title: Selection and characterization of mammalian cell lines with stable over-expression of human pituitary receptors for gonadoliberin.
abstract: The cDNA encoding the receptor for gonadoliberin (GnRH or LH-RH) was isolated from a human pituitary cDNA library and heterologously expressed in the murine fibroblast cell line LTK-. By using a dicistronic expression strategy utilizing the internal ribosomal-entry-site sequence of poliovirus, single cell clones with stable and high expression of human gonadoliberin receptors were selected. In radioligand saturation-binding experiments, the gonadoliberin antagonist Cetrorelix showed high-affinity binding to the heterologously expressed human gonadoliberin receptor with a Kd of 0.1 nM. The pharmacological profile using 125I-Cetrorelix as radioligand and the authentic gonadoliberin or agonistic and antagonistic derivatives as competitors, showed a distinct rank order of binding potencies. Superagonistic gonadoliberin derivatives had more than ten-times higher binding affinities in comparison to gonadoliberin with a Kd of 3.47 nM. The gonadoliberin receptor expressed in stably transfected LTK- cells coupled to the inositol phosphate signal-transduction pathway. Gonadoliberin stimulated the synthesis of inositol 1,4,5-trisphosphate in a dose-dependent way with an EC50 of 5 nM. This stimulatory effect of gonadoliberin was completely antagonized by Cetrorelix in equimolar concentrations, demonstrating the high potency of this competitive receptor antagonist. In growth-arrested cells, a transient expression of the c-fos protooncogene was induced by gonadoliberin or [D-Trp6]gonadoliberin, showing that the gonadoliberin receptor couples to a putative mitogenic signal-transduction pathway in this heterologous cell system.
authors.affiliation: ASTA Medica AG, Frankfurt/Main, Germany.
authors.forename: T
authors.initials: T
authors.lastname: Beckers
authors.forename: K
authors.initials: K
authors.lastname: Marheineke
authors.forename: H
authors.initials: H
authors.lastname: Reiländer
authors.forename: P
authors.initials: P
authors.lastname: Hilgard
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary; D016762:Genes, fos; D007987:Gonadotropin-Releasing Hormone / Q000378:metabolism*; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000096:biosynthesis; D051379:Mice; D008969:Molecular Sequence Data; D010902:Pituitary Gland / Q000378:metabolism*; D011869:Radioligand Assay; D011966:Receptors, LHRH / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D018076:DNA, Complementary; D011966:Receptors, LHRH; D011993:Recombinant Fusion Proteins; D007987:Gonadotropin-Releasing Hormone; D015544:Inositol 1,4,5-Trisphosphate