Adrenergic receptor β3 (ADRβ3) is a member of the rhodopsin-like G protein-coupled receptor family. The binding of the ligand to ADRβ3 activates adenylate cyclase and increases cAMP in the cells. ADRβ3 is highly expressed in white and brown adipocytes and controls key regulatory pathways of lipid metabolism. Trp64Arg (W64R) polymorphism in the ADRβ3 is associated with the early development of type 2 diabetes mellitus, lower resting metabolic rate, abdominal obesity, and insulin resistance. It is unclear how the substitution of W64R affects the functioning of ADRβ3. This study was initiated to functionally characterize this obesity-linked variant of ADRβ3. We evaluated in detail the expression, subcellular distribution, and post-activation behavior of the WT and W64R ADRβ3 using single cell quantitative fluorescence microscopy. When expressed in HEK 293 cells, ADRβ3 shows a typical distribution displayed by other GPCRs with a predominant localization at the cell surface. Unlike adrenergic receptor β2 (ADRβ2), agonist-induced desensitization of ADRβ3 does not involve loss of cell surface expression. WT and W64R variant of ADRβ3 displayed comparable biochemical properties, and there was no significant impact of the substitution of tryptophan with arginine on the expression, cellular distribution, signaling, and post-activation behavior of ADRβ3. The obesity-linked W64R variant of ADRβ3 is indistinguishable from the WT ADRβ3 in terms of expression, cellular distribution, signaling, and post-activation behavior.

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.