Spatiotemporal dynamics of <i>β</i> ‐arrestin‐mediated Src activation in 5‐ <scp>HT7</scp> 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-HT₇R), 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-HT₇R. 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-HT₇R 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-HT₇R-targeted therapeutics.