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

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Abstract

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 pI 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.

Introduction

Many mechanisms of steroid hormone action remain elusive. The classical model for steroid action relies on the interaction of the steroid with an intracellular receptor that modulates transcriptional activity of target genes. This signaling route for steroids is well-characterized and accounts for many of the known effects of steroid hormones. However, some responses to steroids fail to fit the classical genomic model and, instead, appear to utilize mechanisms involving membrane receptors and second messengers. Several recent reviews focus on steroid membrane receptors and non-genomic actions of steroids [1], [2], [3], [4], [5], [6], [7], [8].

Evidence for the existence of membrane steroid receptors comes from several types of observations. First, some effects of steroid hormones are too rapid to be explained by the classical model. For example, electrophysiological studies find that membrane conductance can be modified within seconds of exposure to estradiol [9], progestins [10] and corticosteroids [11]. Second, the pharmacological specificity of rapid responses to steroids does not always match the specificity of steroid binding to known intracellular steroid receptors. The progesterone-induced acrosome reaction in sperm [12], [13], androgenic modulation of second messenger systems in osteoblasts [14], and corticosteroid-induced inhibition of reproductive behaviors [15] are induced by different sets of steroids than those that bind intracellular progesterone, androgen, and glucocorticoid receptors, respectively. Third, steroids can induce rapid responses even when prevented from entering the cell by being conjugated to BSA. Progesterone-BSA can modulate hypothalamic neuronal activity [16] and behavioral responses [17], and estradiol-BSA can potentiate kainate-induced currents in CA1 neurons [18]. Fourth, some steroids can bind to and modulate the actions of known membrane receptors. Progesterone apparently binds to the oxytocin receptor and, thereby, inhibits oxytocin binding and oxytocin induced Ca2+ currents and inositol triphosphate (IP3) turnover [19]. Progesterone metabolites also directly modulate the GABAA receptor/chloride channel [20].

The present study focused on a membrane glucocorticoid receptor (mGR) found in the brain of an amphibian, the roughskin newt (Taricha granulosa). Ligand-binding studies demonstrated that this receptor is localized in neuronal membranes, and binds corticosterone (CORT) with high affinity (sub-nanomolar KD) and a specificity that distinguishes it from classical intracellular glucorticoid receptors [15]. This receptor has characteristics of a G-protein coupled receptor in that [3H]CORT specific binding is enhanced by Mg2+ and inhibited by non-hydrolyzable guanyl nucleotides [21]. Evidence that this mGR regulates brain function and behavior in newts comes from studies showing that the rank-order potency of specific steroids to inhibit [3H]CORT specific binding to neuronal membranes (CORT > cortisol > aldosterone > RU 28,362 > dexamethasone) matches the rank-order potency of these steroids to rapidly inhibit (within 20 min) male reproductive behavior [15]. Similarly, CORT administration, but not dexamethasone, can rapidly inhibit stress-induced increases in locomotor activity [22] and suppress the neuronal activity in medullary neurons in newts [23], [24].

Other studies indicate that a mGR that is similar to the newt mGR may exist in other vertebrates. Ligand-binding studies using membrane preparations from rat brains [25], [26], rat liver tissue [27], and mouse pituitary glands [28] find binding sites with high specificity for corticosteroids. Neurophysiological studies also reveal that corticosteroid administration causes rapid changes in Ca2+ currents in guinea pig hippocampal CA1 neurons and that this rapid response appears to be mediated by a G-protein coupled receptor because it is sensitive to pertussis toxin (PTX) and GDPβS [29]. Similarly, corticosteroid administration induces rapid changes in neuronal activity in rats [11], [30] and cats [31]. Physiological studies in fish show that cortisol administration can rapidly inhibit prolactin secretion and reduce cAMP activity in lactotrophs [32]. Behavioral studies report rapid effects of CORT administration on locomotor activity in birds [33] and rats [34].

Detergent solubilization and chromatographic purification strategies have helped to identify several membrane steroid receptors and binding proteins. The membrane receptor for 1-alpha,25-dihydroxyvitamin D3, which is associated with rapid transport of Ca2+ across intestinal basal-lateral membranes [35], has been solubilized and partially purified from chick intestinal epithelial membranes [36]. The membrane-associated cortisol binding protein in rat liver has been solubilized and purified [27]. The progesterone binding protein in porcine liver membranes has been solubilized, purified, and partially sequenced [37]. When this putative membrane receptor for progesterone was subsequently cloned, it was found to be a single transmembrane protein of 194 amino acids with low sequence similarity to known proteins [38], to be localized to endomembranes [39], and have a pharmacological signature that is reminiscent of the sigma receptor [40].

Other studies have investigated steroid membrane receptors and binding proteins using photoaffinity labels. Photoactive progestins have labeled proteins in plasma membranes of Xenopus oocytes [41], mouse cerebellum [42] and porcine liver [43]. Photoactive aldosterone derivatives have labeled a protein in the plasma membrane of human mononuclear leukocytes [44]. The photoactive glucocorticoid, dexamethasone mesylate, has been used to label membrane proteins from lymphocytes [45]. This approach with photoactive steroids can provide biochemical information about steroid binding proteins and be used in conjunction with purification strategies.

This paper describes the effectiveness of several chromatographic resins for enriching the mGR from newt brains, a biochemical characterization of the mGR, and the development of novel strategies for visualizing proteins labeled with photoactive steroids. These studies used newt brains for the mGR because [3H]CORT has sub-nanomolar affinity for the newt mGR with approximately 85% specific binding at its KD [15], [46]. In contrast, [3H]CORT binding studies in rat brain show a lower affinity of approximately 100 nM with 70% specific binding [25], [26]. Furthermore, previous studies have provided detailed information about the pharmacology and physiology of the newt mGR and have validated procedures for maintaining high-affinity binding after solubilization of this receptor [46].

Section snippets

Materials and methods

All buffers, salts, detergents, DEAE-Sepharose, WGA-agarose and Donkey anti-Sheep IgG monoclonal antibody were purchased from Sigma (St. Louis, MO). CM-Sepharose and EAH-Sepharose were from Pharmacia Biotech (Piscataway, NJ). Macro-Prep Ceramic hydroxylapatite resin and glass chromatography columns with flow adapters were from Bio-Rad (Hercules, CA). Disposable 2.5 ml plastic chromatography columns were from IsoLabs (Akron, OH). Silver staining kit was from Novex (San Diego, CA). Anti-CORT

Ammonium sulfate fractionation

To determine if the newt mGR could be enriched by ammonium sulfate fractionation, solubilized newt neuronal membrane proteins were fractionated by precipitation with increasing concentrations of ammonium sulfate. Pellets from each precipitation step were resuspended for quantification of total protein by absorbance at 280 nm (A280) and quantification of receptor binding activity by [3H]CORT binding assays (Fig. 1A). The greatest amount of receptor binding activity occurred in the 80–100%

Discussion

The current study evaluated and applied methods for purifying and characterizing mGR from newt neuronal membranes. Ammonium sulfate fractionation followed by WGA-agarose and hydroxylapatite chromatographies provided significant enrichment of the newt mGR. Purification of a 63 kDa protein to near homogeneity was completed with the use of a CORT-Sepharose affinity resin. Furthermore, using photoaffinity labeling that was visualized by western blots following 1D or 2D SDS–PAGE gels, differential

Acknowledgments

The work described in this manuscript was supported by NSE grant #IBN-9319633 to Dr. Frank Moore.

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