Binding characteristics of glucocorticoid receptor in synaptic plasma membrane from rat brain

The study was carried out to identify the specific glucocorticoid membrane binding site (GCMB) on synaptic plasma membrane (SPM), and to investigate the ligand binding properties and physico-chemical characteristics of GCMB. The radioligand binding assay showed that tritiated corticosterone ([3H]B) could bind to GCMB specifically with positive cooperativity. For SPM, the binding capacity (R(zero)) was 46.67 +/- 4.62 fmol/mg protein; the dissociation constant (Kd), 97.48 +/- 18.85 nmol/L, and the Hill coefficient, 1.633 +/- 0.082. Various steroids bound with GCMB in the following rank order: RU26752, aldosterone > corticosterone, cortisol > RU26988, progesterone, R5020, triamcinolone acetonide > testosterone, estradiol > RU38486 > dexamethasone. GCMB, which could be solubilized by detergent Triton X-100, showed thermal stability, although it was susceptible to hydrolysis by trypsin and to the reducing agent, mercaptoethanol. The addition of divalent cations (Ca2+, Mg2+) was beneficial to the ligand binding. The results strongly indicate that GCMB, fulfilling the basic criteria for a receptor, constitutes a novel type of glucocorticoid receptor on neuronal membrane which is significantly different from the cytosolic glucocorticoid receptors.