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

Ser 204和Ser 207在跨膜5（TM 5）的？2肾上腺素能 受体（？2AR）在与儿茶酚胺的相互作用中至关重要， 其他的？2AR结合配体。 据信， 这两个氨基酸残基形成氢键，2AR激动剂 和拮抗剂。 在这个研究项目中，重组？2AR， 将构建Ser 204至Cys（S204 C）或S207 C，以sf 9表达 杆状病毒表达系统的细胞。 纯化的受体将是 用硫代儿茶酚胺衍生物放射性标记 （[125 I]-1-（1-氧-3-羟基-4-硫代-苯基） -3-（N-碘-酪胺）-2-丙醇和 （[125 I]-1-（1-氧-3-硫代-4-羟基-苯基）-3-（N-碘-酪胺）-2-丙醇 其具有连接到邻苯二酚苯环上的硫代基团 一个羟基。 此外，本发明还提供了新型光活化放射性化合物， 合成了芴酮和二苯甲酮衍生物， 结合位点探测。 光放射标记的？2AR将被切割 蛋白酶和肽图谱将用多克隆抗体进行分析。 使用蛋白质印迹技术检测针对TM 5的抗体。 通过测序 蛋白水解肽，这些配体的结合位点可以 通过将获得的序列与推导的氨基酸进行比较来鉴定 酸序列的？2AR基因。

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.