Single-cell Analysis of β2-Adrenergic Receptor Dynamics by Quantitative Fluorescence Microscopy

Abstract

Background: G protein-coupled receptors (GPCRs) represent the largest family of surface proteins and are involved in the regulation of key physiological processes. GPCRs are characterized by seven transmembrane domains, an extracellular N-terminus and an intracellular C-terminus. Cellular response of these receptors to their ligands is largely determined by their surface expression and postactivation behavior including expression, desensitization and resensitization.

Objective: To develop a quantitative fluorescence Microscopy assay to study β2- Adrenergic receptor expression and desensitization.

Method: β2-Adrenergic receptor cDNA was engineered to put an HA tag at the extracellular N-terminus and GFP Tag at the intracellular C-terminus. GFP fluorescence serves as a measure of total cellular expression; whereas staining with CY3 conjugated anti-HA antibodies without permeabilizing the cells represents the surface expression of β2-AR. The images are quantified and amount of CY3 (surface) and GFP (total) fluorescence for each cell determined using image processing software.

Results: The method is sensitive and allows for the simultaneous measurement of surface and total expression of β2-AR.

Conclusion: A highly accurate method is described for measuring β2-AR surface and total expression based on single-cell quantitative immunofluorescence. The method can be used to determine agonist-induced desensitization and resensitization process as well as receptor kinetics like endocytosis and exocytosis of β2-Adrenergic receptor and can be applied to essentially any other GPCR.

Keywords: C-terminus; G-protein coupled receptors; fluorescence microscopy; microscopy assay; quantitative immune-flourescence; receptor desensitization..