The orexin neuropeptides promote robust apoptosis in cancer cells. We have recently shown that the 7-transmembrane-spanning orexin receptor OX1R mediates apoptosis through an original mechanism. OX1R is equipped with a tyrosine-based inhibitory motif ITIM, which is tyrosine-phosphorylated on receptor activation, allowing the recruitment and activation of the tyrosine phosphatase SHP-2, leading to apoptosis. We show here that another motif, immunoreceptor tyrosine-based switch motif (ITSM), is present in OX1R and is mandatory for OX1R-mediated apoptosis. This conclusion is based on the following observations: 1) a canonical ITSM sequence is present in the first intracellular loop of OX1R; 2) mutation of Y⁸³ to F within ITSM abolished OX1R-mediated apoptosis but did not alter orexin-induced inositol phosphate formation or calcium transient via coupling of OX1R to G_q protein; 3) mutation of Y⁸³ to F further abolished orexin-induced tyrosine phosphorylation in ITSM and subsequent recruitment of SHP-2 by the receptor. Finally, we developed a structural model of OX1R showing that the spatial localization of phosphotyrosines in ITSM and ITIM in OX1R is compatible with their interaction with the two SH2 domains of SHP-2. These data represent the first evidence for a functional role of an ITSM in a 7-transmembrane-spanning receptor.—El Firar, A., Voisin, T., Rouyer-Fessard, C., Ostuni, M. A., Couvineau, A., Laburthe, M. Discovery of a functional immunoreceptor tyrosine-based switch motif in a 7-transmembrane-spanning receptor: role in the orexin receptor OX1R-driven apoptosis.