Partial purification and biochemical characterization of a membrane glucocorticoid receptor from an amphibian brain

• Published in: The Journal of steroid biochemistry and molecular biology Vol. 72; no. 5; pp. 209 - 221
• Main Authors: Evans, Simon J., Murray, Thomas F., Moore, Frank L.
• Format: Journal Article
• Language: English
• Published: Oxford Elsevier Ltd 01.04.2000; Elsevier Science
• Subjects: Ammonium Sulfate - chemistry; Amphibia; Animals; Biological and medical sciences; Brain - metabolism; Brain Chemistry; Chromatography, Affinity; Chromatography, Agarose - methods; Chromatography, Ion Exchange - methods; Corticosterone - metabolism; corticosterone receptors; Durapatite - chemistry; Freshwater; Fundamental and applied biological sciences. Psychology; Hormones and neuropeptides. Regulation; Hypothalamus. Hypophysis. Epiphysis. Urophysis; Lectins - metabolism; Membrane Proteins - chemistry; Membrane Proteins - isolation & purification; Membrane Proteins - metabolism; Photoaffinity Labels - chemistry; Receptors, Glucocorticoid - chemistry; Receptors, Glucocorticoid - isolation & purification; Receptors, Glucocorticoid - metabolism; Salamandridae; Taricha granulosa; Vertebrates: endocrinology; Wheat Germ Agglutinins - metabolism; Purification; Steroid hormone; Corticosterone; Caudata; Central nervous system; Amphibia; Glucocorticoid; Characterization; Vertebrata; Membrane receptor; Adrenal hormone; Biological receptor; Brain (vertebrata); Hormonal receptor
• ISSN: 0960-0760; 1879-1220
• DOI: 10.1016/S0960-0760(00)00031-5

A membrane receptor for corticosterone (mGR) in the brain of the roughskin newt ( Taricha granulosa) has been previously identified. This manuscript reports the evaluation of several chromatographic resins for enrichment of the newt mGR solubilized from neuronal membranes. A protein with an apparent molecular weight of 63 kDa was purified to near homogeneity following sequential purification using ammonium sulfate fractionation, wheat germ agglutinin (WGA)-agarose chromatography, hydroxylapatite chromatography, and an immobilized ligand affinity resin (Corticosterone-Sepharose). Other studies employed a novel protein differential display strategy and a photoaffinity labeling strategy to visualize candidate receptor proteins following SDS–PAGE. Both of these techniques also identified a 63 kDa protein, agreeing with the estimation of molecular weight from the purification data. Furthermore, the use of 2D SDS–PAGE following the photolabeling procedure showed the candidate 63 kDa protein to have a p I of approximately 5.0. Taken together these data suggest that the newt mGR is an acidic glycoprotein with an apparent molecular weight of 63 kDa. Because these characteristics of newt mGR are inconsistent with the characteristics of intracellular glucocorticoid receptors, these two receptor proteins are apparently distinct.