Publication — IRIC

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

Agonist stimulation of G-protein-coupled receptors (GPCRs) typically leads to phosphorylation of GPCRs and binding to multifunctional proteins called β-arrestins (βarrs). The GPCR-βarr interaction critically contributes to GPCR desensitization, endocytosis, and downstream signaling, and GPCR-βarr complex formation can be used as a generic readout of GPCR and βarr activation. Although several methods are currently available to monitor GPCR-βarr interactions, additional sensors to visualize them may expand the toolbox and complement existing methods. We have previously described antibody fragments (FABs) that recognize activated βarr1 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-βarr1 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 βarr1 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 βarr1, and the intrabodies co-localized with βarr1 at the cell surface and in intracellular vesicles. Interestingly, we discovered that intrabody sensors can also report βarr1 recruitment and trafficking for several unmodified GPCRs. Our characterization of intrabody sensors for βarr1 recruitment and trafficking expands currently available approaches to visualize GPCR-βarr1 binding, which may help decipher additional aspects of GPCR signaling and regulation.

Date de publication
24 juillet 2020
Chercheurs
Baidya M, Kumari P, Dwivedi-Agnihotri H, Pandey S, Sokrat B, Sposini S, Chaturvedi M, Srivastava A, Roy D, Hanyaloglu AC, Bouvier M, Shukla AK
Référence PubMed
J. Biol. Chem. 2020;295(30):10153-10167
ID PubMed
32439801
Affiliation
Department of Biological Sciences and Bioengineering, Indian Institute of Technology, Kanpur, India.