Spatiotemporal dynamics of β ‐arrestin‐mediated Src activation in 5‐ HT7 receptor signaling pathway

G protein‐coupled receptors (GPCRs) initiate G protein‐mediated signaling pathways upon ligand binding and are desensitized via β ‐arrestin‐dependent internalization. However, emerging evidence indicates that β ‐arrestin also mediates G protein‐independent signaling pathways that lead to distinct cellular responses. This signaling complexity has led to the development of GPCR‐biased ligands that selectively modulate either G protein‐ or β ‐arrestin‐dependent pathways for drug discovery, offering precise control of cellular processes with minimal side effects. We have developed biased ligands for the 5‐hydroxytryptamine receptor subtype 7 (5‐HT 7 R), which is implicated in autism spectrum disorder (ASD). Notably, we observed that a G protein‐biased ligand (2b), but not a β ‐arrestin‐biased ligand (1 g), induced a characteristic ASD behavior in mice, suggesting differential signaling pathways between G protein‐ or β ‐arrestin‐mediated pathways. We thus further explored the mechanisms of β ‐arrestin‐biased signaling pathways of 5‐HT 7 R. Our study revealed that 1 g induced a slow but sustained activation of proto‐oncogene tyrosine‐protein kinase Src (Src), demonstrating temporal specificity of β ‐arrestin‐biased signaling. Src was recruited to 5‐HT 7 R via β ‐arrestin‐2, followed by the internalization and accumulation of this receptor complex at endosomes. The sustained colocalization of Src at the internalized receptor complex suggests a mechanism for the slow and sustained Src activation. These results highlight spatiotemporal regulation of Src activation in GPCR‐biased signaling pathways, providing new insights into 5‐HT 7 R‐targeted therapeutics.