCG may be a candidate for tocolytic therapy of preterm labor.

Human chorionic gonadotropin dilates uterine and mesenteric resistance arteries in pregnant and nonpregnant rats.

Membrane receptors for human chorionic gonadotropin (hCG) are expressed in a variety of steroidogenic cells, and also in extragonadal tissues such as vessels of the female genital tract. We examined the possible contribution of hCG to the endocrine control of prearteriolar mesenteric and uterine vessels before and during pregnancy. Lumen diameters of isolated pressurized resistance arteries from Sprague-Dawley rats were measured using a video-electronic system. hCG produced marked and dose-dependent vasodilation. Uterine radial arteries were found to be highly sensitive to hCG (EC50 approximately =60 mU/ml) before and throughout gestation. Second-order mesenteric arteries from nonpregnant animals were even more sensitive (EC50=38 mU/ml), but, in these vessels, responsiveness to hCG was significantly attenuated by the pregnant state. Mechanical removal of the vascular endothelium did not reduce the degree of vasodilation mediated by hCG. The expression of hCG receptor mRNA in intact vessels could be demonstrated using reverse transcriptase polymerase chain reaction (RT-PCR). hCG appears to be an important embryonic signal, which could trigger adaptive cardiovascular changes in early pregnancy, simultaneously preserving a sufficient utero-placental perfusion during the entire gestation period by an endothelium-independent mechanism.

Autoradiographic analysis of follicle-stimulating hormone and human chorionic gonadotropin receptors in the ovary of immature rats treated with equine chorionic gonadotropin.

The gonadotropin-primed immature rat has become the most common model for the study of follicular development and ovulation. In this study, prepubertal female rats, 23 and 24 days old, were injected s. c. with 5 IU eCG, and ovaries were collected for topical autoradiography of FSH and hCG receptors at 48 or 24 h post-eCG, respectively (i.e., Day 25). In a baseline group, on Day 25 (before eCG), even the smallest preantral follicles with 1 layer of granulosa cells (GCs; primary follicles) possessed FSH receptors, but hCG receptors were found only on the theca of follicles with 2 or more layers of GCs. Human CG receptors were especially prominent in the interstitium that intimately surrounds preantral follicles without any distinction between theca and interstitial cells. There was a discrete theca surrounding antral follicles. Occasionally antral follicles had hCG receptors in the interstitium, but the adjacent theca was negative, suggesting that these follicles might be destined for atresia. By 24 h post-eCG, a now-discrete theca layer with hCG receptors surrounded all preantral follicles except for the primary follicles, which never responded to eCG. The interstitium was hypertrophied and epithelioid, as was the theca surrounding nonatretic preantral and antral follicles. Increased mitotic activity characterized the growing preantral follicle, and for the first time, FSH binding in GCs of antral follicles was greater than in the preantral population. By 48 h post-eCG, the primary follicles were still unresponsive to eCG. FSH receptors were even more pronounced in the GCs of large antral follicles, although hCG receptors were present in the GCs of only one third of the antral follicles, reflecting the small dose of eCG administered. By 48 h post-eCG, receptors in the interstitium were barely detectable. Using this model, the following study considers the functional in vitro changes in steroidogenesis in follicles from the smallest preantral follicles to the largest antral follicles.

Synthesis of multi-subunit domain gonadotropin complexes: a model for alpha/beta heterodimer formation.

The human glycoprotein hormones chorionic gonadotropin (CG), thyrotropin (TSH), lutropin (LH), and follitropin (FSH) are heterodimers, composed of a common alpha subunit assembled to a hormone-specific beta subunit. The subunits combine noncovalently early in the secretory pathway and exist as heterodimers, but not as multimers. Little information is available regarding the steps associated with the assembly reaction. It is unclear if the initial alpha beta engagement results either in the formation of only mature heterodimer or if the nascent complex is reversible and can undergo an exchange of subunits or combine transiently with an additional subunit. This is relevant for the case of LH and FSH, because both are synthesized in the same cell (i.e., pituitary gonadotrophs) and several of the alpha subunit sequences required for association with either the LH beta or FSH beta subunits are different. Such features could favor the generation of short-lived, multi-subunit forms prior to completion of assembly. Previously, we showed that the CG beta or FSH beta subunit genes can be genetically fused to the alpha gene to produce biologically active single chains, CG beta alpha and F beta alpha, respectively. Studies using monoclonal antibodies sensitive to the conformation of the hCG subunits suggested that in contrast to the highly compact heterodimer, the interactions between the beta and alpha domains in the single chain are in a more relaxed configuration. That the tethered domains do not interact tightly predicts that they could combine with an additional subunit to form triple domain complexes. We tested this point by cotransfecting CHO cells with the genes encoding F beta alpha and the CG beta subunit or the CG beta alpha and FSH beta monomer. The CG beta subunit combined noncovalently with F beta alpha to form a F beta alpha/CG beta complex. Ternary complex formation was not restricted to a specific set of single chain/monomeric subunit, because a CG beta alpha/FSH beta complex was also detected implying that triple domain intermediates could be transiently generated along the secretory pathway. Monoclonal antibodies specific for the CG heterodimer recognized the F beta alpha/CG beta complex, which suggests that the epitopes unique for dimeric CG were established. In addition, media containing F beta alpha/CG beta displayed high-affinity binding to both CG and FSH receptors. The presence of CG activity is presumptive for the existence of a functional F beta alpha/CG beta complex, because neither F beta alpha nor the uncombined CG beta subunit binds to CG receptor. These data show that the alpha subunit of the tether, although covalently linked to the FSH beta domain, can functionally interact with a different beta subunit implying that the contacts in the nascent alpha beta dimer are reversible. The formation of a functional single chain/subunit complex was not restricted to the FSH single chain/CG beta subunit since CG single chain interacts with the monomeric FSH beta subunit and exhibits FSH activity. The presence of the triple domain configuration does not abolish bioactivity, suggesting that although the gonadotropins are heterodimers, the cognate receptor is capable of recognizing a larger ligand composed of three subunit domains.

Immortalized hippocampal cells contain functional luteinizing hormone/human chorionic gonadotropin receptors

We used immortalized HN33p cells as surrogates for hippocampal neurons to investigate the functional importance of luteinizing hormone (LH)/human chorionic gonadotropin (hCG) receptors. The use of various detection techniques demonstrated that HN33p cells contain LH/hCG receptor transcripts and receptor protein that can bind 125I-hCG. Culturing them with highly purified hCG resulted in a significant, although modest, dose-and time-dependent and hormone specific increase in steady state 5-lipoxygenase (5-LO) mRNA and protein levels. The studies on signaling revealed that treatment of HN33p cells with hCG resulted in a robust dose- and a time-dependent significant increase in media cyclic AMP levels. In addition, treatment with a protein kinase (PK)A inhibitor, isoquinolinesulfonamide (H-89), but not with a PKC inhibitor, bisindolylmaleimide (Bis), prevented hCG from increasing the 5-LO protein levels. Pretreatment of HN33p cells for 48 hrs with 2 μM antisense, but not sense, phosphorothioate oligodeoxy-nucleotides (ODN) synthesized from mouse LH/hCG receptor sequence resulted in a dramatic decrease in LH/hCG receptor protein levels. In the antisense, but not in sense, ODN-treated cells, hCG was unable to increase cyclic AMP and 5-LO protein levels, suggesting that receptors are required for hCG to work in HN33p cells.

Evidence that constitutively active luteinizing hormone/human chorionic gonadotropin receptors are rapidly internalized.

The luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor, which belongs to the family of G-protein coupled receptors, plays an important role in gonadal steroidogenesis. Substitution of aspartic acid 556 of the LH/hCG receptor with glycine (D556G) creates a constitutively active receptor that activates adenylyl cyclase in the absence of hormone. To examine receptor internalization, human embryonic kidney cells (293 T) expressing wild type (WT) or D556G mutant receptors were incubated with [125I]hCG and subsequently analyzed for cell surface bound and internalized radioactivity. Comparison of the rate constants of internalization of the D556G mutant and WT receptors revealed that the rate of internalization of the D556G mutant was five times greater than that of the WT receptor. Although the D556G receptor internalizes [125I]hCG rapidly, a corresponding increase in [125I]hCG degradation was not seen. The internalization of another constitutively active LH/hCG receptor (aspartic acid 556 to tyrosine) was also greater than that of the WT receptor. Internalization of receptor bound [125I]hCG was inhibited by a hypertonic sucrose solution, confirming that the ligand enters the cell by receptor-mediated endocytosis. Furthermore, the constitutively active D556G and D556Y LH/hCG receptors utilize the arrestin dependent internalization pathway. These results suggest that the active state conformation of the constitutively active receptor is conducive to rapid internalization.

Messenger ribonucleic acid for the gonadal luteinizing hormone/human chorionic gonadotropin receptor is not present in human endometrium

Objective: To determine whether messenger RNA for the gonadal LH/hCG receptor is present in human endometrium with the use of reverse-transcriptase polymerase chain reaction. Design: In vitro experiment. Setting: Academic medical center. Patient(s): Premenopausal women who were not receiving hormonally active medications and who were undergoing hysterectomy for uterine leiomyomas, menorrhagia, pelvic pain, or uterine prolapse. Intervention(s): Tissue from hysterectomy specimens was processed for RNA and treated with deoxyribonuclease where appropriate, and RNA was reverse-transcribed to complementary DNA. Main Outcome Measure(s): An appropriately sized band after reverse-transcriptase polymerase chain reaction, followed by sequencing to confirm the results. Result(s): A primer pair that spanned the extracellular domain was unable to amplify receptor complementary DNA from human endometrial tissue. For a primer pair that spanned transmembrane regions 2–6 of the receptor and was contained wholly in exon 11, a 552–base pair fragment was amplified successfully in 19 of 25 human endometrial samples. Conclusion(s): The traditional gonadal LH/hCG receptor does not appear to be present in human endometrial tissue. The presence of a portion of the transmembrane part of the molecule suggests that human endometrium may express a truncated or variant form of the receptor.

Treatment of human endometrial stromal cells with chorionic gonadotropin promotes their morphological and functional differentiation into decidua

Human endometrial stromal cells contain luteinizing hormone (LH)/human chorionic gonadotropin (hCG) receptors and treatment with highly purified hCG results in an up-regulation of cyclooxygenase-2 (COX-2) gene expression and increased production of prostaglandin (PG) E 2. Since PGE 2 promotes the differentiation of endometrial stromal cells into decidua, we tested the hypothesis that LH and hCG themselves may promote this process. The results revealed that these hormones can promote morphological as well as functional differentiation. While their action on morphological differentiation did not require the presence of estradiol (E 2) and progesterone (P 4), they did require them for the functional differentiation. The hCG effect was mimicked by LH, but not by follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH) or α and β subunits of hCG, suggesting that the hCG action was hormone specific and requires the conformation of native hormone. The hCG treatment also increased the steady state PRL mRNA levels. This increase was due to an increase in the transcription rate of the gene rather than a decrease in the degradation of PRL transcripts. In summary, we conclude that hCG and LH can increase the morphological as well as functional differentiation of human endometrial stromal cells into decidua. This is one of the newly discovered actions of LH and hCG that may be important for the implantation of blastocyst and maintenance of pregnancy.

Analysis of the promoter of the luteinizing hormone/human chorionic gonadotropin receptor gene in neuroendocrine cells

We investigated the molecular basis of luteinizing hormone (LH)/human chorionic gonadotropin (hCG) receptor gene transcription in immortalized αT3 gonadotropes, hypothalamic GT1–7 and hippocampal HN33p neurons. Nuclear run-on transcription, as well as transfection assays with fusion constructs of luciferase and the 5′-flanking region of LH/hCG receptor gene, revealed that GT1–7 neurons transcribe more than the αT3 or HN33p cells. Transient transfection of truncated reporter gene constructs and gel mobility shift assays revealed that while all neuroendocrine cells use the same promoter, they contain different levels of promoter binding proteins. Higher levels of these proteins may explain increased transcription of the LH/hCG receptor gene in GT1–7 neurons compared with αT3 and HN33p cells.

Invasion of cytotrophoblastic JEG-3 cells is stimulated by hCG in vitro

Trophoblast invasion into the uterine wall is controlled by many factors. Previously, a human chorionic gonadotropin (hCG) receptor has been found to be expressed on invasive trophoblast as well as on choriocarcinoma cells implying a possible role for the hormone in trophoblast invasion. Therefore, this study examined the role of hCG in the invasion of trophoblastic (JEG-3) cells. Increasing hCG concentrations were applied in a trophoblast invasion model, JEG-3, through matrigel-coated filters. The proliferation was quantified by WST-1 cleavage assay. Cell migration was studied by examining the number of cells that had passed the uncoated porous (8-μn pore size) filters. After staining, filters were examined microscopically for cells on the underside of the membrane. A quantitative protease assay was also performed. Flow cytometric analysis of a5 and a6 integrin subunits, which are essential for interactions between cells and extracellular matrix, was performed. hCG increased significantly ( P<0.01) the in vitro invasion of trophoblastic JEG-3 cells in a dose-dependent manner. Migration was also increased by hCG ( P<0.01). However, cell proliferation remained unchanged. The second messenger analogue dibutyryl CAMP (db cAMP) and the CAMP elevating factor (forskolin) mimicked the effects of hCG by stimulating a dose-dependent increase of trophoblastic cell (JEG-3) invasion. The collagenolytic activity of trophoblastic cells (IEG-3) was increased by hCG stimulation. No changes were shown in the expression of α5 and α6 integrin subunits on JEG-3 cells. In vitro hCG is a regulatory factor of invasion and migration in trophoblastic JEG-3 cells, whereas proliferation is not influenced. The endogenous production of hCG by the trophoblast in vivo implies an autocrine control of invasion processes by hCG.