Determining the oligomeric state of G-protein-coupled receptors via single-molecule fluorescence microscopy

Abstract

In membrane science, the stoichiometry of cell-surface proteins is fundamental to cellular signaling and function. Many membrane receptors including G-protein-coupled receptors (GPCRs) have been proposed to form dimeric or higher order oligomeric complexes and hence certain functional states. Therefore, GPCRs have become of great interest for both scientists and pharmacologists over the past decades. In this work, single-molecule studies of the Glutamate receptor - a constitutive dimer serving as a model system of a protein with known subunit stoichiometry - are presented. In future experiments, results based on mGlu2 dimerization will be used to characterize the unknown oligomeric state of the Dopamine receptor D2s. The Dopamine receptor is a member of the rhodopsin-like GPCRs and mediates the physiological function of the neurotransmitter dopamine. Abnormalities in dopaminergic neurotransmission are associated with various neuropsychiatric and neurodegenerative disorders, e.g. schizophrenia, depression, bipolar disorder, Parkinson's disease, Huntington's disease, drug addiction and alcohol dependence. Recently, single particle tracking and FRET (Förster Resonance Energy Transfer) experiments on mGlu2 and D2s have been performed by our collaborator at low receptor surface densities. Results suggested, that dopamine receptors form dimeric or higher-order oligomeric complexes with distinctive signaling profiles and functions, while the pure dimeric nature of mGlu2 was confirmed. In order to be able to characterize these receptors at physiologically high surface densities, we utilized in-house developed single-molecule methods. The TOCCSL (Thinning Out Clusters while Conserving Stoichiometry of Labeling) method is a single-molecule technique for counting subunits of proteins in live cellular membranes. TOCCSL transiently and virtually dilutes the surface density of fluorophores in a subregion of the plasma membrane without altering their properties. It is based on photobleaching of a small region of interest and subsequent imaging at the onset of the recovery process. A two-color TOCCSL approach was utilized in order to identify the subunit stoichiometry of the glutamate receptor at different surface densities by colocalization analysis. In addition, single-molecule FRET between differently labeled molecules was used to support the two-color TOCCSL based results.