Endoplasmic Reticulum Degradation Impedes Olfactory G-protein Coupled Receptor Functional Expression

Research Article

Endoplasmic Reticulum Degradation Impedes Olfactory G-protein Coupled Receptor Functional Expression

Claire Dubois^1*, Henry Adams² and Lily Cooper²

1Department of Virology, University of Chicago, Chicago, USA
2Department of Biochemistry, Imperial College London, London, UK

Published: 14 April 2015

Abstract

Background: Olfactory Receptors (ORs), belonging to the G-Protein Coupled Receptor (GPCR) superfamily, constitute the largest gene family in mammals and are responsible for detecting odorants. However, achieving functional expression of ORs on the plasma membrane of heterologous cells is notoriously inefficient, with most receptors being retained intracellularly. This severely hampers biochemical and functional studies. The Endoplasmic Reticulum (ER) Possesses Stringent Quality Control (ERQC) mechanisms that target misfolded or unassembled proteins for degradation via the ER-Associated Degradation (ERAD) pathway.

Objective: This study investigated the hypothesis that ERAD actively contributes to the poor surface expression of ORs by targeting improperly folded or retained receptors for proteasomal degradation.

Methods: Several representative mouse ORs, tagged with epitope markers (e.g., FLAG or Rho tag), were transiently expressed in human embryonic kidney (HEK293) cells. Subcellular localization was determined by immunofluorescence microscopy and co-staining with ER markers (Calnexin, PDI). Cell surface expression was quantified using cell surface ELISA on non-permeabilized cells and surface biotinylation assays. The ubiquitination status of ORs was assessed by immunoprecipitation followed by Western blotting for ubiquitin. The role of ERAD was investigated by treating cells with proteasome inhibitors (MG132, lactacystin) and examining their effects on total OR protein levels, ubiquitination, localization, stability (using pulse-chase analysis), and cell surface expression

Results: The majority of expressed ORs were retained intracellularly and showed significant co-localization with ER markers. Only a small fraction (<5-10%) of total OR protein was detected on the cell surface. Immunoprecipitation revealed that intracellularly retained ORs were poly-ubiquitinated. Treatment with proteasome inhibitors MG132 or lactacystin led to a significant increase in total OR protein levels and accumulation of poly-ubiquitinated OR species, indicating that ORs are substrates for proteasomal degradation. Pulse-chase analysis confirmed that ORs were relatively unstable proteins with rapid turnover, which was significantly slowed by proteasome inhibition. Importantly, inhibiting proteasomal degradation resulted in a modest but statistically significant increase (e.g., 1.5 to 2.5 fold) in the amount of OR protein detected at the cell surface.

Conclusion: The olfactory receptors expressed in heterologous cells are inefficiently processed and are actively targeted for degradation by the ERAD pathway via ubiquitination and proteasomal degradation. This ERAD process represents a major limiting factor, actively clearing retained ORs and thus significantly impeding their functional expression at the cell surface. Overcoming ER retention and subsequent ERAD is likely crucial for efficient OR trafficking and function.

Keywords: Olfactory Receptors (ORs); G-protein Coupled Receptors (GPCRs); Endoplasmic Reticulum (ER); ER-Associated Degradation (ERAD); Protein Folding; Quality Control; Ubiquitination; Proteasome; Heterologous Expression; Protein Trafficking.