Abstract
Organization of G protein‐coupled receptors at the plasma membrane has been the focus of much recent attention. Advanced microscopy techniques have shown that these receptors can be localized to discrete microdomains and reorganization upon ligand activation is crucial in orchestrating their signaling. Here, we have compared the membrane organization and downstream signaling of a mutant (R108A, R3.50A) of the adenosine A3 receptor (A3AR) to that of the wild‐type receptor. Fluorescence Correlation Spectroscopy (FCS) studies with a fluorescent agonist (ABEA‐X‐BY630) demonstrated that both wild‐type and mutant receptors bind agonist with high affinity but in subsequent downstream signaling assays the R108A mutation abolished agonist‐mediated inhibition of cAMP production and ERK phosphorylation. In further FCS studies, both A3AR and A3AR R108A underwent similar agonist‐induced increases in receptor density and molecular brightness which were accompanied by a decrease in membrane diffusion after agonist treatment. Using bimolecular fluorescence complementation, experiments showed that the R108A mutant retained the ability to recruit β‐arrestin and these receptor/arrestin complexes displayed similar membrane diffusion and organization to that observed with wild‐type receptors. These data demonstrate that effective G protein signaling is not a prerequisite for agonist‐stimulated β‐arrestin recruitment and membrane reorganization of the A3AR.
Highlights
The adenosine A3 receptor (A3AR) belongs to a subfamily of four G protein-coupled receptors (GPCRs) that are all activated by their cognate ligand adenosine.[1]
Through the use of advanced microscopy techniques, it is clear that some GPCRs are localized to discrete microdomains, and reorganization occurs upon ligand activation.[26]
The residue mutated in this study, R108 or R3.50 according to the numbering of Ballesteros and Weinstein,[19] is one of the most conserved residues within family A GPCRs, with Arg being present in 95% of receptors.[28]
Summary
The adenosine A3 receptor (A3AR) belongs to a subfamily of four G protein-coupled receptors (GPCRs) that are all activated by their cognate ligand adenosine.[1]. According to the ternary complex model of GPCR activation proposed in 1980, GPCRs can exist in resting (R) or activated (R*) conformations, the latter of which may be coupled to a G protein (R*G) with agonists binding preferentially, and with high affinity, to the active conformations.[15] In 1993, it was found that replacement of the third intracellular loop of the β2 adrenergic receptor with the equivalent sequence of the α1B adrenergic receptor resulted in a receptor that signaled in the absence of ligand. Using a combination of second messenger assays and FCS, we have studied the signaling, diffusion, and organization of this mutant receptor at the cell surface under a variety of different conditions, including its agonist and β-arrestin bound states
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