Title:Single-cell Analysis of β2-Adrenergic Receptor Dynamics by Quantitative Fluorescence Microscopy
Volume: 20
Issue: 6
关键词:
G蛋白偶联受体，受体脱敏，定量免疫荧光，荧光显微镜，C末端，显微镜检测。
摘要:
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.
