Chloroethylclonidine and 2-Aminoethyl Methanethiosulfonate Recognize Two Different Conformations of the Human α2A-Adrenergic Receptor

Abstract

The substituted cysteine-accessibility method and two sulfhydryl-specific reagents, the methane-thiosulfonate derivative 2-aminoethyl methanethiosulfonate (MTSEA) and the ␣ 2 -adrenergic receptor (␣ 2 -AR) agonist chloroethylclonidine (CEC), were used to determine the relative accessibility of engineered cysteines in the fifth transmembrane domain of the human ␣ 2A -AR (H␣2A). The second-order rate constants for the reaction of the receptor with MTSEA and CEC were determined with the wild type H␣2A (cysteine at position 201) and receptor mutants containing accessible cysteines at other positions within the binding-site crevice (positions 197, 200, and 204). The rate of reaction of CEC was similar to that of MTSEA at residues Cys-197, Cys-201, and Cys-204. The rate of reaction of CEC with Cys-200, however, was more than 5 times that of MTSEA, suggesting that these compounds may interact with two different receptor conformations. MTSEA, having no recognition specificity for the receptor, likely reacts with the predominant inactive receptor conformation (R), whereas the agonist CEC may stabilize and react preferentially with the active receptor conformation (R*). This hypothesis was consistent with threedimensional receptor-ligand models, which further suggest that ␣ 2A -AR activation may involve the clockwise rotation of transmembrane domain 5.␣ 2 -Adrenergic receptors (␣ 2 -ARs) 1 belong to the family of G-protein-coupled receptors and are integral cell membrane proteins with seven transmembrane (TM) domains (1). The TM regions are predicted to be ␣-helical and to form a wateraccessible crevice containing the binding site for receptor ligands (2). Some of the amino acid residues forming the surface of this crevice directly interact with ␣ 2 -AR agonists and/or antagonists, whereas some others in the TM domains may affect ligand binding indirectly (3, 4). G-protein-coupled receptors are thought to exist in an equilibrium between two (or more) conformations or allosteric states, R and R* (5). In the absence of ligand, the inactive state R predominates. Agonist binding stabilizes the receptor protein in its active state R*, promoting its coupling with G-proteins. The resulting G-protein activation initiates a cascade of intracellular events leading to physiological responses.Recently, a technique utilizing charged methanethiosulfonate derivatives to probe the accessibility of substituted cysteine residues has been used to study the structure of the dopamine D2 and the ␤ 2 -adrenergic receptors (4, 6 -8). This method identifies residues that form the surface of the binding-site crevice by replacing consecutive amino acid residues in the membranespanning segments with cysteine, one at a time. The sulfhydryl side chain of a cysteine can face the water-accessible bindingsite crevice, the interior of the protein, or the lipid bilayer. Sulfhydryls facing the binding-site crevice react rapidly with sulfhydryl-specific methanethiosulfonate derivatives such as 2-aminoethyl methanethiosulfonate (MTSEA), with formation of a co...

Phenoxybenzamine Binding Reveals the Helical Orientation of the Third Transmembrane Domain of Adrenergic Receptors

“…All G-protein-coupled receptors are thought to exist in an equilibrium between two or more conformations or allosteric states, R and R* (29 -31). Being antagonists, the ligands tested in this study mainly interact with the receptor in its predominant, inactive conformation (R) (30). The orientation of the TM domains may change upon receptor activation and agonist binding (1,21,30).…”

“…Being antagonists, the ligands tested in this study mainly interact with the receptor in its predominant, inactive conformation (R) (30). The orientation of the TM domains may change upon receptor activation and agonist binding (1,21,30). Nevertheless, results obtained with catecholamine agonists (9,22,32) indicate that D 3.32 indeed is accessible also in the active conformation (R*), and if TM3 has typical ␣-helical periodicity, then C 3.36 would be expected to be exposed also in the active conformation (R*).…”

Synergistic Contributions of the Functional Groups of Epinephrine to Its Affinity and Efficacy at the β2Adrenergic Receptor

“…TM3, TM5, and TM6, have been proposed based on biophysical and biochemical studies in adrenergic and rhodopsin receptors Farrens et al, 1996;Gether et al, 1997;Javitch et al, 1997;Marjamaki et al, 1999;.…”

Three-dimensional Models of α2A-Adrenergic Receptor Complexes Provide a Structural Explanation for Ligand Binding

“…) was estimated by determining the extent of the reaction after a fixed time, 15 min (CEC) or 2 min (MTSEA), with 7 concentrations of CEC (0.5, 1, 5, 10, 50, 100, and 500 M) or MTSEA (5,20,50,100,150,200, and 250 M) as previously reported (9,12).…”

“…Residues within this cavity directly participate in ligand binding, which leads to alteration of the receptor structure with subsequent activation of downstream signaling (7,8). Recently, we have combined targeted mutagenesis experiments with structural modeling to show that two molecules that covalently bind to ␣ 2A -AR, chloroethylclonidine (CEC) and 2-aminoethyl methanethiosulfonate hydrobromide (MTSEA), recognize two different receptor conformations (9). Here, we show that our most recent model, based on the vertebrate rhodopsin template (5), provides a better explanation for CEC and MTSEA binding that does another model constructed by us using the 2.5-Å bacteriorhodopsin structure (3).…”