Two highly related odorant receptors specifically detect α-bile acid pheromones in sea lamprey (Petromyzon marinus).

Pheromones play critical roles in habitat identification and reproductive behavior synchronization in the sea lamprey (Petromyzon marinus). The bile acid 3-keto petromyzonol sulfate (3kPZS) is a major component of the sex pheromone mixture from male sea lamprey that induces specific olfactory and behavioral responses in conspecific individuals. Olfactory receptors interact directly with pheromones, which is the first step in their detection, but identifying the cognate receptors of specific pheromones is often challenging. Here, we deorphanized two highly related odorant receptors (ORs), OR320a and OR320b, of P. marinus that respond to 3kPZS. In a heterologous expression system coupled to a cAMP-responsive CRE-luciferase, OR320a and OR320b specifically responded to C24 5α-bile acids, and both receptors were activated by the same set of 3kPZS analogs. OR320a displayed larger responses to all 3kPZS analogs than did OR320b. This difference appeared to be largely determined by a single amino acid residue, Cys-792.56, the C-terminal sixth residue relative to the most conserved residue in the second transmembrane domain (2.56) of OR320a. This region of TM2 residues 2.56-2.60 apparently is critical for the detection of steroid compounds by odorant receptors in lamprey, zebrafish, and humans. Finally, we identified OR320 orthologs in Japanese lamprey (Lethenteron camtschaticum), suggesting that the OR320 family may be widely present in lamprey species and that OR320 may be under purifying selection. Our results provide a system to examine the origin of olfactory steroid detection in vertebrates and to define a highly conserved molecular mechanism for steroid-ligand detection by G protein-coupled receptors.