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Curr Mol Med. 2019 Dec 16. doi: 10.2174/1566524020666191216125825. [Epub ahead of print]

Single cell analysis of β2-Adrenergic receptor dynamics by quantitative fluorescence Microscopy.

Author information

1
Experimental Medicine, King Abdullah International Medical Research Center (KAIMRC), King Saud Bin Abdulaziz University for Health Sciences (KSAU-HS), Ministry of National Guard Health Affairs (NGHA), Riyadh, Saudi Arabia.

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 genetically 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:

Drug targets; GPCR; Quantitative immunoflourescence; Receptor desentization; Receptor signaling; Receptor trafficking

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