Functional Characterization of the Obesity-Linked Variant of the β3-Adrenergic Receptor

Abstract

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.

Keywords: G-protein coupled receptors; beta-3-adrenergic receptor; receptor desensitization.

Figure 1

Engineering of HA-ADRβ3-GFP reporter construct and its functional validation. (A) HA-ADRβ3-GFP reporter construct was generated by PCR cloning as described in the method section. (B) HEK293 cells stably expressing HA-ADRβ3-GFP and visualized under fluorescence microscope (GFP channel). (C) β3 agonist-induced cAMP formation in cells expressing empty vector, untagged ADRβ3, or HA-ADRβ3-GFP.

Figure 2

Total and cell surface expression of WT and W64R variant of ADRβ3. (A) Imaging showing GFP (total) and CY5 (cell surface) expression of WT and W64R ADRβ3 (B,C) Single cell analysis showing total (B) and cell surface expression (C) of WT and W64R ADRβ3. Each graph represents analysis of at least 100 cells across multiple experiments.

Figure 3

Agonist-induced cAMP formation in cells expressing WT and W64R variant of ADRβ3. (Dose dependent cAMP formation in cells expressing WT and W64R ADRβ3. Each bar represents an average of three independent experiments. Statistical analysis was carried out by students’ t-test ^ns p > 0.05.

Figure 4

Agonist-induced desensitization of ADRβ2 and ADRβ3. (A) Experimental outline to measure agonist-induced desensitization of ADRβ2 and ADRβ3. (B) cAMP formation in cells expressing ADRβ2 after 1st or 2nd challenge with isoproterenol. (C) cAMP formation in cells expressing ADRβ3 after 1st or 2nd challenge with SR-58611A. (D) Fluorescent images of cells showing expression of HA-ADRβ2-GFP and HA-ADRβ3-GFP without and with agonist stimulation. (E) Graph showing surface to total cellular expression (Cy5/GFP) of ADRβ2 and ADRβ3 with or without agonist stimulation. Each bar represents an average of three independent experiments. ** p < 0.01 and ^ns p > 0.05.

Figure 5

Agonist-induced desensitization of WT and W64R ADRβ3. (A) Agonist-induced cAMP formation in cells expressing ADRβ2, WT ADRβ3, and W64R ADRβ3. (B) Fluorescent images of cells showing expression HA-ADRβ2-GFP, WT HA-ADRβ3-GFP, and W64R HA-ADRβ3-GFP without and with agonist stimulation. (C) Graph showing surface to total cellular expression (Cy5/GFP) of ADRβ2, WT ADRβ3, and W64R ADRβ3 with or without agonist stimulation. Each bar represents an average of three independent experiments. ** p < 0.01 and ^ns p > 0.05.