Orexin/hypocretin receptor chimaeras reveal structural features important for orexin peptide distinction

We wanted to analyze the basis for the distinction between OX1 and OX2 orexin receptors by the known agonists, orexin-A, orexin-B and Ala11, d-Leu15-orexin-B, of which the latter two show some selectivity for OX2. For this, chimaeric OX1/OX2 and OX2/OX1 orexin receptors were generated. The receptors were transiently expressed in HEK-293 cells, and potencies of the agonists to elicit cytosolic Ca2+ elevation were measured. The results show that the N-terminal regions of the receptor are most important, and the exchange of the area from the C-terminal part of the transmembrane helix 2 to the transmembrane helix 4 is enough to lead to an almost total change of the receptor’s ligand profile.

► Receptor interaction with orexin peptides investigated.
► Construction of chimaeric OX1–OX2 receptors.
► Evaluation of the potency of orexin-A, orexin-B and Ala11, d-Leu15-orexin-B.
► Receptor’s N-terminal half is most important for distinction between peptides.
► Nearly entirely controlled by the area between transmembrane helices 2 and 4.