Solubilization and characterization of the rat pituitary gonadotrophin-releasing hormone receptor.
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.
Iwashita
M
M
Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, Bethesda, Maryland 20892.
Hirota
K
K
Izumi
S I
SI
Chen
H C
HC
Catt
K J
KJ
D000345:Affinity Labels; D000818:Animals; D001386:Azides; D001667:Binding, Competitive; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002793:Cholic Acids; D003902:Detergents; D008297:Male; D008970:Molecular Weight; D010902:Pituitary Gland / Q000378:metabolism*; D011869:Radioligand Assay; D051381:Rats; D011919:Rats, Inbred Strains; D011966:Receptors, LHRH / Q000302:isolation & purification* / Q000378:metabolism
D000345:Affinity Labels; D001386:Azides; D002793:Cholic Acids; D003902:Detergents; D011966:Receptors, LHRH; C029534:hydroxysuccinimidyl-4-azidobenzoate; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate