Solubilization and biochemical characterization of the human myometrial corticotrophin-releasing hormone receptor.
We have solubilized an active form of the myometrial corticotrophin-releasing hormone (CRH) receptor using 1% w/v digitonin. The solubilized receptor retains its capacity for high-affinity binding as demonstrated by Scatchard analysis, although there was a shift in dissociation constant (Kd) from 83.6 +/- 15-195 +/- 35 pM for the membrane-bound and soluble receptor respectively. There was no difference in the maximum binding site concentrations (Bmax) of 13 +/- 5 and 21.5 +/- 6 fmol/mg protein for the membrane-bound and soluble receptor respectively. Sauvagine unlike CRH had no effect on radiolabeled CRH binding which suggests that the CRH-R2 receptor is not present in the myometrium. The solubilized receptor did not retain guanine-nucleotide sensitivity. The isoelectric focusing (IEF) profile of the human myometrial CRH receptors was significantly different from that of the rat cerebral cortex. Furthermore, solubilization of human myometrial membrane proteins followed by gel filtration and SDS-PAGE revealed a specifically labeled protein with an apparent molecular weight of 42000-47000 kDa. Our results suggest that during solubilization the human myometrial CRH receptor is dissociated from the guanine nucleotide-binding protein (Gs) and that high affinity binding for soluble CRH receptors is not dependent on the coupling of a guanine nucleotide-binding protein.
Department of Biological Sciences, The University of Warwick, Coventry, UK.
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Grammatopoulos
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Hillhouse
D000818:Animals; D001665:Binding Sites; D002540:Cerebral Cortex / Q000378:metabolism; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004072:Digitonin; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D007525:Isoelectric Focusing; D007700:Kinetics; D008970:Molecular Weight; D009215:Myometrium / Q000378:metabolism*; D051381:Rats; D018019:Receptors, Corticotropin-Releasing Hormone / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility
D018019:Receptors, Corticotropin-Releasing Hormone; D004072:Digitonin