Pérodin Jacqueline, Deraët Maud, Auger-Messier Mannix, Boucard Antony A, Rihakova Lenka, Beaulieu Marie-Eve, Lavigne Pierre, Parent Jean-Luc, Guillemette Gaétan, Leduc Richard, Escher Emanuel
Département de pharmacologie, Faculté de médecine, Université de Sherbrooke, 3001 12e Avenue Nord, Sherbrooke, Québec, J1H 5N4, Canada.
Biochemistry. 2002 Dec 3;41(48):14348-56. doi: 10.1021/bi0258602.
The human angiotensin II type 1 receptor (hAT(1)) was photolabeled with a high-affinity radiolabeled photoreactive analogue of AngII, (125)I-[Sar(1), Val(5), p-Benzoyl-L-phenylalanine(8)]AngII ((125)I-[Sar(1),Bpa(8)]AngII). Chemical cleavage with CNBr produced a 7 kDa fragment (285-334) of the C-terminal portion of the hAT(1). Manual Edman radiosequencing of photolabeled, per-acetylated, and CNBr-fragmented receptor showed that ligand incorporation occurred through Phe(293) and Asn(294) within the seventh transmembrane domain of the hAT(1). Receptor mutants with Met introduced at the presumed contact residues, F293M and N294M, were photolabeled and then digested with CNBr. SDS-PAGE analysis of those digested mutant receptors confirmed the contact positions 293 and 294 through ligand release induced by CNBr digestion. Additional receptor mutants with Met residues introduced into the N- and C-terminal proximity of those residues 293 and 294 of the hAT(1) produced, upon photolabeling and CNBr digestion, fragmentation patterns compatible only with the above contact residues. These data indicate that the C-terminal residue of AngII interacts with residues 293 and 294 of the seventh transmembrane domain of the human AT(1) receptor. Taking into account a second receptor-ligand contact at the second extracellular loop and residue 3 of AngII (Boucard, A. A., Wilkes, B. C., Laporte, S. A., Escher, E., Guillemette, G., and Leduc, R. (2000) Biochemistry 39, 9662-70) the Ang II molecule must adopt an extended structure in the AngII binding pocket.
人血管紧张素II 1型受体（hAT(1)）用血管紧张素II的高亲和力放射性标记光反应类似物(125)I-[Sar(1), Val(5), p-苯甲酰基-L-苯丙氨酸(8)]血管紧张素II((125)I-[Sar(1),Bpa(8)]血管紧张素II)进行光标记。用溴化氰进行化学裂解产生了hAT(1) C末端部分的一个7 kDa片段（285 - 334）。对光标记、全乙酰化且经溴化氰裂解的受体进行手动埃德曼放射性测序表明，配体通过hAT(1)第七跨膜结构域内的苯丙氨酸(293)和天冬酰胺(294)掺入。在假定的接触残基处引入甲硫氨酸的受体突变体F293M和N294M进行光标记，然后用溴化氰消化。对那些消化后的突变体受体进行SDS - PAGE分析，通过溴化氰消化诱导的配体释放证实了接触位置293和294。将甲硫氨酸残基引入hAT(1)的那些残基293和294的N末端和C末端附近的其他受体突变体，在光标记和溴化氰消化后，产生的片段模式仅与上述接触残基相符。这些数据表明，血管紧张素II 的C末端残基与人AT(1)受体第七跨膜结构域的残基293和294相互作用。考虑到在第二个细胞外环处的第二个受体 - 配体接触以及血管紧张素II的残基3（Boucard, A. A., Wilkes, B. C., Laporte, S. A., Escher, E., Guillemette, G., and Leduc, R. (2000) Biochemistry 39, 9662 - 70），血管紧张素II分子在血管紧张素II结合口袋中必须采取伸展结构。
