The P2Y₂ Receptor C-Terminal Tail Modulates but Is Dispensable for β-Arrestin Recruitment

The P2Y₂ receptor (P2Y₂R) is a G protein-coupled receptor that is activated by extracellular ATP and UTP, to a similar extent. This allows it to play roles in the cell's response to the (increased) release of these nucleotides, e.g., in response to stress situations, including mechanical stress and oxygen deprivation. However, despite its involvement in important (patho)physiological processes, the intracellular signaling induced by the P2Y₂R remains incompletely described. Therefore, this study implemented a NanoBiT^® functional complementation assay to shed more light on the recruitment of β-arrestins (βarr1 and βarr2) upon receptor activation. More specifically, upon determination of the optimal configuration in this assay system, the effect of different (receptor) residues/regions on βarr recruitment to the receptor in response to ATP or UTP was estimated. To this end, the linker was shortened, the C-terminal tail was truncated, and phosphorylatable residues in the third intracellular loop of the receptor were mutated, in either singly or multiply adapted constructs. The results showed that none of the introduced adaptations entirely abolished the recruitment of either βarr, although EC₅₀ values differed and time-luminescence profiles appeared to be qualitatively altered. The results hint at the C-terminal tail modulating the interaction with βarr, while not being indispensable.