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Updated: Oct 7, 2025
High-throughput Analysis of Mammalian Olfactory Receptors: Measurement of Receptor Activation via Luciferase Activity
Published on: June 2, 2014
Hot Spot Mutagenesis Improves the Functional Expression of Unique Mammalian Odorant Receptors
Yosuke Fukutani1, Yuko Nakamura1, Nonoko Muto1
1Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Tokyo 184-8588, Japan.
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.
Insights
Specific amino acid residues enhance olfactory receptor (OR) expression in cells. These findings improve understanding of G protein-coupled receptor (GPCR) function and odor detection in vertebrates.
Area of Science:
- Olfactory receptor (OR) research
- G protein-coupled receptor (GPCR) biology
- Molecular biology and cell expression systems
Background:
- Vertebrate animals detect odors using olfactory receptors (ORs), which are G protein-coupled receptors (GPCRs).
- Heterologous expression of ORs is challenging, hindering studies on their odor recognition mechanisms.
- Functional expression of many ORs requires co-expression with receptor transporting proteins (RTPs), though some ORs are RTP-independent.
Purpose of the Study:
- To investigate if conserved amino acid residues in RTP-independent ORs can enhance the functional expression of ORs in heterologous cells.
- To determine if these conserved residues impact the membrane expression of both RTP-independent and RTP-dependent ORs.
- To assess whether modifications at these sites affect the odorant responsiveness of ORs.
Main Methods:
- Investigated the role of conserved amino acid residues in RTP-independent ORs.
- Performed single amino acid substitutions at specific sites (N3.39 and 3.43) within ORs.
- Assessed functional expression and membrane expression of ORs in heterologous cells, with and without RTP co-expression.
- Evaluated odorant responsiveness of modified ORs.
Main Results:
- A single amino acid substitution at sites N3.39 (E) and 3.43 (L) significantly improved the functional expression of ORs in heterologous cells.
- These substitutions also enhanced the membrane expression of RTP-dependent ORs, even without RTP.
- The identified amino acid changes did not alter the odorant responsiveness of the tested ORs.
Conclusions:
- Specific amino acid residues within transmembrane domains play a crucial role in regulating the membrane expression of certain ORs.
- These findings offer insights into improving OR expression for future functional studies.
- The study identifies key sites for enhancing GPCR membrane trafficking and expression.
