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.
OBJECTIVE
Orexins (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.


METHODS
The 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.


RESULTS
Alanine 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
The 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.
Faculté de Médecine Site Bichat, INSERM U1149/Inflammation Research Center (CRI), Paris-Diderot University, DHU UNITY, 16, rue H. Huchard, 75018, Paris, France.
Pascal
P
Nicole
Faculté de Médecine Site Bichat, INSERM U1149/Inflammation Research Center (CRI), Paris-Diderot University, DHU UNITY, 16, rue H. Huchard, 75018, Paris, France.
Pierre
P
Couvineau
Laboratoire des Protéines et Systèmes Membranaires, CEA, iBiTecS, I2BC, F-91191, Gif-sur-Yvette Cedex, France.
Nadege
N
Jamin
Faculté de Médecine Site Bichat, INSERM U1149/Inflammation Research Center (CRI), Paris-Diderot University, DHU UNITY, 16, rue H. Huchard, 75018, Paris, France.
Thierry
T
Voisin
Faculté de Médecine Site Bichat, INSERM U1149/Inflammation Research Center (CRI), Paris-Diderot University, DHU UNITY, 16, rue H. Huchard, 75018, Paris, France.
Alain
A
Couvineau
D000409:Alanine / Q000737:chemistry*; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D064446:Orexin Receptors / Q000502:physiology*; D000068797:Orexins / Q000737:chemistry / Q000502:physiology*; D013329:Structure-Activity Relationship
D064446:Orexin Receptors; D000068797:Orexins; D000409:Alanine