Alignment of transmembrane domains of alpha2 adrenoceptors

G蛋白偶联受体家族(人的A2A型肾上腺素受体是其中的一员)揭示了一个共同的模式，即7个疏水区，它们以螺旋的形式跨越细胞膜(跨膜区，TMD)。在本研究中，我们利用重组DNA技术构建了人α2A和β2受体嵌合蛋白，揭示了TMD I如何与TMD VII结合。方法：利用编码人α2A和β2肾上腺素受体的基因构建人α2A和人β2受体的定点突变体和/或嵌合体。将新构建的基因导入COS-7细胞。免疫细胞化学证实这种新型受体在细胞膜上得到了充分的表达和适当的定位。结果：β受体TMD VII上的312^TH氨基酸被α2受体上的苯丙氨酸取代，阻止了错误折叠蛋白的正常“转运”。当折叠被恢复时，通过用其阿尔法2对应物替换该结构中的TMD I和II，贩运被正常化。当该结构的前40个氨基酸残基被β2肾上腺素能受体对应的残基取代时，这种结构是错误折叠的。然而，β序列仅延伸到第39位，合成了一个功能完整的受体，表明TMD I上的40^TH氨基酸与TMD VII上的312^TH氨基酸相互作用。

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.