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High-throughput Analysis of Mammalian Olfactory Receptors: Measurement of Receptor Activation via Luciferase Activity
Published on: June 2, 2014
A specific heat shock protein enhances the expression of mammalian olfactory receptor proteins
Eva M Neuhaus1, Anastasia Mashukova, Weiyi Zhang
1Department of Cell Physiology, Ruhr-Universitaet Bochum, Universitaetsstrasse 150, 44780 Bochum, Germany. eva.neuhaus@rub.de
Abstract:
Multiple trials failed to express significant amounts of olfactory receptors in heterologous cells as they are typically retained in the endoplasmic reticulum (ER). Evidence is accumulating that cell-type-specific accessory proteins regulate the folding of olfactory receptors, their exit from the ER, and the trafficking to the plasma membrane of the olfactory cilia where the receptors gain access to odorants. We found Hsc70t, a testis-enriched variant of the Hsp70 family of heat shock proteins which is specifically expressed in post-meiotic germ cells, in the olfactory epithelium of mouse and human. Cotransfected HEK293 cells with Hsc70t and different green fluorescent protein-tagged odorant receptors (ORs) from mouse and man showed a significantly enhanced OR expression. Hsc70t expression also changed the amount of cells functionally expressing olfactory receptors at the cell surface as the number of cells responding to odorants in Ca2+-imaging experiments significantly increased. Our results show that Hsc70t helps expression of ORs in heterologous cell systems and helped the characterization of an "orphan" human olfactory receptor.
Insights
Heat shock protein Hsc70t enhances olfactory receptor (OR) expression in cells. This finding aids in studying ORs and characterizing previously unknown human olfactory receptors.
Area of Science:
- Molecular biology
- Neuroscience
- Cell biology
Background:
- Olfactory receptors (ORs) are difficult to express in heterologous cells due to retention in the endoplasmic reticulum (ER).
- Cell-type-specific accessory proteins are implicated in OR folding, ER exit, and trafficking to cilia.
- Understanding OR expression is crucial for olfactory research and drug development.
Purpose of the Study:
- To investigate the role of Hsc70t, a testis-enriched heat shock protein, in enhancing olfactory receptor expression.
- To determine if Hsc70t facilitates the cell surface expression and functional activity of ORs in heterologous systems.
- To aid in the characterization of orphan olfactory receptors.
Main Methods:
- Identified Hsc70t in mouse and human olfactory epithelium.
- Cotransfected HEK293 cells with Hsc70t and various green fluorescent protein-tagged ORs.
- Assessed OR expression levels using quantitative methods.
- Evaluated functional OR expression via calcium (Ca2+) imaging experiments in response to odorants.
Main Results:
- Hsc70t significantly enhanced the expression of multiple mouse and human olfactory receptors in cotransfected HEK293 cells.
- Hsc70t expression increased the number of cells functionally expressing ORs at the cell surface.
- Calcium imaging revealed a significant increase in odorant-evoked responses in cells expressing Hsc70t and ORs.
- Facilitated the characterization of an orphan human olfactory receptor.
Conclusions:
- Hsc70t acts as a crucial facilitator for expressing olfactory receptors in heterologous cell systems.
- Hsc70t improves the folding, ER exit, and cell surface trafficking of ORs.
- This study provides a valuable tool for studying OR function and characterizing orphan receptors.
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