PROBING LIGAND BINDING SITE OF 2 ADRENERGIC RECEPTOR W/ PHOTOAFFINITY LABELS

β2肾上腺素能跨膜蛋白5(TM5)上的Ser204和Ser207 受体(？2AR)在与儿茶酚胺和 其他？2AR结合配体。据信，氢氧化物中的羟基 这两个氨基酸残基与？2AR激动剂形成氢键。 和敌手。在这项研究项目中，一种重组的？2AR 将构建Ser204到Cys(S204C)或S207C，以sf9表示 杆状病毒表达系统的细胞。纯化的受体将是 硫代儿茶酚胺类化合物的放射性标记 ([125I]-1-(1-氧-3-羟基-4-硫代苯基)) -3-(N-碘酪胺)-2-丙醇和 ([125I]-1-(1-oxy-3-thio-4-hydroxy-phenyl)-3-(N-iodo-tyramine)-2-propanol 它们具有连接在邻苯二酚苯环上的硫基 羟基的。此外，新的光活化放射性 合成了荧酮类和二苯甲酮类化合物。 结合部位探测。光放射性标记的？2AR将被切割 蛋白水解酶和多肽图谱将用多克隆进行分析 用免疫印迹技术检测抗TM5抗体。通过测序 蛋白解肽，这些配体的结合位置可以是 通过将获得的序列与推导的氨基酸进行比较来鉴定 C DNA中？2AR的氨基酸序列分析。

Ser204 and Ser207 on Transmembrane 5 (TM5) of the ?2 adrenergic receptor (?2AR) are critical in interactions with catecholamines and other ?2AR binding ligands. It is believed that the hydroxyls of these two amino acid residues form hydrogen bonds with ?2AR agonists and antagonists. In this research project, a recombinant ?2AR with Ser204 to Cys (S204C) or S207C will be constructed, expressed in sf9 cells of baculovirus expression system. The purified receptor will be radiolabeled with thiocatecholamine derivatives ([125I]-1-(1-oxy-3-hydroxy-4-thio-phenyl) -3-(N-iodo-tyramine)-2-propanol and ([125I]-1-(1-oxy-3-thio-4-hydroxy-phenyl)-3-(N-iodo-tyramine)-2-propanol which have a thio group attached to the catechol phenyl ring instead of a hydroxyl group. In addition, novel photoactivable radioactive fluorenone and benzophenone derivatives have been synthesized for the binding site probing. The photoradiolabeled ?2AR will be cleaved by proteases and the peptide mapping will be analyzed with polyclonal antibodies to TM5 using Western blotting techniques. By sequencing the protealytic peptides, the binding sites for those ligands can be identified by comparing the obtained sequence with the deduced amino acid sequence of the ?2AR from the cDNA.