Adhesion GPCR ADGRL1/Latrophilin-1 mobilizes β-arrestins as a prerequisite for endosomal restriction of G protein activation and splice-dependent scaffolds

Sustained signaling mediated by β-arrestins (βarr) in endosomal compartments has been described for several classical G protein-coupled receptors (GPCR), yet whether adhesion GPCRs—many of which exhibit constitutive activity driven by intrinsic agonism—engage similar βarr-dependent signaling mechanisms, remains unresolved. Here, we investigated two splice variants of the synapse-organizing adhesion GPCR ADGRL1/Latrophilin-1 using overexpression approaches and G-protein biosensors in HEK293 cells. Both ADGRL1 splice variants exhibited conserved βarr recognition motifs, displayed βarr-dependent trafficking properties, recruited βarr intrinsically at the plasma membrane and early endosomes, and assembled into βarr-containing complexes. Prolonged stimulation with neurexin1β promoted receptor internalization into βarr-positive vesicles with splice variant-dependent trafficking kinetics. In contrast to the canonical paradigm of sustained signaling set by classical GPCRs, knockdown- and knockout-mediated depletion of endogenous βarr suppressed ADGRL1-mediated activation of biosensors representing all four G-protein families, phenocopying dynamin inhibition. Endosome-targeted biosensors revealed splice variant-specific βarr- and dynamin-dependent G-protein trafficking profiles, and a marked endosomal retention of active G-proteins in the absence of βarr. Consistent with a role for βarr as G-protein adaptors in endosomes, ADGRL1 activity generated splice-dependent patterns of βarr/G-protein complex assembly, shared for Gs but divergent for Gq and G13. Collectively, these data identify β-arrestins as key organizers of adhesion GPCR-mediated signaling acting through receptor endosomal priming, select complex assembly and spatiotemporal control of G protein trafficking. • Adhesion GPCR ADGRL1 exhibits a high level of intrinsic interaction with β-arrestins. • Sustained signaling elicited by ADGRL1 occurs predominantly through endosomal pathways. • β-arrestins promote endosomal G protein signaling induced by ADGRL1. • ADGRL1-mediated trafficking of active G proteins in endosomes relies on β-arrestins • Alternative splicing of the ADGRL1 cytoplasmic tail can direct distinct signaling profiles and β-arrestins/G protein complexes.