A3 Adenosine Receptors are Localized to Plaque‐like Microdomains in Chemotaxing Cells

In human neutrophils, chemoattractant stimulation promotes a polarized release of ATP. The released ATP is hydrolyzed to adenosine, which facilitates cell migration through the autocrine activation of G protein‐coupled A 3 adenosine receptors (A 3 ARs); however, the precise role of A 3 ARs in cell migration is controversial. Using fluorescent A 3 AR ligands, we show that endogenous A 3 ARs are localized to plaque‐like microdomains on the surface of neutrophil‐like HL60 cells. Pre‐incubation with a selective A 3 AR antagonist blocked the binding of the fluorescent ligands, and cholesterol depletion dissociated the plaques. Furthermore, HL60 cells expressing an RFP‐tagged actin binding protein (LifeAct) that does not interfere with actin polymerization revealed that the plaques are strongly co‐localized with pseudopod‐ (leading‐edge) like structures. Fluorescence correlation spectroscopy (FCS) and total internal fluorescence microscopy (TIRFM) were used to further characterize the plaques, and studies using transwell chambers, microscopy and 3D‐chemotaxis gels documented the role of the A 3 AR in migration. Our results reveal a clear role of A 3 ARs in chemotaxis; an increased understanding of adenosine receptors in this process could reveal new therapeutic targets for infectious and autoimmune diseases. This work was funded by the Medical Research Council/British Pharmacological Society.