Alignment of transmembrane domains of alpha2 adrenoceptors

α2 肾上腺素受体跨膜域的对齐

• 批准号: 11671494
• 负责人: HAYASHI Yukio
• 金额: $ 1.98万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11671494/
• 关键词: adrenoceptors; mutant-receptor; three-dimensional structure

G protein-coupled receptor surerfamily (of which the human a2A adrenoceptor is one member) reveal a common pattern of 7 hydrophobic regions which span the membrane as a helices (transmembrane domain, TMD). In this studv using recombinant DNA technology involving alpha2A/beta2 chimeric adrenoceptor protein, we showed how TMD I aligns with TMD VII.Methods : Site-directed mutants and/or chimerae of the human alpha2A and human beta2 adrenoceptors were constructed from genes encoding human alpha2A and beta 2 adrenocertors. COS-7 cells were transfected with new constructs. Immunocytochemistry confirmed adequate transfection and appropriate localization of the novel receptor in the plasma membrane. Cells were harvested and membranes prepared for radiolabeled ligand binding.Results : Substitution of 312^<th> amino acid on TMD VII of the beta adrenocertor by phenylalanine, its counterpart on the alpha 2 adrenoceptor prevents normal "trafficking" of the resultant malfolded protein. Trafficking is normalized when folding is recovered by replacing TMDs I and II in this construct with their alpha 2 counterparts. When the first 40 amino acid residues of this construct are substituted by the residues of beta2 adrenoceptor counterparts, such construct is malfolded. However, the beta sequence extends only as far as residue 39, a fully functional receptor is synthesized, indicating that the 40^<th> amino acid on TMD I is facing to and interacts with 312^<th> amino acid on TMD VII.

G蛋白偶联受体跨膜结构域（transmembranedomain，TMD）是G蛋白偶联受体跨膜结构域家族（人α 2A肾上腺素受体是其中一员）的一个共有的结构模式，由7个疏水区组成，呈螺旋状跨膜。本研究利用重组DNA技术，将α 2A/β 2嵌合肾上腺素能受体蛋白与TMD Ⅰ和TMD Ⅶ进行了同源性比对。方法：从编码人α 2A和β 2肾上腺素能受体的基因中，构建了人α 2A和β 2肾上腺素能受体的定点突变体和/或嵌合体。用新的构建体转染COS-7细胞。免疫细胞化学证实了足够的转染和适当的定位在质膜的新受体。结果：<th>β肾上腺素受体TMD VII上的312个氨基酸被α 2肾上腺素受体上的对应物苯丙氨酸取代，阻止了所产生的错误折叠蛋白的正常“运输”。当通过用它们的α 2对应物替换该构建体中的TMD I和II来恢复折叠时，运输被标准化。当该构建体的前40个氨基酸残基被β 2肾上腺素受体对应物的残基取代时，这种构建体是错误折叠的。然而，β序列仅延伸至残基39，合成了全功能受体，表明TMD I上的40个<th>氨基酸与TMD VII上的312个氨基酸相对并相互作用<th>。