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 (ScFvs) 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.

Key phosphorylation sites in GPCR s orchestrate the contribution of β‐Arrestin 1 in ERK 1/2 activation

“…patterns (Ahn et al, 2004;Srivastava et al, 2015). Not only the differences in the phosphorylation patterns of different GPCRs can fine tune receptor-specific conformations and functional capabilities of barrs (Lee et al, 2016;Nuber et al, 2016;Baidya et al, 2020) but also, differential phosphorylation patterns of a given GPCR can impart distinct conformational changes in recruited barrs resulting in different functional responses (Charest et al, 2005;Shukla et al, 2008;Nobles et al, 2011;Zimmerman et al, 2012). These examples suggest that different GPCR-barr complexes formed in cells, which may grossly look similar, do not necessarily encode identical functional outcomes.…”

“…For transfection, 50-60% confluent cells were transfected with desired DNA using PolyEthylenImine (PEI) as transfection reagent at the DNA:PEI ratio of 1:3. Expression plasmids for V 2 R, B 2 R, AT 1a R, Ib30-YFP, barr1 shRNA, and barr1-mCherry have been described previously (Kumari et al, 2016(Kumari et al, , 2017Ghosh et al, 2017Ghosh et al, , 2019Pandey et al, 2019a;Baidya et al, 2020). Receptor mutants were generated using site-directed mutagenesis kit (NEB) and sequenced (Macrogen) before use in the experiments.…”

“…In order to gain additional mechanistic insight into differential contribution of barr1 in ERK1/2 activation for V 2 R and B 2 R, we used a previously described intrabody-based sensor of barr1 conformation in cellular context (Ghosh et al, 2019;Baidya et al, 2020). This sensor is based on a synthetic antibody fragment referred to as Fab30 that selectively recognizes V 2 R-bound barr1 conformation in vitro (Shukla et al, 2013(Shukla et al, , 2014Kumari et al, 2016Kumari et al, , 2017Ghosh et al, 2019).…”

“…Moreover, an intrabody version of Fab30, referred to as Ib30, also recognizes barr1 upon agonist stimulation of V 2 R in cellular context (Ghosh et al, 2019). Recently, a YFP-tagged version of Ib30 has also been generated and used as a conformational probe for measuring the interaction and trafficking of barr1 in cells upon GPCR stimulation (Baidya et al, 2020). As previously reported, we observed that Ib30-YFP efficiently recognizes V 2 R-bound barr1 and follows the trafficking pattern of barr1 upon agonist stimulation (Figs 4A and EV3), but fails to recognize barr1 upon stimulation of B 2 R (Figs 4B and EV3).…”

miR-7 Regulates GLP-1-Mediated Insulin Release by Targeting β-Arrestin 1

“…Experimental evidence has recently shown that β-arrestin 1 (βARR1), a protein known to be involved in the regulation of signal transduction of GPCRs [ 23 , 24 , 25 , 26 , 27 , 28 , 29 ], plays a key role in the desensitization of GLP-1 receptor in β cells [ 30 ]. Therefore, targeting βARR1 by miR intervention could be a promising strategy for the treatment of diabetes mellitus.…”