Expression and functional characterization of Metabotropic Glutamate Receptor Type 6 (mGluR6) in detergent micelles

Abstract

Metabotropic glutamate receptors (mGluRs) are G protein coupled receptors which are implicated in different brain functions and dysfunctions including learning, memory, pain perception, neurodegeneration, schizophrenia and addiction. Metabotropic glutamate receptor type 6 (mGluR6) is a subtype of mGluRs which is exclusively expressed in ON-bipolar cells and is involved in night vision. Recent genome-wide association studies have discovered involvement of mGluR6 in heroin addiction. mGluR6 is emerging as a new drug target but structure-function relationships of this receptor and in general of mGluRs are poorly understood. These receptors have very low expression levels in their native cells and are only active in a membrane environment. The major problem in studying these receptors with biophysical approaches is in obtaining sufficient quantities of functional protein. To overcome this, we have constructed a tetracycline inducible mammalian stable cell line expressing full length human mGluR6. We optimized the detergent and buffer conditions required for mGluR6 purification. We are developing a reliable, quantitative in vitro assay for verifying the function of purified mGluR6 in different detergents. We have been successful in developing a medium scale expression and purification system of mGluR6. Preliminary data suggests that this receptor responds to a number of ligands relevant to its implicated role in vision and addiction with changes in activity and structure as evidenced by G protein binding and cysteine accessibility measurements, respectively.