Alignment of transmembrane domains of alpha2 adrenoceptors

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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.