Genetically encoded intrabody sensors report the interaction and trafficking of β-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 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.
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BibTeX
@article{baidya2026genetically,
title = {{Genetically encoded intrabody sensors report the interaction and trafficking of β-arrestin 1 upon activation of G-protein–coupled receptors}},
author = {Mithu Baidya and Punita Kumari and Hemlata Dwivedi-Agnihotri and Shubhi Pandey and Badr Sokrat and Silvia Sposini and Madhu Chaturvedi and Ashish Srivastava and Debarati Roy and Aylin Hanyaloglu and Michel Bouvier and Arun Shukla},
journal = {Journal of Biological Chemistry},
year = {2026},
month = sep,
doi = {10.1074/jbc.ra120.013470},
url = {https://hal.science/hal-05767761v1},
note = {CC BY},
}
RIS
TY - JOUR
TI - Genetically encoded intrabody sensors report the interaction and trafficking of β-arrestin 1 upon activation of G-protein–coupled receptors
AU - Mithu Baidya
AU - Punita Kumari
AU - Hemlata Dwivedi-Agnihotri
AU - Shubhi Pandey
AU - Badr Sokrat
AU - Silvia Sposini
AU - Madhu Chaturvedi
AU - Ashish Srivastava
AU - Debarati Roy
AU - Aylin Hanyaloglu
AU - Michel Bouvier
AU - Arun Shukla
PY - 2026
DA - 2026/09/28
JO - Journal of Biological Chemistry
DO - 10.1074/jbc.ra120.013470
UR - https://hal.science/hal-05767761v1
AB - 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.
SN - 0021-9258
SN - 1083-351X
ER -