Solubilization and characterization of pituitary thyrotropin-releasing hormone receptors.
Thyrotropin-releasing hormone (TRH) receptors were solubilized from a rat pituitary tumor cell line, GH4C1, with digitonin. Convenient assays were developed based on the ability of hydroxylapatite and polyethyleneimine-soaked glass fiber filters to adsorb the solubilized [3H]methyl-TRH-receptor complex but not free [3H]methyl-TRH. The kinetics of [3H]methyl-TRH binding to solubilized receptors were extremely temperature dependent. Binding reached equilibrium at 10-20 nM [3H]methyl-TRH in 30 min at 23 degrees and 6 hr at 0 degree. The half-times for dissociation were less than 5 min at 23 degrees and 7.6 hr at 0 degree. Equilibrium binding experiments yielded linear Scatchard plots at 0 degree with Kd = 3 nM, whereas the Kd was greater than 20 nM at 23 degrees. A series of TRH congeners displaced [3H]methyl-TRH with the rank order reported for membrane receptors, N3-methyl-HisTRH greater than or equal to TRH greater than pGlu-N3-methyl-HisProNH(CH2)6NH2 greater than or equal to pGluHisProNH(CH2)6NH2 greater than pGluHisTyrNH2 much greater than TRH free acid. The antagonist chlordiazepoxide exhibited an IC50 of 10 microM. [3H]methyl-TRH binding to solubilized receptors displayed a broad pH optimum, from 6.5 to 7.5. The solubilized receptor could be obtained from cultured GH4C1 cells and in much larger quantities from GH4C1 tumors. Tumors from 12 rats yielded greater than 700 pmol of specific soluble TRH binding activity (1 g of protein). The solubilized receptor could be purified 10-20-fold by chromatography on wheat germ agglutinin columns and could be concentrated by adsorption on either DEAE-Sephadex or hydroxylapatite. The procedures outlined allow the solubilization of pituitary TRH receptors from a rich and abundant source, the rapid and reproducible assay of [3H]methyl-TRH binding, and substantial enrichment of receptor activity. These findings should be valuable for the purification and identification of the TRH receptor protein.
Phillips
W J
WJ
Department of Pharmacology, University of Rochester School of Medicine and Dentistry, New York.
Hinkle
P M
PM
D000818:Animals; D002460:Cell Line; D002845:Chromatography; D004072:Digitonin; D066298:In Vitro Techniques; D007700:Kinetics; D009374:Neoplasms, Experimental / Q000032:analysis; D010902:Pituitary Gland / Q000032:analysis*; D051381:Rats; D017981:Receptors, Neurotransmitter / Q000032:analysis* / Q000378:metabolism; D018025:Receptors, Thyrotropin-Releasing Hormone; D012995:Solubility; D013973:Thyrotropin-Releasing Hormone / Q000502:physiology*
D017981:Receptors, Neurotransmitter; D018025:Receptors, Thyrotropin-Releasing Hormone; D013973:Thyrotropin-Releasing Hormone; D004072:Digitonin