Abstract 5299: The C-terminus of orexin plays a crucial role in the cellular apoptosis induction mediated by OX1 receptor
Authors: Couvineau Alain , Nicole Pascal , Voisin Thierry , Couvineau Pierre 
Published: May 2015

Orexins (orexin-A and orexin-B) are hypothalamic peptides involved in the sleep/wake control which interact with two GPCR sub-types, OX1R and OX2R. Our group has demonstrated that OX1R was highly expressed in colonic cancer cell lines (HT29, Caco-2, SW480, Lovo…) in which orexins: 1) induce a strong mitochondrial apoptosis; 2) induce a strong inhibition of tumor growth in nude mice xenografted with those cells. This effect was mediated by an original mechanism involving: i) the presence of two ITIMs (immunoreceptor tyrosine inhibitory motif) in the OX1R sequence; ii) the recruitment and activation of the tyrosine phosphatase SHP-2. Moreover, OX1R was expressed in resected colonic tumors and in liver metastases but not in normal tissue. The present study explores the structure-function relationships between orexin-B and its OX1R. The contribution of each side chains of orexin-B were investigated by systematic single alanine (alanine-scanning) exchange of each residue of the native peptides. All modified orexin-B peptides were analyzed for their binding affinities and abilities to induce apoptosis in CHO cells stably transfected with recombinant human OX1R (CHO-OX1R). The data shows that the substitution of L11, L15, I25, G24, L26 and M28 in alanine and A22 in leucine resulted in the loss of binding affinity of orexin-B to its OX1R. Moreover, the substitution of these residues and some others including Q16, A17, S18, N20 and T27 localized mainly in the C-terminal part of orexin-B, strongly alter the ability of the peptide to induce mitochondrial apoptosis in CHO-OX1R. To determine which OX1R domains are involved in orexin-B-induced apoptosis, we have constructed a 3D-molecular model of OX1R derived of structural data recently obtained for beta2 adrenergic receptor. Based on this 3D-model, we have docked the C-terminal end of orexin-B (sequence 20-28) into the whole receptor and performed the substitution of residues presents in transmembrane domains (TM) by directed mutagenesis. Our data reveal that K120, P123, Y124, F340, T341, H344 and W345 localized in TM2, TM3, TM6 and TM7 of OX1R residues play a role in orexin-B recognition and in orexin-B-induced apoptosis. These results strongly suggest that 1) the C-terminal end of orexin-B plays an important role in the pro-apoptotic effect of the peptide; 2) the C-terminal end of orexin-B interacts with some residues localized into the TM of receptor. In conclusion, this study defines the structure-function relationship of ligand recognition and orexin-B-induced apoptosis of human OX1R and should allow the development of new agonist molecules.

Citation Format: Alain Couvineau, Pascal Nicole, Thierry Voisin, Pierre Couvineau. The C-terminus of orexin plays a crucial role in the cellular apoptosis induction mediated by OX1 receptor. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5299. doi:10.1158/1538-7445.AM2014-5299