AbdAlla S, Jarnagin K, Müller-Esterl W, Quitterer U
Institute of Physiological Chemistry and Pathobiochemistry, University of Mainz, 55099 Germany.
J Biol Chem. 1996 Nov 1;271(44):27382-7. doi: 10.1074/jbc.271.44.27382.
To obtain data of the bradykinin B2 receptor's agonist binding site, we used a combined approach of affinity labeling and "immunoidentification" of receptor fragments generated by cyanogen bromide cleavage. Domain-specific antibodies to the various extracellular receptor domains were applied to detect receptor fragments with covalently attached [125I-Tyr8]bradykinin. As a cross-linker we used the homobifunctional reagent disuccinimidyl tartarate (DST), which reacts preferentially with primary amines. With this technique a [125I-Tyr8]bradykinin-labeled receptor fragment derived from the third extracellular domain was identified. The epsilon-amino group of lysine (Lys172) of the human B2 receptor provides the only primary amino group within this receptor fragment. This strongly suggests that DST attached the N-terminal amino group of [Tyr8]bradykinin to Lys172 of the human B2 receptor. Next we asked whether DST attaches [Tyr8]bradykinin to the analogous residue, Lys174 of the rat B2 receptor, which is 81% identical to the human B2 receptor, and we attempted to label the wild-type rat B2 receptor and a rat B2 receptor mutant where Lys174 had been exchanged for alanine. Affinity labeling of the wild-type rat B2 receptor worked efficiently, whereas DST did not attach detectable amounts of [125I-Tyr8]bradykinin to the K174A rat B2 receptor mutant. Taken together these observations indicate that the N-terminal amino group of [Tyr8]bradykinin is bound to analogous positions of the rat and of the human B2 receptor, i.e. [Tyr8]bradykinin's N terminus is bound adjacent to Lys172 of the human and Lys174 of the rat B2 receptor.
为了获取缓激肽B2受体激动剂结合位点的数据，我们采用了亲和标记与对溴化氰裂解产生的受体片段进行“免疫鉴定”相结合的方法。针对各个细胞外受体结构域的结构域特异性抗体被用于检测共价连接了[125I - Tyr8]缓激肽的受体片段。我们使用同双功能试剂酒石酸二琥珀酰亚胺酯（DST）作为交联剂，它优先与伯胺反应。通过这项技术，鉴定出了一个源自第三个细胞外结构域的[125I - Tyr8]缓激肽标记的受体片段。人B2受体赖氨酸（Lys172）的ε - 氨基是该受体片段内唯一的伯氨基。这有力地表明DST将[酪氨酸8]缓激肽的N末端氨基连接到了人B2受体的Lys172上。接下来我们探究DST是否将[酪氨酸8]缓激肽连接到大鼠B2受体的类似残基Lys174上，大鼠B2受体与人类B2受体有81%的同源性，我们尝试对野生型大鼠B2受体和Lys174被丙氨酸替换的大鼠B2受体突变体进行标记。野生型大鼠B2受体的亲和标记效果良好，而DST未将可检测量的[125I - Tyr8]缓激肽连接到K174A大鼠B2受体突变体上。综合这些观察结果表明，[酪氨酸8]缓激肽的N末端氨基与大鼠和人类B2受体的类似位置结合，即[酪氨酸8]缓激肽的N末端与人类B2受体的Lys172以及大鼠B2受体的Lys174相邻结合。
