Deorphanizing Renal Olfactory Receptor 1393

Abstract

Olfactory receptors (ORs) are G protein‐coupled receptors which serve to detect odorants in the nose. Recently, ORs have been shown to play novel roles beyond odorant detection in a number of tissues, and we previously determined that a subset of ORs are expressed in the kidney. One of these receptors is Olfr1393, an orphan receptor with no known ligand. In order to determine the physiological role of Olfr1393 within the kidney, it is important to understand its ligand profile. Unfortunately, most ORs are orphan receptors, due in large part to the inability to functionally express ORs in heterologous cell systems; ORs are retained in the ER and degraded when heterologously expressed. Luckily, with the assistance of chaperone proteins and a novel cleavable tag that we developed (Shepard, et al PLoS One 2013), we were able to express Olfr1393 on the cell surface of HEK293T cells. Thus, we undertook a large scale ligand screen using a cAMP‐dependent luciferase reporter assay. We began our unbiased screen using different odorant mixtures, each containing a variety of chemicals clustered by functional group such that the mixes, in sum, cover a wide “odorant space”. However, we did not observe any Olfr1393 activation using these libraries. The murine OR gene family (consisting of ~1000 ORs) is subdivided into subfamilies, grouped based on phylogenetic clustering and protein sequence identity, and ORs within a subfamily often share similar ligands. Olfr1393 belongs to the MOR256 subfamily, and several other MOR256 family members have been previously deorphanized. Thus, we screened Olfr1393 against known MOR256 odorants and found that Olfr1393 does respond to one of these chemicals – cyclohexanone. Using cyclohexanone as a guide, we broadened our search by using a multidimensional physiochemical metric for odorant prediction and identified a total of 8 different ligands. All 8 of these ligands are structurally similar, and can be broadly described as pre‐constrained rings containing either ketone or alcohol functional groups. Notably, if the ligand is modified even slightly (with the addition or removal of a functional group) it will no longer activate the receptor. Further unbiased screens ‐including the use of a 1280 compound chemical library ‐ did not reveal any additional ligands for Olfr1393 indicating the narrow range of its ligand profile. Efforts are currently underway to determine the physiological relevance of these 8 ligands using targeted metabolomics of murine urine and plasma; discovering the source of these ligands will contribute to the understanding that Olfr1393 plays in the kidney.Support or Funding InformationNIDDK