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Assaying Surface Expression of Chemosensory Receptors in Heterologous Cells
Published on: February 23, 2011
A protocol for heterologous expression and functional assay for mouse pheromone receptors
Sandeepa Dey1, Senmiao Zhan, Hiroaki Matsunami
1Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC, USA.
Methods in Molecular Biology (Clifton, N.J.)
|September 10, 2013
Summary
Researchers overcame a major hurdle in understanding mouse social behaviors by developing a new method to study vomeronasal receptor type 2 (V2Rs). This breakthrough enables functional expression of V2Rs on cell surfaces for ligand identification.
Area of Science:
- Neuroscience
- Molecular Biology
- Biochemistry
Background:
- Innate social behaviors in mice, such as aggression and mating, are crucial for species survival.
- These behaviors are thought to be mediated by peptide pheromones detected by vomeronasal receptor type 2 (V2Rs) in the vomeronasal organ (VNO).
- Identifying V2R ligands is essential for understanding pheromone action but is hindered by V2Rs' inability to surface-express in heterologous systems.
Purpose of the Study:
- To develop a method for functional expression of V2Rs on the surface of heterologous cells.
- To enable receptor deorphanization and identification of cognate pheromone ligands.
- To provide protocols for maintaining a specific cell line and performing V2R functional assays.
Main Methods:
- Investigated the role of calreticulin, a chaperone protein, in V2R trafficking.
- Developed a stable knockdown of calreticulin in a HEK293T-derived cell line (R24 cells).
- Established protocols for R24 cell line maintenance, expansion, and V2R functional assays.
Main Results:
- Demonstrated that calreticulin knockdown in R24 cells facilitates surface expression of V2Rs.
- Successfully enabled functional V2R expression in a heterologous cell system.
- Provided detailed protocols for utilizing this system for V2R research.
Conclusions:
- Stable knockdown of calreticulin is a key strategy for achieving functional V2R surface expression in heterologous cells.
- The developed R24 cell line and associated protocols offer a valuable tool for V2R deorphanization and ligand discovery.
- This advancement significantly aids in understanding the molecular basis of pheromone-mediated social behaviors in mice.
