The mouse olfactory receptor Olfr544 is expressed in several non-olfactory tissues and has been suggested as a functional receptor regulating different signaling pathways. However, the molecular interaction between Olfr544 and its natural ligand, azelaic acid (AzA), remains poorly characterized, primarily due to difficulties in the heterologous expression of the receptor protein on the cell membrane and lack of entire protein structure. In this report, we describe the molecular determinants of Olfr544 activation by AzA. N-terminal lucy-flag-rho tag ensured the heterologous expression of Olfr544 on the Hana3A cell surface. Molecular modeling and docking combined with mutational analysis identified amino acid residues in the Olfr544 for the interaction with AzA. Our data demonstrated that the Y109 residue in transmembrane helix 3 forms a hydrogen bond with AzA, which is crucial for the receptor-ligand interaction and activation. Y109 is required for the Olfr544 activation by AzA which, in turn, stimulates the Olfr544-dependent CREB-PGC-1 a signaling axis and is followed by the induction of mitochondrial biogenesis in Olfr544 wild-type transfected Hana3A cells, but not in mock or Y109A mutant transfected cells. Collectively, these data indicated that a hydrogen bond between Y109 residue and AzA is a major determinant of the Olfr544-AzA interaction and activation. (C) 2017 Elsevier Inc. All rights reserved.