Efficient G protein coupling is not required for agonist‐mediated internalization and membrane reorganization of the adenosine A3 receptor. Issue 4 (12th March 2021)

• Record Type: Journal Article
• Authors: Stoddart, Leigh A.; Kilpatrick, Laura E.; Corriden, Ross; Kellam, Barrie; Briddon, Stephen J.; Hill, Stephen J.
• Is Part Of: FASEB journal. Volume 35:Issue 4(2021)
• Page Start: n/a
• Page End: n/a
• Publication Date: 2021-03-12
• Subjects: β‐arrestin -- adenosine A3 receptor -- fluorescence correlation spectroscopy -- G protein‐coupled receptor -- internalization -- membrane organization; Biology -- Periodicals; Biology, Experimental -- Periodicals; 570
• DOI: 10.1096/fj.202001729RR
• Languages: English
• ISSNs: 0892-6638

Abstract

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 (A3 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 A3 AR and A3 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 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 A3 AR.