Efficient G protein coupling is not required for agonist-mediated internalization and membrane reorganization of the adenosine A3 receptor

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 A(3) receptor (A(3)AR) 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 A(3)AR and A(3)AR 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 beta-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 beta-arrestin recruitment and membrane reorganization of the A(3)AR.

Automated summary

The organization of G protein-coupled receptors at the plasma membrane has been a recent focus. Studies on the adenosine A(3) receptor and its mutant R108A reveal differences in membrane organization and downstream signaling, but both receptors can recruit beta-arrestin upon agonist stimulation, showing that effective G protein signaling is not necessary for membrane reorganization and beta-arrestin recruitment.