Javitch J A, Fu D, Liapakis G, Chen J
Center for Molecular Recognition, Columbia University College of Physicians & Surgeons, New York, New York 10032, USA.
J Biol Chem. 1997 Jul 25;272(30):18546-9. doi: 10.1074/jbc.272.30.18546.
The binding site of the beta2 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 beta2 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 beta2 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 beta2 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.
β2肾上腺素能受体的结合位点，与其他同源G蛋白偶联受体的结合位点一样，位于其七个跨膜片段之间形成的一个可被水接触的裂隙内。甲硫基磺酸盐乙铵（MTSEA）是一种带电荷、亲水性、疏脂性、巯基特异性试剂，对激动剂或拮抗剂与HEK 293细胞中表达的野生型β2受体的结合没有影响。这表明在结合位点裂隙中没有内源性半胱氨酸可被接触到。相比之下，在组成型活性β2受体中，MTSEA显著抑制拮抗剂结合，而异丙肾上腺素减缓了MTSEA的反应速率。这意味着在组成型活性β2受体的结合位点裂隙中至少有一个内源性半胱氨酸变得可被接触到。位于第六个跨膜片段中的半胱氨酸-285负责MTSEA对配体与组成型活性突变体结合的抑制作用。组成型活性突变体中半胱氨酸-285获得的可接触性可能是由于与受体激活相关的第六个跨膜片段的旋转和/或倾斜。这种重排可能会使半胱氨酸-285到达结合位点裂隙的边缘，在那里它可被MTSEA接触到。
