The Journal of Steroid Biochemistry and Molecular Biology
Solubilization and pharmacological characterization of a glucocorticoid membrane receptor from an amphibian brain
Introduction
Steroid hormones use intracellular receptors that bind DNA in a sequence specific manner to modulate transcriptional activity of target genes. Evidence for rapid steroid actions through plasma membrane receptors has accumulated in recent years (review by Wehling[1]) and comes from pharmacological, electrophysiological and behavioral experiments.
Progestins2, 3, glucocorticoids4, 5, 6, estrogens[7], mineralocorticoids8, 9and 1,25(OH)2D3[10]have all been shown to bind specifically to cellular membrane sites. Rapid influences on electrolyte movement across cellular membranes are also induced by progesterone[11], glucocorticoids12, 13, estradiol14, 15, aldosterone[8], 1,25(OH)2D3[16]and testosterone[17]. Steroid hormones can also effect rapid behavioral changes, such as the rapid inhibition of mating behavior in male newts4, 18.
Some steroid membrane receptors may use G-proteins in the signal transduction machinery. Cortisol-induced inhibition of Ca2+ currents in guinea pig is diminished by pertussis toxin treatment or the presence of GDPβS[12]. Corticosterone binding in newt brains is inhibited by guanyl nucleotide analogs in a concentration dependent manner[19]. Aldosterone effects on HML cells involve 1,4,5-inositol trisphosphate and are sensitive to pertussis toxin, but not cholera toxin[8]. Estradiol inhibition of Ca2+ currents in rat neostriatal neurons is reversed by GTPγS[15]. Testosterone stimulation of Ca2+ currents and induction of IP3 and DAG in rat osteoblasts is blocked by pertussis toxin[17]. All of these observations are both consistent with and characteristic of G-protein coupled signal transduction mechanisms.
A receptor for corticosterone found in newt (Taricha granulosa) neuronal membranes has been identified and characterized[4]. This protein also possesses characteristics of a G-protein coupled receptor[19]. A single intraperitoneal injection of corticosterone inhibits courtship behavior with a latency of less than 8 min[4]and inhibits stress-induced locomotion in a similar time frame[20]. The affinities of an array of steroids for this [3H]corticosterone binding site are correlated with their respective potencies in newt behavioral assays[4]. The receptor is enriched in plasma membrane fractions prepared by sucrose density centrifugation and does not bind dexamethasone and other ligands which have high affinity for intracellular glucocorticoid receptors[4]. These data suggest that there is a membrane corticosterone receptor which is distinct from the intracellular corticosterone receptor.
Detergent solubilization followed by affinity purification of membrane receptors has proven useful in advancing molecular and biochemical understanding of various receptor systems. We have extended the work with the newt corticosterone membrane receptor by solubilizing the receptor in neuronal membranes using non-ionic detergents. We have characterized the solubilized receptor and identified ligands that can be used during affinity purification.
Section snippets
Materials
Male T. granulosa were collected locally and used immediately or stored in tanks containing de-chlorinated water for up to three weeks. Stored animals were feed tubifix worms on a three day cycle and were handled in compliance with accepted animal use guidelines.
[3H]corticosterone (71.07 Ci/mmol) was purchased from NEN/Dupont (Boston, MA). Corticosterone, aldosterone, dexamethasone, digitonin, sodium cholate, bacitracin, trypsin inhibitor, dioxane and Sephadex were from Sigma (St. Louis, MO).
Preparation of neuronal membranes
Synaptosomes were prepared from newt brains by the method of Whittaker[21]with modifications by Orchinik[22]. Briefly, newt brains were homogenized in 40 vol. (per original weight) 0.32 M sucrose, 5 mM HEPES, pH 7.45 using a Teflon on glass tissue homogenizer. Homogenate was centrifuged at 1,000×g for 10 min at 4°C using a Beckman J2-21 centrifuge. Pellet (P1) was discarded and supernatant centrifuged at 16,000×g for 40 min at 4°C. Pellet (P2) was quick frozen and held at −70°C for at least 30 min
[3H]corticosterone association and dissociation kinetics
Kinetic experiments were performed to determine that [3H]corticosterone binding was reversible and to establish parameters for subsequent equilibrium experiments (Fig. 1). The kinetic data were indicative of a simple bimolecular reaction with a monophasic association and dissociation. The half-time for equilibrium association at a 0.5 nM radioligand concentration was 45 min with a kobs of 0.015±0.002 min−1. Dissociation had a half-time of 60 min with a k−1 of 0.012±0.001 min−1.
Equilibrium saturation analysis
Saturation experiments
Discussion
We have described the pharmacological characterization of a corticosterone membrane receptor solubilized from newt neuronal membranes. This is the first example of solubilization of a steroid membrane receptor from neuronal tissue. A cortisol-binding protein has been purified from rat hepatic plasma membranes[25], a progesterone-binding site has been purified from porcine liver membranes[26]and cloned from porcine vascular smooth muscle cells[27], and a 1,25(OH)2D3-binding protein has been
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