Characterizing Novel Olfactory Receptors Expressed in the Renal Cortex

Abstract

A primary function of the kidney is to maintain homeostasis. Recent studies have shown that specialized G‐protein coupled receptors, such as olfactory receptors (ORs), play essential roles in modulating kidney function. However, ORs are often orphan receptors with unknown ligands. In a recent RNA seq screen we identified 11 ORs as expressed in the renal cortex, including 5 ORs we had previously published as being expressed in the kidney, and 6 novel ORs. We have previously published the results of ligand screening for 4 of these ORs (Olfr31, Olfr78, Olfr1392, Olfr1393), but have not yet explored the ligands of the remaining 7 (Olfr56, Olfr90, Olfr461, Olfr558, Olfr1033, Olfr1034, Olfr1396). In order to better understand the physiological relevance of these ORs in the kidney, we cloned these 7 receptors from the kidney and attempted to determine ligands that activate these receptors to provide insight into physiological function. Ligand‐screening studies were performed using a cAMP dependent luciferase reporter assay, for which it is crucial that the ORs traffic to the cell surface. Because surface expression is often problematic for heterologously expressed ORs, we first performed immunofluorescence imaging utilizing an N‐terminal flag tag to determine the surface expression of each OR. Surface labeling for flag antibody in live, unpermeabilized cells revealed that 5 ORs (Olfr90, Olfr461, Olfr558, Olfr1034, Olfr1396) demonstrated robust surface trafficking, while 2 ORs (Olfr56 and Olfr1033) were very poorly trafficked to the cell surface. Thus, we screened the 5 ORs which trafficked to the cell surface using a ligand library of 85 compounds with diverse functional groups, divided into 3 categories: 1) compounds known to activate a large percentage of isolated olfactory sensory neurons, 2) sibling ligands, or compounds known to activate ORs within the same sub‐family as these uncharacterized ORs, particularly those known to be present in biofluids including blood and urine, and 3) other odorant and small molecules known to exist in the body produced either endogenously or by microorganisms. Olfr558 has previously reported ligands, and we were able to confirm activation by both butyric acid and nonanoic acid using our luciferase assay (p<0.05 vs control media); to date, none of the other chemicals screened have activated this receptor. Using this library of odorant compounds, we identified 10 novel ligands that activated Olfr90 (p<0.05 vs control media) in a dose dependent manner, none of which activated unrelated ORs or cells transfected with vector only (1‐octen‐3‐ol, 2‐methyl‐4‐propyl‐1,3‐oxathiane, 2‐pentylfuran, allyl benzene, amyl acetate, cinnamaldehyde, 2‐octanone, 3‐octanol, benzyl cyanide, linalool). Interestingly, 6 out of 10 of these ligands are known to be of fungal origin, strongly suggesting that this receptor senses fungal metabolites. To date, none of the compounds screened against Olfr461, Olfr1034, or Olfr1396 have resulted in OR activation. In sum, we have examined 7 ORs expressed in the renal cortex, and have identified novel ligands for Olfr90, which could provide a better understanding into both host‐fungus interactions and the overall physiological function of these receptors.Support or Funding InformationF31 DK104454‐01A1This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.