Human Luteinizing Hormone Receptor Research Articles

Expression of Human Luteinizing Hormone (LH) Receptor: Interaction with LH and Chorionic Gonadotropin from Human but not Equine, Rat, and Ovine Species

Studies on human LH receptors are difficult due to the limited availability of clinical samples. Recent cloning of rat and porcine LH receptor cDNAs indicated that these binding sites are single polypeptides of the G-protein-coupled receptor family with seven transmembrane domains. Based on the conserved sequences of rat and porcine receptors, we performed reverse transcription polymerase chain reaction, using human ovarian mRNA as template and obtained partial human LH receptor cDNA clones. Further screening of a human ovary cDNA library and subsequent ligation of individual cDNA clones generated a human LH receptor cDNA containing the entire amino acid-coding region. Sequence analysis indicated that the human receptor cDNA displays 89% and 82% homology at the nucleotide level with its porcine and rat counterparts, respectively. A region spanning the second extracellular and third transmembrane domains is highly conserved among the human LH, FSH, and TSH receptors. The ovarian LH receptor clone is, however, significantly different from an incompletely spliced LH receptor cDNA recently obtained from a human thyroid library. Unlike the thyroid clone, the ovarian LH receptor cDNA could be expressed in the human fetal kidney cell line (293), and radioligand receptor assay identified high affinity (Kd, 1.2 x 10(-10) M) LH/hCG-binding sites on the plasma membrane. Binding specificity of the human LH receptor was studied using recombinant human CG, LH, and FSH secreted by CHO cells transfected with the respective genes. Human CG and LH displaced [125I]hCG binding with an ED50 of 4.3 and 4.8 ng/ml, respectively. In contrast, recombinant FSH was not effective. Treatment of transfected cells with recombinant gonadotropins also induced dose-dependent increases in extracellular cAMP production (hCG = LH much greater than FSH; ED50 25, 10, and greater than 3000 ng/ml). Although equine, rat, and ovine LH as well as equine CG competed effectively for rat testicular LH receptor binding, these hormones were unable to displace [125I]hCG binding to the human receptor, suggesting evolutionary changes in receptor binding specificity and the importance of using human receptors for clinical studies. Thus, the cloning and expression of the human LH receptor cDNA allowed analysis of interactions between human LH receptor and gonadotropins from diverse species. The present work should provide the basis for future design of therapeutic agents capable of interacting with the human receptor and for understanding the structural basis for LH receptor binding to different gonadotropins.

Structure of the Luteinizing Hormone Receptor Gene and Multiple Exons of the Coding Sequence*

The genomic structure of the LH receptor is important to our understanding of its expression mechanisms, functional domains, relationships with other hormone receptors, and evolution. We have isolated four overlapping cosmid clones and six subgenomic clones of the rat LH receptor gene. They span a total of 95.6 kilobases (kb) and extend from 23 kb upstream of the translation start site to 13 kb down-stream of the stop codon. In addition, part of the human LH receptor gene has been isolated. The coding region of the rat hormone receptor gene spans over 60 kb and consists of 11 exons and 10 introns. Southern blots hybridized with exon 1 and exon 11 probes as well as gene dose analyses demonstrate that a single copy gene encodes the rat LH receptor. Sequence comparison suggests that the porcine and human LH receptor genes have similar, if not identical, exon-intron structures. There is no consensus cAMP-responsive element within 600 basepairs up-stream of the translation start site in spite of the cAMP responsiveness of the LH receptor gene. There are, however, unconventional cAMP-responsive elements in the region: one which is identical, several which are homologous to the activating protein-2-binding elements, CCCCAGGC, and several sequences which are similar to the G-rich cAMP-responsive element found in P450c21, a steroid 21-hydroxylase. The first 10 exons encode the N-terminal half of the molecule, while exon 11 encodes the C-terminal half of the molecule. This last exon is the same in the rat and human genes. The DNA and amino acid sequences of the first 10 exons show significant similarities and reveal repetitive sequence motifs. They have similar sizes which occur in the range of 69 and 183 bases; 8 of them are from 69-81 bases. Despite these remarkable similarities, structural predictions of exons 1-10 show a diversity of structures. The N-terminal half of the LH receptor appears to have a folded structure, with frequent turns and an extensive surface area. Part of the surface is predicted to be covered by amphiphilic helices and beta structures, types of secondary structure frequently found at the interfaces between subunits or between 2 interacting molecules. The introns dividing these exons also share many similarities.(ABSTRACT TRUNCATED AT 400 WORDS)

Localization of the human luteinizing hormone/choriogonadotropin receptor gene (LHCGR) to chromosome 2p21

Probes corresponding to human and porcine LH (luteinizing hormone) receptor cDNA were used for in situ hybridization to human chromosomes. This allowed us to assign the LH receptor gene to chromosome 2p21.

Solubilization of luteinizing hormone receptor from human corpora lutea in a stable form and identification of the hormone-binding unit by ligand blotting.

LH receptors were solubilized from human corpora lutea in phosphate-buffered saline containing 1% Triton X-100, 20% glycerol, and protease inhibitors. The presence of 20% (vol/vol) glycerol was necessary for quantitative preservation of [125I]hCG-binding activity in detergent solution. The solubilized receptors were stable for several weeks at -20 C and at -80 C and for at least 18 h at 4 C. Binding of [125I]hCG to the soluble LH receptors was time and temperature dependent and varied linearly with the amount of soluble protein. Equilibrium binding studies revealed a single class of high affinity [125I]hCG-binding sites with an equilibrium dissociation constant (Kd) of 4.3 x 10(-10) mol/L (at 20 C). The molecular size of the human LH receptors was analyzed by ligand blotting. Solubilized receptors were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions and transferred to nitrocellulose. Incubation of the protein blot with [125I]hCG to the 85/90K mol wt species was inhibited by unlabeled hCG. These results indicate that LH receptors can be solubilized in nonionic detergent while maintaining their hormone-binding activity and demonstrate that the receptors contain 85/90K hormone-binding species.

Human testicular LH receptors: correlations with circulating gonadotrophins and testicular steroid secretion.

Testicular androgen production is regulated by circulating LH, the effect of which is mediated by its testicular membrane receptors. In the present study, testicular [125I]hCG binding in man was investigated and found to be characterized by saturability and high affinity (mean KD in testes of 21 patients with prostatic carcinoma = 1.64 X 10(-10) M), and stimulated testosterone production in dispersed interstitial cells in vitro. The concentrations of the LH receptors correlated with serum FSH concentrations (r = 0.52, P less than 0.05) but not with circulating LH levels. Neither had they any correlation with intratesticular testosterone, 5 alpha-dihydrotestosterone, androstenedione, progesterone, 17-hydroxyprogesterone or pregnenolone concentrations, which may be due to the fact that LH receptors are confined to Leydig cells, whereas steroids may be unevenly distributed in the different cells of the testis. In contrast, the concentrations of the LH receptor displayed positive correlations with the concentrations of testosterone (r = 0.81, P less than 0.001), androstenedione (r = 0.54, P less than 0.05), 17-hydroxyprogesterone (r = 0.76, P less than 0.001) and progesterone (r = 0.60, P less than 0.05) in the spermatic vein serum of the patients. Our data suggest that the Leydig cells mainly responsible for steroid secretion into the blood are under gonadotrophic control exerted via their receptors, whereas Leydig cell function is not rapidly reflected in steroid concentrations within the testis itself.

Regulation of Primate Testicular Luteinizing Hormone Receptors and Steroidogenesis

The testicular luteinizing hormone (LH) receptors of the rhesus monkey and human have many features in common, including high equilibrium association constant, marked species specificity, and relatively low binding capacity. We have, therefore, used rhesus monkeys as models for human LH-receptor regulation in vivo during gonadotropin treatment. In four adult male monkeys, treated with 10,000 IU human chorionic gonadotropin (hCG), serum and testicular steroidogenic responses were monitored at 24-h intervals during the following 4 d, and LH-receptor concentrations were measured by (125)I-hCG binding to 15,000-g particles prepared from testis biopsy specimens. In treated animals, serum hCG was maximal on day 1 at 322+/-16 ng/ml and declined to 24.4+/-2.3 ng/ml by day 4. Serum testosterone was increased threefold during the subsequent 4 d (from 6.5+/-2.0 to 18.6+/-4.4 ng/ml) but serum progesterone remained unchanged. In contrast, serum 17alpha-hydroxyprogesterone increased 12-fold to 5.5+/-0.5 ng/ml at day 1 and was increased fourfold during the subsequent 3 d. The LH-receptor binding capacity of the primate testis was reduced by 18.3+/-6.0% on day 1, 51.7+/-7.4% on day 2, and 45.3+/-2.4% on day 4. Occupancy of the LH receptors by endogenously bound hCG was significant on day 1 but was negligible by day 4. These data demonstrate that gonadotropin-induced LH-receptor depletion occurs in the rhesus testis and indicate that primate gonadotropin receptors are susceptible to the regulatory processes recently described in the rat. In addition, the simultaneous and disproportionate accumulation of 17alpha-hydroxyprogesterone indicates that 17,20-desmolase was rate-limiting under these conditions in the primate testis Leydig cell.