SELECTION AND CHARACTERIZATION OF MAMMALIAN-CELL LINES WITH STABLE OVER-EXPRESSION OF HUMAN PITUITARY RECEPTORS FOR GONADOLIBERIN

The cDNA encoding the receptor for gonadoliberin (GnRH or LH-RH) was i solated from a human pituitary cDNA library and heterologously express ed in the murine fibroblast cell line LTK(-). By using a dicistronic e xpression strategy utilizing the internal ribosomal-entry-site sequenc e of poliovirus, single cell clones with stable and high expression of human gonadoliberin receptors were selected. In radioligand saturatio n-binding experiments, the gonadoliberin antagonist Cetrorelix showed high-affinity binding to the heterologously expressed human gonadolibe rin receptor with a K-d of 0.1 nM. The pharmacological profile using I -125-Cetrorelix as radioligand and the authentic gonadoliberin or agon istic and antagonistic derivatives as competitors, showed a distinct r ank order of binding potencies. Superagonistic gonadoliberin derivativ es had more than ten-times higher binding affinities in comparison to gonadoliberin with a K-d of 3.47 nM. The gonadoliberin receptor expres sed in stably transfected LTK(-) cells coupled to the inositol phospha te signal-transduction pathway. Gonadoliberin stimulated the synthesis of inositol 1,4,5-trisphosphate in a dose-dependent way with an EC(50 ) of 5 nM. This stimulatory effect of gonadoliberin was completely ant agonized by Cetrorelix in equimolar concentrations, demonstrating the high potency of this competitive receptor antagonist. In growth-arrest ed cells, a transient expression of the c-fos protooncogene was induce d by gonadoliberin or [D-Trp6]gonadoliberin, showing that the gonadoli berin receptor couples to a putative mitogenic signal-transduction pat hway in this heterologous cell system.