Solubilization and pharmacological characterization of a glucocorticoid membrane receptor from an amphibian brain.
Physiological functions of steroid hormones involve activation of intracellular receptors as well as poorly understood membrane receptors. We report the pharmacological characterization of a solubilized corticosterone receptor from neuronal membranes. This receptor previously was shown to localize with plasma membrane subcellular fractions and to be involved in the modulation of courtship behaviors in the roughskin newt (Taricha granulosa). We describe procedures with non-ionic detergents that solubilize the receptor and maintain high affinity [3H]corticosterone binding. The pharmacology of the solubilized corticosterone receptor resembles that of the membrane receptor with high affinity for [3H]corticosterone and an identical rank-order potency for other steroid ligands (corticosterone>cortisol>aldosterone>dexamethasone). Unlike binding in membrane preparations, [3H]corticosterone binding to the solubilized receptor is insensitive to negative modulation by guanyl nucleotides and only modestly sensitive to the presence of Mg2+. We also identified two ligands that exhibit high affinity binding to the solubilized receptor and have the potential to be used in an affinity purification scheme. They are corticosterone-3-carboxymethyloxime (CORT-3-CMO), which may be covalently attached to a Sepharose resin, and a derivitized azide form of CORT-3-CMO which can be covalently coupled to the solubilized receptor itself. The stability of the solubilized [3H]corticosterone receptor in the detergent system will facilitate further purification and molecular characterization.
Molecular and Cellular Biology Program, Oregon State University, Corvallis 97331, USA. evanss@bcc.orst.edu
S J
SJ
Evans
F L
FL
Moore
T F
TF
Murray
D000345:Affinity Labels; D000818:Animals; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism*; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism; D003345:Corticosterone / Q000378:metabolism; D006150:Guanine Nucleotides / Q000494:pharmacology; D007700:Kinetics; D015636:Magnesium Chloride / Q000494:pharmacology; D008297:Male; D009474:Neurons / Q000378:metabolism*; D011869:Radioligand Assay; D011987:Receptors, Steroid / Q000302:isolation & purification* / Q000378:metabolism*; D012455:Salamandridae; D012995:Solubility; D013574:Synaptosomes / Q000378:metabolism*; D014316:Tritium
D000345:Affinity Labels; D006150:Guanine Nucleotides; D011987:Receptors, Steroid; C024357:corticosterone receptor; D015636:Magnesium Chloride; D014316:Tritium; D003345:Corticosterone