β‐arrestin1 phosphorylation by GRK5 regulates G protein‐independent 5‐HT4 receptor signalling

G protein‐coupled receptors (GPCRs) have been found to trigger G protein‐independent signalling. However, the regulation of G protein‐independent pathways, especially their desensitization, is poorly characterized. Here, we show that the G protein‐independent 5‐HT₄ receptor (5‐HT₄R)‐operated Src/ERK (extracellular signal‐regulated kinase) pathway, but not the G_s pathway, is inhibited by GPCR kinase 5 (GRK5), physically associated with the proximal region of receptor’ C‐terminus in both human embryonic kidney (HEK)‐293 cells and colliculi neurons. This inhibition required two sequences of events: the association of β–arrestin1 to a phosphorylated serine/threonine cluster located within the receptor C‐t domain and the phosphorylation, by GRK5, of β–arrestin1 (at Ser⁴¹²) bound to the receptor. Phosphorylated β‐arrestin1 in turn prevented activation of Src constitutively bound to 5‐HT₄Rs, a necessary step in receptor‐stimulated ERK signalling. This is the first demonstration that β‐arrestin1 phosphorylation by GRK5 regulates G protein‐independent signalling.