Characterizing novel olfactory receptors expressed in the murine 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 c...

Ectopically expressed olfactory receptors OR51E1 and OR51E2 suppress proliferation and promote cell death in a prostate cancer cell line

“…Overall, our results on activation of OR51E1 by aliphatic acids but not structurally similar compounds ( Fig. 4 ) were consistent with those earlier studies where the OR expression at the protein level was convincingly demonstrated ( 11 , 18 ). Having established the functional activity of the overexpressed OR51E1 and OR51E2, we focused on their cancer-related properties, i.e.…”

“…Olfr558 appears to be responsible for sensing nutrients such as butyrate and isovaleric acid in the gut ( 17 ). The human ortholog OR51E1 is also activated by butyrate and other aliphatic acids ( 18 ). Some investigators reported activation of OR51E2 by other chemicals, e.g.…”

The Sensing Liver: Localization and Ligands for Hepatic Murine Olfactory and Taste Receptors

“…Recently, these proteins have also been shown to have important roles in other tissues as well. For example, ectopically expressed ORs have been found throughout the body including the testis ( Feldmesser et al, 2006 ; Kang and Koo, 2012 ), heart ( Feldmesser et al, 2006 ; Kang and Koo, 2012 ; Flegel et al, 2013 ), kidney ( Feldmesser et al, 2006 ; Kang and Koo, 2012 ; Pluznick and Caplan, 2012 ; Flegel et al, 2013 ; Rajkumar et al, 2014 ; Halperin Kuhns et al, 2019 ), brain ( Feldmesser et al, 2006 ; Kang and Koo, 2012 ; Flegel et al, 2013 ), thyroid ( Feldmesser et al, 2006 ; Flegel et al, 2013 ), and pancreas ( Feldmesser et al, 2006 ; Kang and Koo, 2012 ), while TRs and Opns have also been identified in the airway epithelium ( Li, 2013 ; Gilca and Dragos, 2017 ), large and small intestine ( Li, 2013 ; Gilca and Dragos, 2017 ), adrenal glands ( Halford et al, 2001 ; Li, 2013 ), and kidney ( Halford et al, 2001 ). While the physiological relevance of these receptors is continuing to emerge, many of these ‘extra sensory’ localizations have been linked to physiological and pathophysiological functions ( Spehr et al, 2003 ; Neuhaus et al, 2009 ; Sharma et al, 2017 ; Abaffy et al, 2018 ; Massberg and Hatt, 2018 ; Lee et al, 2019 ; Tole et al, 2019 ).…”

Systems and classical biology approaches unraveled role of olfactory receptors in progression of kidney fibrosis

“…One of the tissues, in which the presence and function of ORs is investigated, is the kidney (Halperin Kuhns et al, 2019; Miyamoto et al, 2016; Peti-Peterdi, Kishore, & Pluznick, 2016; Pluznick et al, 2009; Rajkumar, Aisenberg, Acres, Protzko, & Pluznick, 2014; Shepard & Pluznick, 2017). Studies using unsupervised high-throughput techniques have discovered the existence of these receptors in the kidney (Feldmesser et al, 2006; Flegel, Manteniotis, Osthold, Hatt, & Gisselmann, 2013).…”

“…Furthermore, Shepard et al demonstrated that Olfr1393 participates in glucose transportation in both normal and pathological states of the kidney (Shepard et al, 2016; Shepard, Koepsell, & Pluznick, 2019). Considering the magnitude of this gene family (around 1000 genes in the mouse and 400 genes in the human) as well as their role in chemo-sensation, more investigations are needed to uncover the role of other OR subtypes in kidney function and hemostasis (Halperin Kuhns et al, 2019; Shepard & Pluznick, 2016).…”