title: Cloning and functional expression of the equine luteinizing hormone/chorionic gonadotrophin receptor.
abstract: Pituitary equine luteinizing hormone (eLH) and fetal chorionic gonadotrophin (eCG) have identical polypeptidic chains, but different linked carbohydrates. In equine tissues, eCG and eLH bind only to the LH/CG receptor (eLH/CG-R) and have no FSH activity. However, radio-receptor assays on equine luteal or testicular tissues have shown that eCG binds to the eLH/CG-R with only 2-4% of the binding activity of eLH. In order to study the structure-function relationship of eLH and eCG in a homologous system, we undertook the cloning and functional expression of the eLH/CG-R. Based on sequence homologies among mammalian sequences for the LH/CG-R, overlapping partial fragments of LH/CG-R cDNAs were obtained from mare luteal RNA using reverse transcription-PCR and 5'-rapid amplification of cDNA ends. Ligations of the partial cDNA fragments encoded a part of the signal peptide followed by a putative 672 amino acid eLH/CG-R mature protein. The mature eLH/CG-R displayed 88.2-92.8% overall sequence homology with the other mammalian LH/CG-Rs and contained one unique seventh N-glycosylation site in its extracellular domain. COS-7 cells were transiently transfected with a cDNA construct encoding an engineered complete signal peptide and the mature eLH/CG-R. Membrane preparations from transfected COS-7 cells bound 125I-eLH with high affinity (Kd 3.8 x 10(-10) M). On a molar basis, eCG competed with 125I-eLH on membrane preparations with only 12.4% of the eLH binding activity. In transfected COS-7, both eLH and eCG increased the extracellular cAMP concentration in a dose-dependent manner, whereas eFSH did not. Furthermore, on a molar basis, eCG stimulated cAMP production with only 13.9% of the eLH stimulating activity. We conclude that the cloned cDNA encodes a The differences functional eLH/CG-R. between eLH and eCG activities towards this receptor will be useful in studies of the influence of carbohydrates on gonadotrophin receptor binding and activation.
authors.affiliation: Unité de Physiologie de la Reproduction et des Comportements, UMR 6175 INRA-CNRS-Université F. Rabelais de Tours-Haras Nationaux, 37 380 Nouzilly, France. stdizier@inapg.fr
authors.forename: Marie
authors.initials: M
authors.lastname: Saint-Dizier
authors.forename: Florence
authors.initials: F
authors.lastname: Foulon-Gauze
authors.forename: François
authors.initials: F
authors.lastname: Lecompte
authors.forename: Yves
authors.initials: Y
authors.lastname: Combarnous
authors.forename: Maryse
authors.initials: M
authors.lastname: Chopineau
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002417:Cattle; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005260:Female; D015870:Gene Expression; D006736:Horses; D006801:Humans; D007986:Luteinizing Hormone / Q000494:pharmacology; D008969:Molecular Sequence Data; D051381:Rats; D011974:Receptors, LH / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D013552:Swine; D014162:Transfection
chemical_list: D011974:Receptors, LH; D007986:Luteinizing Hormone; D000242:Cyclic AMP