Crucial role of the orexin-B C-terminus in the induction of OX₁ receptor-mediated apoptosis: analysis by alanine scanning, molecular modelling and site-directed mutagenesis

作     者：Nicole, Pascal Couvineau, Pierre Jamin, Nadege Voisin, Thierry Couvineau, Alain 

作者机构：Paris Diderot Univ CRI INSERM U1149DHU UNITYFac Med Site Bichat F-75018 Paris France CEA Lab Prot & Syst Membranaires iBiTecS I2BC F-91191 Gif Sur Yvette France 

出 版 物：《BRITISH JOURNAL OF PHARMACOLOGY》 (英国药理学杂志)

年 卷 期：2015年第172卷第21期

页      面：5211-5223页

核心收录：

学科分类：1007[医学-药学(可授医学、理学学位)] 10[医学] 

基　　金：INSERM/The Inflammation Research Center (CRI) [U1149] Institut National du Cancer (INCA) [2013-213] Ligue contre le Cancer [RS 12/75-64] 

主　　题：Alanine Site-Directed Mutagenesis Orexin A Receptor Structure-function relationships molecular model Orexin-B Cancer of Colon Orexins C-Terminus 

摘      要：Background and PurposeOrexins (A and B) are hypothalamic peptides that interact with OX1 and OX2 receptors and are involved in the sleep/wake cycle. We previously demonstrated that OX1 receptors are highly expressed in colon cancer tumours and colonic cancer cell lines where orexins induce apoptosis and inhibit tumour growth in preclinical animal models. The present study explored the structure-function relationships of orexin-B and OX1 receptors. Experimental ApproachThe contribution of all orexin-B residues in orexin-B-induced apoptosis was investigated by alanine scanning. To determine which OX1 receptor domains are involved in orexin-B binding and apoptosis, a 3D model of OX1 receptor docked to the orexin-B C-terminus (AA-20-28) was developed. Substitution of residues present in OX1 receptor transmembrane (TM) domains by site-directed mutagenesis was performed. Key ResultsAlanine substitution of orexin-B residues, L-11, L-15, A(22), G(24), I-25, L-26 and M-28, altered orexin-B s binding affinity. Substitution of these residues and of the Q(16), A(17), S-18, N-20 and T-27 residues inhibited apoptosis in CHO-S-OX1 receptor cells. The K-120, P-123, Y-124, N-318, K-321, F-340, T-341, H-344 and W-345 residues localized in TM2, TM3, TM6 and TM7 of OX1 receptors were shown to play a role in orexin-B recognition and orexin-B/OX1 receptor-induced apoptosis. Conclusions and ImplicationsThe C-terminus of orexin-B (i) plays an important role in its pro-apoptotic effect;and (ii) interacts with some residues localized in the OX1 receptor TM. This study defines the structure-function relationship for orexin-B recognition by human OX1 receptors and orexin-B/OX1 receptor-induced apoptosis, an important step for the future development of new agonist molecules.