Single-Molecule Fluorescence Imaging Reveals GABAB Receptor Aggregation State Changes.

Fang Luo,Lina Wang,Gege Qin,Xiaohong Fang

doi:10.3389/fchem.2021.779940

Abstract

The GABAB receptor is a typical G protein–coupled receptor, and its functional impairment is related to a variety of diseases. While the premise of GABAB receptor activation is the formation of heterodimers, the receptor also forms a tetramer on the cell membrane. Thus, it is important to study the effect of the GABAB receptor aggregation state on its activation and signaling. In this study, we have applied single-molecule photobleaching step counting and single-molecule tracking methods to investigate the formation and change of GABAB dimers and tetramers. A single-molecule stoichiometry assay of the wild-type and mutant receptors revealed the key sites on the interface of ligand-binding domains of the receptor for its dimerization. Moreover, we found that the receptor showed different aggregation behaviors at different conditions. Our results offered new evidence for a better understanding of the molecular basis for GABAB receptor aggregation and activation.

Highlights

G protein–coupled receptors (GPCRs) are a large family of cell surface signaling proteins which play a central physiological role and represent more than one-third of current drug targets (Cornwell and Feigin, 2020; Wang et al, 2021)
Their study was based on the fluorescence resonance energy transfer (FRET) analysis of association and exchange between two populations of the GBR1–GBR2 complex, one is the donor fluorophore–labeled GBR1–GBR2 population presented at the cell surface, and the other is acceptor fluorophore–labeled GBR1-GBR2 population originally retained in the endoplasmic reticulum (ER) but targeted to the plasma membrane after drug application
With the imaging of the GABAB receptor on the cell membrane by using a total internal reflection fluorescence microscope (TIRFM), we revealed the key sites on the hydrophobic interface for GABAB receptor heterodimerization

Summary

INTRODUCTION

G protein–coupled receptors (GPCRs) are a large family of cell surface signaling proteins which play a central physiological role and represent more than one-third of current drug targets (Cornwell and Feigin, 2020; Wang et al, 2021). GBR1 is retained in the endoplasmic reticulum after expression Once it binds with GBR2 to form a heterodimer, the GBR1–GBR2 complex docks to the cell membrane for signaling. It is of great interest to study the aggregation states and their dynamic changes of GABAB receptors under different conditions It has been confirmed by in vitro biochemical assays that GABAB receptors form a heterodimer GBR1–GBR2 in the cell (Burmakina et al, 2014). Their study was based on the fluorescence resonance energy transfer (FRET) analysis of association and exchange between two populations of the GBR1–GBR2 complex, one is the donor fluorophore–labeled GBR1–GBR2 population presented at the cell surface, and the other is acceptor fluorophore–labeled GBR1-GBR2 population originally retained in the endoplasmic reticulum (ER) but targeted to the plasma membrane after drug application. Our results elucidate the molecular base for GABAB receptor dimerization and its influencing factors, providing new reference for a deeper understanding of GABAB receptor signaling under different conditions

MATERIALS AND METHODS

RESULTS AND DISCUSSION

DATA AVAILABILITY STATEMENT