Hepatic Olfactory Receptors Respond to Pyrazines and Terpenes

Abstract

Sensory receptors, including olfactory receptors (ORs), taste receptors (TRs), and opsins (Opns) have recently been found in a variety of non‐sensory tissues where they have important physiological functions. As G protein‐coupled receptors (GPCRs), these proteins can serve as chemosensors to interpret chemical cues in their native environment. We reasoned that the liver, the largest metabolic organ in the body, is primed to take advantage of some of these receptors in order to sense and regulate its internal environment. To identify the complete complement of sensory receptors expressed in murine liver, we began by performing an unbiased screen of whole liver using custom‐made TaqMan qPCR array cards. This screen identified a total of 17 ORs, 10 TRs, and 3 Opns expressed in both male and female murine liver. Subsequent RT‐PCR using gene‐specific and full‐length primer sets confirmed the expression of these receptors in multiple liver samples (n=4). To understand the physiological function of these receptors, we began by focusing on ORs as they are known to respond to small molecules found in the human body. Unfortunately, the vast majority of ORs remain orphan receptors with no known ligands, and our identified hepatic ORs are no exception. This is due, in part, to the inability to functionally express ORs in heterologous cell systems. To deorphanize the hepatic ORs, we began by cloning the five highest expressing ORs (Olfr99, Olfr267, Olfr1366, Olfr57, and Olfr177) in a vector containing an N‐terminal Flag tag (for detection) and a cleavable LUCY tag (for surface expression). HEK293T cells expressing the ORs of interest were live‐cell surface labeled to identify conditions that promote functional expression. We were unable to identify conditions that allow for surface expression of Olfr267 and Olfr1366; however, Olfr57, Olfr99 and Olfr177 were found on the plasma membrane when they were expressed either alone (Olfr57) or in combination with two chaperone proteins, RTP1s and Ric8b (Olfr99 and Olfr177). With expression conditions established, we performed a large‐scale ligand screen using a cAMP‐driven Dual Luciferase assay. We began by screening each OR against mixtures of small odorant molecules as well as a common odorant library to cover a wide chemical space. We identified one ligand for Olfr177: 2‐ethyl‐3,5 dimethylpyrazine (EC50: 1096mM), a pyrazine associated with food odors and pheromones. Additionally, we have identified two ligands for Olfr57: camphor (EC50: 680mM) and fenchone (EC50: 94mM), both monoterpenes. To date, structurally similar terpenes including camphene and norcamphor did not induce activation of Olfr57 suggesting a potentially narrow binding pocket. Monoterpenes are commonly found in herbal extracts which possess antioxidant capabilities. Additionally, these compounds have also been reported as volatile organic compounds produced by fungus. The responses to these ligands is currently being confirmed in the human orthologs of Olfr177 and Olfr57. In addition, using RNAscope to localize individual mRNA transcripts, we found that Olfr57 is localized to the hepatocytes of the liver. Thus, ongoing studies are being conducted to investigate the physiological relevance of both terpenes and pyrazines in hepatocytes.Support or Funding InformationNIDDK