Solubilization and characterization of the rat pituitary gonadotrophin-releasing hormone receptor

Abstract

Specific receptors for gonadotrophin-releasing hormone (GnRH) were extracted from the rat pituitary gland with several detergents and characterized by binding studies with the potent GnRH antagonist [Ac-D-pCl-Phe1.2, D-Trp3, D-Lys6, D-Ala10]-GnRH (GnRHant). The particulate GnRH receptors were most effectively solubilized with 5 mM 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulphonate (CHAPS), which extracted 63% of the original membrane binding activity when assayed with 125I-labelled GnRHant. The binding affinities of particulate and CHAPS-solubilized receptors analysed with 125I-labelled GnRHant were 1.5 +/- 0.4 x 10(9) M-1 and 1.2 +/- 0.2 x 10(9) M-1 respectively. Gel filtration of the CHAPS-solubilized receptor revealed a major peak of specific binding activity with Mr of about 700,000. A hormone-receptor complex of similar Mr was observed when CHAPS-solubilized receptors were labelled with photoreactive radioiodinated [D-Lys6]-des-Gly10-GnRH-N-ethyl-amide and then analysed by gel chromatography. However, when pituitary particles were photolabelled and solubilized in 2% Triton X-100 before analysis on Sephacryl S-300, the Mr of the receptor was approximately 250,000, similar to the value obtained by sucrose density gradient centrifugation of the CHAPS-solubilized receptor. After solubilization in sodium dodecyl sulphate (SDS) the photolabelled receptor was eluted from Sephacryl S-300 as a 60 kDa peak which on SDS-gel electrophoresis contained a 52 kDa component, corresponding to the major binding subunit extracted directly from photolabelled pituitary membranes. The difference in higher molecular weight forms observed under non-denaturing and denaturing conditions could reflect the need for additional membrane components to maintain the active conformation of the GnRH receptor site. Whereas the minimum Mr of the solubilized receptor is about 250,000 under non-denaturing conditions, analysis of the photolabelled GnRH-receptor complex by SDS chromatography and electrophoresis indicates that a binding subunit with Mr of 50,000-60,000 is present in the GnRH holoreceptor.

The Gonadotropin-Releasing Hormone Receptor: Signals Involved in Gonadotropin Secretion and Biosynthesis

“…Down-regulation of GnRH receptors represents most likely only one aspect of the phenomenon of desensitization which is caused by prolonged exposure to GnRH in vivo and in vitro. Uncoupling of the GnRH transducer (G protein) and/or the effector (phospholipase C; PLC) is probably the ma jor biochemical process contributing to desensitization.A 60-kilodalton membrane protein is presumed to be the GnRH receptor as revealed by photoaffinity labeling and ligand immunoblotting [15][16][17], Similar binding sites are present in rat gonads and human placenta, but the extrapituitary effects of GnRH and its analogues are still not clear [18][19][20][21][22], The GnRH receptor was solubi lized recently and purified from pituitary membranes, and monoclonal antibodies against the receptor were raised [23][24][25], Further studies will, therefore, concen trate on isolation of the GnRH receptor gene. Unlike thyrotropin-releasing hormone or somatostatin which interact with various pituitary' cells, GnRH binds to and activates only the gonadotrophs as evident from bio chemical, morphological and cytochemical studies [26][27][28][29],…”