Novel ligands for the affinity labelling of luteinizing hormone releasing hormone receptors.

A number of novel luteinizing hormone releasing hormone (LHRH) analogues incorporating biotin together with potential covalent attachment sites have been synthesized. Those based on the des-Gly10-[D-Lys6]-LHRH ethylamide peptide backbone resulted in the most useful characteristics of binding to the LHRH receptor in rat anterior pituitary gland membranes. Of these, des-Gly10-[biotinyl-aminoethylglycyl-D-Lys6]-LHRH ethylamide (XBAL) gave the best specific: non-specific binding ratio, with 44 +/- 6% (+/- S.E.M.) of total binding being specific with a Kd of 131 +/- 16 pM (+/- S.E.M., n = 4) as determined by Scatchard analysis. Two methods have been used to covalently crosslink these analogues with the LHRH receptor; photoaffinity labelling and the use of homobifunctional N-hydroxysuccinimide ester crosslinkers. The photoaffinity analogues gave poor specific: non-specific binding ratios. Of the chemical crosslinkers tested, ethylene glycolbis(succinimidylsuccinate) (EGS) was found to be the most efficient at covalently linking the 125I-XBAL bound to the LHRH receptor site. At an EGS concentration of 5 mM, 23 +/- 3% (+/- S.E.M.) of the specific binding of 125I-XBAL was covalently crosslinked.

Facilitated calcium mobilization and inositol phosphate production in the priming effect of LH-releasing hormone in the rat.

The ability of LHRH to induce Ca2+ mobilization and production of inositol phosphates in rat anterior pituitary tissue in vitro was investigated in relation to the self-priming effect of LHRH. Prior exposure to LHRH (which caused a characteristic potentiation of subsequent secretory responses) specifically enhanced LHRH-induced inositol phosphate production and mobilization of intracellular Ca2+ stores. LHRH-induced influx of Ca2+ through dihydropyridine-sensitive Ca2+ channels was unaltered, as was ligand binding to LHRH receptors. These data suggest that a novel facilitation of signalling may occur in the phospho-inositide-Ca2+ mobilization response mechanism during LHRH priming, and that this may represent an important means of regulating cellular responsiveness in gonadotrophs.

Solubilization of a large molecular weight form of the rat LHRH receptor.

The LHRH receptor has been solubilized from male rat anterior pituitary glands, using the zwitterionic detergent 3-((3-cholamidopropyl)-dimethylammonio)-1-propanesulphonate in the presence of a high concentration of sodium chloride. This method gave high yields (up to greater than 70%) of the LHRH-binding site from the membrane preparation. Ligand binding studies using LHRH analogues were carried out to determine dissociation constants for LHRH receptors both in situ in the membrane preparation and for solubilized LHRH receptors. For all the analogues the binding characteristics were similar in both preparations, suggesting that the solubilization procedure left the LHRH receptor undenatured. Gel filtration revealed an apparent molecular weight for the LHRH receptor of 100,000-160,000, with the mean value being approximately twice that found by others using sodium dodecyl sulphate-polyacrylamide gel electrophoretic techniques. The results indicate that the LHRH receptor probably exists in gonadotroph membranes as a large complex of more than one subunit.