Genetically encoded intrabody sensors report the interaction and trafficking of b-arrestin 1 upon activation of G-protein–coupled receptors

Abstract

Agonist stimulation of G-protein–coupled receptors (GPCRs) typically leads to phosphorylation of GPCRs and binding to multifunctional proteins called b-arrestins (barrs). The GPCR–b arr interaction critically contributes to GPCR desensitization, endocytosis, and downstream signaling, and GPCR–barr complex formation can be used as a generic readout of GPCR and barr activation. Although several methods are currently available to monitor GPCR–barr interactions, additional sensors to visualize them may expand the toolbox and complement existing methods. We have previously described antibody fragments (FABs) that recognize activated barr1 upon its interaction with the vasopressin V2 receptor C-terminal phosphopeptide (V2Rpp). Here, we demonstrate that these FABs efficiently report the formation of a GPCR–barr1 complex for a broad set of chimeric GPCRs harboring the V2R C terminus. We adapted these FABs to an intrabody format by converting them to single-chain variable fragments and used them to monitor the localization and trafficking of barr1 in live cells. We observed that upon agonist simulation of cells expressing chimeric GPCRs, these intrabodies first translocate to the cell surface, followed by trafficking into intracellular vesicles. The translocation pattern of intrabodies mirrored that of barr1, and the intrabodies co-localized with barr1 at the cell surface and in intracellular vesicles. Interestingly, we discovered that intrabody sensors can also report barr1 recruitment and trafficking for several unmodified GPCRs. Our characterization of intrabody sensors for barr1 recruitment and trafficking expands currently available approaches to visualize GPCR–barr1 binding, which may help decipher additional aspects of GPCR signaling and regulation.