Hot Spot Mutagenesis Improves the Functional Expression of Unique Mammalian Odorant Receptors.

Yosuke Fukutani,Ikuroh Ohsawa,Kentaro Ikegami,Keiichi Noguchi,Reina Kanemaki,Shunta Miyanaga,Yuko Nakamura,Hiroaki Matsunami,Nonoko Muto,Masafumi Yohda

doi:10.3390/ijms23010277

Abstract

Vertebrate animals detect odors through olfactory receptors (ORs), members of the G protein-coupled receptor (GPCR) family. Due to the difficulty in the heterologous expression of ORs, studies of their odor molecule recognition mechanisms have progressed poorly. Functional expression of most ORs in heterologous cells requires the co-expression of their chaperone proteins, receptor transporting proteins (RTPs). Yet, some ORs were found to be functionally expressed without the support of RTP (RTP-independent ORs). In this study, we investigated whether amino acid residues highly conserved among RTP-independent ORs improve the functional expression of ORs in heterologous cells. We found that a single amino acid substitution at one of two sites (NBW3.39 and 3.43) in their conserved residues (E and L, respectively) significantly improved the functional expression of ORs in heterologous cells. E3.39 and L3.43 also enhanced the membrane expression of RTP-dependent ORs in the absence of RTP. These changes did not alter the odorant responsiveness of the tested ORs. Our results showed that specific sites within transmembrane domains regulate the membrane expression of some ORs.

Highlights

Olfaction is an essential sensation for animals to continue carrying out their daily life activities while experiencing changes in the surrounding environment
We hypothesized that highly conserved amino acid residues in receptor transporting proteins (RTPs)-independent olfactory receptors (ORs) play essential roles in OR trafficking in heterologous cells
Sequence (Figure 1A and Supplementary Data S1). We hypothesized that these ORs that have non-conserved amino acid residues at these positions might show higher levels of expression when these residues are changed to conserved ones

Summary

Introduction

Olfaction is an essential sensation for animals to continue carrying out their daily life activities while experiencing changes in the surrounding environment. Functional analysis of ORs is far behind that of other canonical Class A GPCRs. the flexible and selective odor molecule recognition mechanism of ORs is almost unknown. The flexible and selective odor molecule recognition mechanism of ORs is almost unknown To fill this gap in knowledge, it is necessary to develop a method for improving OR expression without changing the function of ORs. The use of a Rho tag or IL6 tag attached to the N-terminus of ORs has been suggested as a method due to the technology’s ability to promote the enhancement of surface expression of ORs [8,9,10]. Co-expression of chaperone proteins, Hsp70t or Ric-8B, is effective for the functional expression of some ORs [11,12] Both modifications only enhance the expression of a limited number of ORs

Methods

Results

Conclusion