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.
Department of Reproductive Medicine, University of California-San Diego, La Jolla 92093.
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D006063:Chorionic Gonadotropin / Q000378:metabolism*; D003001:Cloning, Molecular; D005260:Female; D015723:Gene Library; D006736:Horses; D006801:Humans; D007700:Kinetics; D007986:Luteinizing Hormone / Q000378:metabolism*; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010053:Ovary / Q000502:physiology; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011974:Receptors, LH / Q000235:genetics* / Q000378:metabolism; D011988:Receptors, Thyroid Hormone / Q000235:genetics; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D012756:Sheep; D013045:Species Specificity; D013961:Thyroid Gland / Q000502:physiology; D014162:Transfection
D006063:Chorionic Gonadotropin; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D011974:Receptors, LH; D011988:Receptors, Thyroid Hormone; D007986:Luteinizing Hormone