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 post-activation behavior including expression, desensitization and resensitization.
Objective: To develop a quantitative fluorescence Microscopy assay to study beta(2)-Adrenergic receptor expression and desensitization.
Method: beta(2)-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 beta(2)-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 beta(2)-AR.
Conclusion: A highly accurate method is described for measuring beta(2)-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 beta(2)-Adrenergic receptor and can be applied to essentially any other GPCR.