Selection and characterization of mammalian cell lines with stable over-expression of human pituitary receptors for gonadoliberin.
In: European Journal of Biochemistry, Jg. 231 (1995-08-01), Heft 3, S. 535-543
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Zugriff:
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 stimutatory effect of gonadotiberin 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. [ABSTRACT FROM AUTHOR]
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Selection and characterization of mammalian cell lines with stable over-expression of human pituitary receptors for gonadoliberin.
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Autor/in / Beteiligte Person: | Beckers, Thomas ; Marheineke, Kathrin ; Reiländer, Helmut ; Hilgard, Peter |
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Zeitschrift: | European Journal of Biochemistry, Jg. 231 (1995-08-01), Heft 3, S. 535-543 |
Veröffentlichung: | 1995 |
Medientyp: | academicJournal |
ISSN: | 0014-2956 (print) |
DOI: | 10.1111/j.1432-1033.1995.0535d.x |
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