The binding site of the ␤ 2 adrenergic receptor, like that of other homologous G-protein-coupled receptors, is contained within a water-accessible crevice formed among its seven membrane-spanning segments. Methanethiosulfonate ethylammonium (MTSEA), a charged, hydrophilic, lipophobic, sulfhydryl-specific reagent, had no effect on the binding of agonist or antagonist to wild-type ␤ 2 receptor expressed in HEK 293 cells. This suggested that no endogenous cysteines are accessible in the binding site crevice. In contrast, in a constitutively active ␤ 2 receptor, MTSEA significantly inhibited antagonist binding, and isoproterenol slowed the rate of reaction of MTSEA. This implies that at least one endogenous cysteine becomes accessible in the binding site crevice of the constitutively active ␤ 2 receptor. Cys-285, in the sixth membrane-spanning segment, is responsible for the inhibitory effect of MTSEA on ligand binding to the constitutively active mutant. The acquired accessibility of Cys-285 in the constitutively active mutant may result from a rotation and/or tilting of the sixth membrane-spanning segment associated with activation of the receptor. This rearrangement could bring Cys-285 to the margin of the binding site crevice where it becomes accessible to MTSEA.The interaction of a diverse array of signals, including neurotransmitters, peptides, hormones, light, and odorants, with G-protein-coupled receptors results in a conformational change which enhances their interaction with heterotrimeric G-proteins and thereby promotes GDP release and subsequent GTP binding and G-protein activation (1). In rhodopsin, for example, photoisomerization of retinal leads to rigid body movements of the third (M3) 1 and sixth (M6) membrane-spanning segments relative to each other (2). Moreover, disulfide cross-linking of these membrane-spanning segments prevents the activation of transducin, the G-protein associated with rhodopsin, further supporting the relevance of their movement in the activation of rhodopsin. The generality of this movement to other G-proteincoupled receptors, however, is not known. Moreover, additional details of the structural mechanisms of receptor activation are necessary to better understand this process. The binding sites of the ␤ 2 adrenergic receptor and of the homologous receptors for other biogenic amines are formed among their seven, mostly hydrophobic, membrane-spanning segments (1, 3) and are accessible to charged, water-soluble agonists like norepinephrine. Thus, for each of these receptors, the binding site is contained within a water-accessible crevice, the binding site crevice, extending from the extracellular surface of the receptor into the plane of the membrane. The surface of this crevice is formed by residues that can contact specific agonists and/or antagonists and by other residues that may play a structural role and affect binding indirectly.To identify the residues that form the surface of the binding site crevice in the dopamine D2 receptor and the ␤ 2 adrenergic receptor, we have ...

The binding site of the dopamine D2 receptor, like that of other homologous G protein-coupled receptors, is contained within a water-accessible crevice formed among its seven membrane-spanning segments. We have developed a method to map systematically all the residues forming the surface of this binding-site crevice, and we have applied this method to the third membrane-spanning segment (M3). We mutated, one at a time, 23 residues in and flanking M3 to cysteine and expressed the mutant receptors heterologously. Ten of these mutants reacted with charged, hydrophilic, lipophobic, sulfhydryl-specific reagents, added extracellularly, and were protected from reaction by a reversible dopamine antagonist. Thus, the side chains of these residues are exposed in the binding-site crevice, which like M3 extends from the extracellular to the intracellular side of the membrane. The pattern of exposure is consistent with a short loop followed by six turns of an alpha helix.

The binding site of the dopamine D2 receptor, like that of other homologous G-protein-coupled receptors, is contained within a water-accessible crevice formed among its seven membrane-spanning segments. Using the substituted-cysteine accessibility method, we previously mapped the residues that form the surface of the binding-site crevice in the third and fifth membrane-spanning segments (M3 and M5). We have now mutated to cysteine, one at a time, 26 consecutive residues in and flanking the seventh membrane-spanning segment (M7) and expressed the mutant receptors in HEK 293 cells. Nine of these mutants reacted with charged, hydrophilic, lipophobic, sulfhydryl-specific reagents, added extracellularly, and were protected from reaction by a reversible dopamine antagonist, sulpiride. Thus, we infer that the side chains of these residues are in the water-accessible surface of the binding-site crevice. The pattern of accessibility of the cysteine-substitution mutants is consistent with M7 being a kinked alpha-helix.

The binding site of the dopamine D2 receptor, like that of other homologous G-protein-coupled receptors, is contained within a water-accessible crevice formed among its seven membrane-spanning segments. Using the substituted-cysteine accessibility method, we previously mapped the residues in the third membrane-spanning segment (M3) that are exposed in the biding site crevice [Javitch et al. (1995) Neuron 14, 825]. We have now mutated, one at a time, 24 consecutive residues in and flanking the fifth membrane-spanning segment (M5) to cysteine and expressed the mutant receptors in HEK 293 cells. Thirteen of these mutants reacted with charged, hydrophilic, lipophobic, sulfhydryl-specific reagents, added extracellularly, and were protected from reaction by another reversible dopamine antagonist, sulpiride. Thus, the side chains of these residues are exposed in the binding-site crevice. Of the 13 exposed residues, 10 are consecutive, from Phe189 to Phe198. This pattern of exposure is inconsistent with the expectation that M5, like M3, forms a fixed alpha-helix, one side of which is exposed in the binding-site crevice. The exposed region of M5, which contains the serines likely to bind agonist [Strader et al. (1989) J. Biol. Chem. 264, 13752], might loop out into the lumen of the binding-site crevice and be completely accessible to water and thus to MTSEA. Alternatively, the exposed region of M5 might be embedded in the membrane and also in contact with other membrane-spanning segments. At any instant, only a limited set of residues might be exposed in the binding-site crevice; however, M5 might move rapidly to expose different sets of residues.