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. 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. G-protein–coupled receptors (GPCRs) recognize a diverse set of ligands and initiate a broad spectrum of downstream signaling responses (1Bockaert J. Pin J.P. Molecular tinkering of G protein–coupled receptors: an evolutionary success.EMBO J. 1999; 18 (10202136): 1723-172910.1093/emboj/18.7.1723Crossref PubMed Scopus (1182) Google Scholar). Upon agonist stimulation, GPCRs couple to three major subfamilies of cellular proteins namely, the heterotrimeric G-proteins, GPCR kinases, and β-arrestins (βarrs) (1Bockaert J. Pin J.P. Molecular tinkering of G protein–coupled receptors: an evolutionary success.EMBO J. 1999; 18 (10202136): 1723-172910.1093/emboj/18.7.1723Crossref PubMed Scopus (1182) Google Scholar). Of these, βarrs are multifunctional adaptor proteins, which play a central role in regulatory and signaling paradigms of GPCRs (2Kang D.S. Tian X. Benovic J.L. Role of β-arrestins and arrestin domain–containing proteins in G protein–coupled receptor trafficking.Curr. Opin. Cell Biol. 2014; 27 (24680432): 63-7110.1016/j.ceb.2013.11.005Crossref PubMed Scopus (132) Google Scholar, 3Lefkowitz R.J. Shenoy S.K. Transduction of receptor signals by β-arrestins.Science. 2005; 308 (15845844): 512-51710.1126/science.1109237Crossref PubMed Scopus (1313) Google Scholar). βarrs are evenly distributed in the cytoplasm under basal condition, and upon agonist stimulation, they typically translocate to the plasma membrane to interact with activated and phosphorylated receptors (4Oakley R.H. Laporte S.A. Holt J.A. Caron M.G. Barak L.S. Differential affinities of visual arrestin, beta arrestin1, and beta arrestin2 for G protein–coupled receptors delineate two major classes of receptors.J. Biol. Chem. 2000; 275 (10748214): 17201-1721010.1074/jbc.M910348199Abstract Full Text Full Text PDF PubMed Scopus (634) Google Scholar). Binding of βarrs to GPCRs at the plasma membrane results in termination of G-protein coupling and desensitization of receptors through a steric hindrance-based mechanism (5Freedman N.J. Lefkowitz R.J. Desensitization of G protein–coupled receptors.Recent Prog. Horm. Res. 1996; 51 (8701085): 319-351PubMed Google Scholar). Subsequently, βarrs either dissociate from the receptors and relocalize back in the cytoplasm or traffic into endosomal vesicles in complex with the receptors (2Kang D.S. Tian X. Benovic J.L. Role of β-arrestins and arrestin domain–containing proteins in G protein–coupled receptor trafficking.Curr. Opin. Cell Biol. 2014; 27 (24680432): 63-7110.1016/j.ceb.2013.11.005Crossref PubMed Scopus (132) Google Scholar, 4Oakley R.H. Laporte S.A. Holt J.A. Caron M.G. Barak L.S. Differential affinities of visual arrestin, beta arrestin1, and beta arrestin2 for G protein–coupled receptors delineate two major classes of receptors.J. Biol. Chem. 2000; 275 (10748214): 17201-1721010.1074/jbc.M910348199Abstract Full Text Full Text PDF PubMed Scopus (634) Google Scholar). These two different patterns are referred to as “class A” and “class B,” respectively (4Oakley R.H. Laporte S.A. Holt J.A. Caron M.G. Barak L.S. Differential affinities of visual arrestin, beta arrestin1, and beta arrestin2 for G protein–coupled receptors delineate two major classes of receptors.J. Biol. Chem. 2000; 275 (10748214): 17201-1721010.1074/jbc.M910348199Abstract Full Text Full Text PDF PubMed Scopus (634) Google Scholar). βarrs also contribute in a number of downstream GPCR signaling pathways such as ERK1/2 MAP kinase activation, although strict G-protein independence of such mechanisms are currently being discussed and debated (6Grundmann M. Merten N. Malfacini D. Inoue A. Preis P. Simon K. Rüttiger N. Ziegler N. Benkel T. Schmitt N.K. Ishida S. Müller I. Reher R. Kawakami K. Inoue A. et al.Lack of beta-arrestin signaling in the absence of active G proteins.Nat. Commun. 2018; 9 (29362459): 34110.1038/s41467-017-02661-3Crossref PubMed Scopus (156) Google Scholar, 7Gurevich V.V. Gurevich E.V. Arrestin-mediated signaling: Is there a controversy?.World J. Biol. Chem. 2018; 9 (30595812): 25-3510.4331/wjbc.v9.i3.25Crossref PubMed Google Scholar, 8Gutkind J.S. Kostenis E. Arrestins as rheostats of GPCR signalling.Nat. Rev. Mol. Cell Biol. 2018; 19 (30026541): 615-61610.1038/s41580-018-0041-yCrossref PubMed Scopus (21) Google Scholar, 9Luttrell L.M. Wang J. Plouffe B. Smith J.S. Yamani L. Kaur S. Jean-Charles P.Y. Gauthier C. Lee M.H. Pani B. Kim J. Ahn S. Rajagopal S. Reiter E. Bouvier M. et al.Manifold roles of β-arrestins in GPCR signaling elucidated with siRNA and CRISPR/Cas9.Sci. Signal. 2018; 11 (30254056): eaat765010.1126/scisignal.aat7650Crossref PubMed Scopus (86) Google Scholar). Considering the multifaceted roles of βarrs, understanding the details of their interaction with GPCRs continues to be a frontier area in GPCR research (10Ranjan R. Dwivedi H. Baidya M. Kumar M. Shukla A.K. Novel structural insights into GPCR–β-arrestin interaction and signaling.Trends Cell Biol. 2017; 27 (28651823): 851-86210.1016/j.tcb.2017.05.008Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). The interaction of βarrs with GPCRs involves two distinct components (11Gurevich V.V. Gurevich E.V. The molecular acrobatics of arrestin activation.Trends Pharmacol. Sci. 2004; 25 (15102497): 105-11110.1016/j.tips.2003.12.008Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 12Shukla A.K. Westfield G.H. Xiao K. Reis R.I. Huang L.Y. Tripathi-Shukla P. Qian J. Li S. Blanc A. Oleskie A.N. Dosey A.M. Su M. Liang C.R. Gu L.L. Shan J.M. et al.Visualization of arrestin recruitment by a G-protein–coupled receptor.Nature. 2014; 512 (25043026): 218-22210.1038/nature13430Crossref PubMed Scopus (315) Google Scholar). One is receptor phosphorylation, primarily in the C terminus but also in the intracellular loops, and the other is the intracellular side of receptor transmembrane bundle, referred to as the receptor core (11Gurevich V.V. Gurevich E.V. The molecular acrobatics of arrestin activation.Trends Pharmacol. Sci. 2004; 25 (15102497): 105-11110.1016/j.tips.2003.12.008Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 12Shukla A.K. Westfield G.H. Xiao K. Reis R.I. Huang L.Y. Tripathi-Shukla P. Qian J. Li S. Blanc A. Oleskie A.N. Dosey A.M. Su M. Liang C.R. Gu L.L. Shan J.M. et al.Visualization of arrestin recruitment by a G-protein–coupled receptor.Nature. 2014; 512 (25043026): 218-22210.1038/nature13430Crossref PubMed Scopus (315) Google Scholar). There are several assays that are currently used to measure GPCR–βarr interaction, including those based on resonance energy transfer (13Angers S. Salahpour A. Joly E. Hilairet S. Chelsky D. Dennis M. Bouvier M. Detection of β2-adrenergic receptor dimerization in living cells using bioluminescence resonance energy transfer (BRET).Proc. Natl. Acad. Sci. U.S.A. 2000; 97 (10725388): 3684-368910.1073/pnas.97.7.3684PubMed Google Scholar, 14Charest P.G. Terrillon S. Bouvier M. Monitoring agonist-promoted conformational changes of β-arrestin in living cells by intramolecular BRET.EMBO Rep. 2005; 6 (15776020): 334-34010.1038/sj.embor.7400373Crossref PubMed Scopus (139) Google Scholar, 15Namkung Y. Le Gouill C. Lukashova V. Kobayashi H. Hogue M. Khoury E. Song M. Bouvier M. Laporte S.A. Monitoring G protein–coupled receptor and β-arrestin trafficking in live cells using enhanced bystander BRET.Nat. Commun. 2016; 7 (27397672): 1217810.1038/ncomms12178Crossref PubMed Scopus (101) Google Scholar), enzyme complementation (16Bassoni D.L. Raab W.J. Achacoso P.L. Loh C.Y. Wehrman T.S. Measurements of β-arrestin recruitment to activated transmembrane receptors using enzyme Mol. Biol. PubMed Scopus Google Scholar), and responses Y. J. B. R. Lee The of signaling to receptor Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar, Huang K. N. as an for of the Mol. Biol. PubMed Scopus Google Scholar). sensors is to expand the currently available toolbox and complement the existing have that receptor phosphorylation is to βarr binding, but can also βarr of receptor and signaling P. A. R. E. P. R. X. B. C. D. Shukla A.K. of a Commun. 2016; 7 PubMed Scopus (86) Google Scholar, P. A. E. R. S. Shukla A.K. with β-arrestin is for vasopressin receptor and Biol. 2017; PubMed Scopus Google Scholar, Plouffe B. Huang L.Y. D.L. S. Shukla A.K. B. et of GPCR–β-arrestin desensitization, signaling, and Natl. Acad. Sci. U.S.A. 2017; PubMed Scopus Google Scholar). These the that such as which recognize βarr by the interaction of phosphorylated may as sensors for βarr recruitment and Here, we and intrabody sensors from antibody fragments (FABs) βarr1 that report the formation of GPCR–βarr1 and to monitor βarr1 trafficking in cellular receptor phosphorylation is a for βarr recruitment (11Gurevich V.V. Gurevich E.V. The molecular acrobatics of arrestin activation.Trends Pharmacol. Sci. 2004; 25 (15102497): 105-11110.1016/j.tips.2003.12.008Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). phosphopeptide to the C terminus of the vasopressin V2 referred to as used as a to active βarr in K. Shenoy S.K. K. Lefkowitz R.J. conformational changes in β-arrestin Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, Xiao K. Lefkowitz R.J. The active of for the in the and conformational in the active of and Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A.K. A. Xiao K. Reis R.I. D. S. Huang L.Y. M. Tripathi-Shukla P. A. S. et of active to a G-protein–coupled receptor PubMed Scopus Google Scholar, E. Dwivedi H. Baidya M. A. P. T. Kim Lee M.H. J. M. D. S. J. R. L.M. et sensors and structural and β-arrestin Rep. Full Text Full Text PDF PubMed Scopus Google Scholar). We have previously and a set of FABs that recognize βarr1 E. A. Baidya M. P. Dwivedi H. K. R. S. A. S. Shukla A.K. and generic of GPCR 2017; PubMed Google Scholar). We have also used of these referred to as to monitor the interaction of βarr1 with a chimeric β2-adrenergic receptor harboring V2R C terminus to as and V2R E. Dwivedi H. Baidya M. A. P. T. Kim Lee M.H. J. M. D. S. J. R. L.M. et sensors and structural and β-arrestin Rep. Full Text Full Text PDF PubMed Scopus Google Scholar). the first these FABs as sensors of GPCR–βarr interaction and we first their to report the formation of complex in Here, we used from cells expressing with βarr1 and followed by and of the receptor as a readout of complex We observed that and the additional FABs upon agonist stimulation through the formation of complex that interact with βarr1 to in the and to generic intrabody sensors from these we their to recognize βarr1 complex with other GPCRs. Considering that these FABs βarr1, we that they βarr1 complex for other chimeric GPCRs harboring the V2R C to that in We different chimeric GPCRs including the from different such as complement and and of these such as and intracellular loops, have intracellular We the of which the to report the formation of complex in for these in we observed that efficiently βarr1 for chimeric GPCR to that of to that these FABs as generic intrabody sensors of βarr1 interaction and trafficking in cellular for a broad set of chimeric GPCRs. these FABs into cellular sensors of βarr1 and is to them in in the cytoplasm as We the FABs into single-chain variable fragments by the variable of their and through a previously A.K. Westfield G.H. Xiao K. Reis R.I. Huang L.Y. Tripathi-Shukla P. Qian J. Li S. Blanc A. Oleskie A.N. Dosey A.M. Su M. Liang C.R. Gu L.L. Shan J.M. et al.Visualization of arrestin recruitment by a G-protein–coupled receptor.Nature. 2014; 512 (25043026): 218-22210.1038/nature13430Crossref PubMed Scopus (315) Google and them in cells as either with a C-terminal or as We observed of two of these intrabodies and in we observed as as localization The for localization of the intrabodies is to although a also localization of an intrabody β2-adrenergic receptor R. M. J.P. J. H. Huang B. M. GPCR from PubMed Scopus Google Scholar). We intrabodies can report the formation of complex in a cellular We first βarr1, and intrabodies in the cells with either an agonist or agonist and the intrabodies using the We observed that and the complex upon agonist stimulation, although and We also the of to recognize the complex upon stimulation of the receptor with a set of ligands with we observed that the of of the complex by the of the ligands C and the of to report the formation of complex and its as a of the of intrabodies to monitor βarr1 trafficking upon receptor stimulation, we and intrabodies in cells and followed the localization of βarr1 and intrabodies using agonist and of in a pattern of βarr1 and the intrabodies followed the localization of βarr1 and and We observed that and first to the cell surface from the and upon agonist stimulation, they in the intracellular vesicles. these demonstrate the of intrabodies as to monitor the formation of the complex in and βarr1 trafficking in the cellular the intrabodies are from FABs βarr1, we that they be to report βarr1 interaction and trafficking for V2R as we the of and to the formation of the complex in and report translocation of βarr1 in a cellular We observed a pattern to that of described in the and and complex upon agonist stimulation and followed the localization pattern of βarr1 upon agonist stimulation as by translocation to the cell surface first followed by localization in intracellular vesicles. additional observed on the in the which the V2R but its is currently to We also the of to recognize βarr1 upon agonist stimulation of V2R and observed a interaction in and we the translocation pattern of upon agonist stimulation for and V2R in cells βarr1 is in to intracellular vesicles agonist stimulation, which is of the translocation pattern of βarr1 for these These the of intrabody sensors described in βarr1 recruitment and the intrabodies to be sensors of βarr recruitment and is that they βarr receptor endocytosis, and G-protein we first recruitment of βarr1 to V2R in of either a intrabody or using an in we in βarr1 to V2R is co-localized with and βarr1 on intracellular we on cells expressing and agonist stimulation we observed a of βarr1, and on intracellular that the trafficking pattern of complex in a cellular is by the pattern of V2R with the endosomal and which in of C and we also βarr1 trafficking to upon V2R using an enhanced bystander Y. Le Gouill C. Lukashova V. Kobayashi H. Hogue M. Khoury E. Song M. Bouvier M. Laporte S.A. Monitoring G protein–coupled receptor and β-arrestin trafficking in live cells using enhanced bystander BRET.Nat. Commun. 2016; 7 (27397672): 1217810.1038/ncomms12178Crossref PubMed Scopus (101) Google in of either or Although we a in to endosomal localization of βarr1 as by is the intrabody sensors are used in the of receptor they receptor to the plasma and be to in We the of intrabodies on coupling to the V2R using as a we in or for and we also the of intrabodies on ERK1/2 MAP kinase activation, a readout of V2R signaling, and a by the intrabodies C and these that intrabodies have a major on coupling and receptor endocytosis, them sensors to βarr1 interaction and trafficking for GPCRs. from the of to recognize βarr1 complex with several chimeric GPCRs as in we as a to report βarr1 trafficking for these chimeric GPCRs in cellular to we the chimeric receptors with and in cells and followed the localization of βarr1 and intrabodies using agonist We observed that to followed βarr1 translocation pattern by first to the cell surface followed by trafficking into intracellular vesicles for of these chimeric receptors is that the receptors used in 9 of the phosphorylation in their C their intracellular are the other receptors in 9 a intracellular which also of the phosphorylation and their C terminus is the in 9 demonstrate the of as a to monitor βarr1 recruitment and trafficking for chimeric GPCRs but also its for receptors in of their C terminus and intracellular we the of the to report the trafficking of βarr1 for a set of GPCRs the of We observed that followed translocation pattern of βarr1 for several different receptors including the complement receptor the receptor the receptor and the receptor We also the of to recognize βarr1 for and by and These that can as a for βarr1 translocation for at GPCRs with their C terminus as Interestingly, we observed that βarr1 translocation for the receptor upon agonist stimulation although there translocation of βarr1, first to the plasma membrane and in intracellular vesicles. these at conformational in GPCR–βarr1 the recruitment patterns are on conformational in different GPCR–βarr may additional insights and the conformational to Monitoring βarr interaction and trafficking used to the and regulatory of GPCRs. number of approaches are for including of proteins to βarrs (4Oakley R.H. Laporte S.A. Holt J.A. Caron M.G. Barak L.S. Differential affinities of visual arrestin, beta arrestin1, and beta arrestin2 for G protein–coupled receptors delineate two major classes of receptors.J. Biol. Chem. 2000; 275 (10748214): 17201-1721010.1074/jbc.M910348199Abstract Full Text Full Text PDF PubMed Scopus (634) Google Scholar), resonance energy transfer assays P.G. Terrillon S. Bouvier M. Monitoring agonist-promoted conformational changes of β-arrestin in living cells by intramolecular BRET.EMBO Rep. 2005; 6 (15776020): 334-34010.1038/sj.embor.7400373Crossref PubMed Scopus (139) Google Scholar, A. C. or to insights from and Opin. Cell Biol. PubMed Scopus Google Scholar), enzyme complementation methods (16Bassoni D.L. Raab W.J. Achacoso P.L. Loh C.Y. Wehrman T.S. Measurements of β-arrestin recruitment to activated transmembrane receptors using enzyme Mol. Biol. PubMed Scopus Google Scholar), and assays Y. J. B. R. Lee The of signaling to receptor Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar, Huang K. N. as an for of the Mol. Biol. PubMed Scopus Google Scholar). of these methods a and of the the or sensors described recognize βarr1 and report its trafficking in cellular the for of Although we that the intrabody sensors are of βarr1 for several GPCRs the of their C a is that they are to be for GPCR as for in the other these intrabody sensors are to recognize βarr1 in the of chimeric GPCRs harboring the V2R C terminus. is that a can be for other GPCRs as by for from the is also that of the βarr assays such as also chimeric GPCRs with V2R C terminus Huang K. N. as an for of the Mol. Biol. PubMed Scopus Google Scholar). V2R typically a pattern on GPCRs and the of βarr1 interaction with the unmodified receptors R.H. Laporte S.A. Holt J.A. Barak L.S. Caron M.G. Molecular the formation of intracellular G protein–coupled receptor Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). is also to that of different FABs two efficiently as intrabodies in the with a number of FABs may be to intrabodies in Considering that the of intrabodies to interact with βarr1 and their is also that they can be adapted in resonance energy transfer or in for of may of these intrabody sensors with approaches although the intrabody sensors are to βarr1 E. Dwivedi H. Baidya M. A. P. T. Kim Lee M.H. J. M. D. S. J. R. L.M. et sensors and structural and β-arrestin Rep. Full Text Full Text PDF PubMed Scopus Google Scholar), is to and intrabodies for as an may help insights into the of the two βarr A. B. C. Shukla A.K. of β-arrestin in GPCR Full Text Full Text PDF PubMed Scopus Google Scholar). of GPCR–βarr1 interaction is the of receptor phosphorylation patterns to distinct in βarrs A.K. D. Shenoy S.K. Lefkowitz R.J. conformational changes in β-arrestin report at Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar, Xiao K. Ahn S. Shukla A.K. Rajagopal S. Huang Shenoy S.K. Lefkowitz R.J. phosphorylation on the β2-adrenergic receptor a that of Signal. PubMed Scopus Google Scholar). several GPCRs, different phosphorylation patterns in cell and have and with βarr R. P. J.M. A. S. Differential G-protein–coupled receptor phosphorylation for a signaling Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, E. Ahn S. Shukla A.K. Lefkowitz R.J. Molecular mechanism of at Rev. Pharmacol. PubMed Scopus Google Scholar, GPCR phosphorylation a mechanism for Pharmacol. Sci. Full Text Full Text PDF PubMed Scopus Google Scholar). is to that intrabodies different from a receptor may of receptor signaling and in expands the currently available toolbox to monitor GPCR–βarr interaction and and the intrabody sensors described insights into GPCR signaling and regulatory cells in and at in of using and the cells typically The have described previously E. Dwivedi H. Baidya M. A. P. T. Kim Lee M.H. J. M. D. S. J. R. L.M. et sensors and structural and β-arrestin Rep. Full Text Full Text PDF PubMed Scopus Google Scholar). intrabodies by their in The chimeric GPCRs by the at in in in in in and in by The from Cell and and from and a previously D. A. Baidya M. H. A. Shukla A.K. of and its interaction with PubMed Scopus Google Scholar). the of FABs cells expressing receptor and with βarr1 and the in the receptor and βarr1 in cells. the cells for with by and with for at Subsequently, the complex with for and to the and of additional of the three with and with on and the receptors using the FABs using the of intrabodies to report the formation of complex and cells expressing the βarr1, and intrabodies with of ligands for at the cells in followed by with of for at The three with with and proteins by antibody at and from at monitor the translocation of βarr1 and intrabodies by C and and and and cells with the and the cells with the cells for to stimulation with of live cell we used and on a with a and a with with a at at and in the for set of and for two by and using the and the in from by the using in S. into in PubMed Scopus Google Scholar). three of cell and the of are in the with the number of cells and and with endosomal receptor of live or cells by cells with antibody in to agonist cells three in to antibody to the surface using at and using antibody followed by or of with endosomal the cells as with either of the antibody from Cell or antibody from Cell The cells using a with a and of as using or and by the using in as measure the of intrabodies on we in a previously described E. Dwivedi H. Baidya M. A. P. T. Kim Lee M.H. J. M. D. S. J. R. L.M. et sensors and structural and β-arrestin Rep. Full Text Full Text PDF PubMed Scopus Google Scholar). cells with the the and the the and the cells and in and of Cell and such as to cells in The cells at for to the and the in the of in the the cells in a the at for followed by an additional of at Subsequently, of the to the and the using a phosphorylation of ERK1/2 MAP kinase by a previously described P. Dwivedi H. Baidya M. Shukla A.K. MAP kinase phosphorylation for G-protein–coupled Biol. PubMed Scopus Google Scholar). βarr1 recruitment and endosomal localization by and on cells in using at a of monitor interaction, we used and described previously E. S. A. M. Bouvier M. characterization of vasopressin receptor to and of for Pharmacol. PubMed Scopus Google Scholar). monitor endosomal translocation of βarr1, we used enhanced bystander in which the is to the from to and βarr1 with the Y. Le Gouill C. Lukashova V. Kobayashi H. Hogue M. Khoury E. Song M. Bouvier M. Laporte S.A. Monitoring G protein–coupled receptor and β-arrestin trafficking in live cells using enhanced bystander BRET.Nat. Commun. 2016; 7 (27397672): 1217810.1038/ncomms12178Crossref PubMed Scopus (101) Google Scholar). the and the cells with and by the cells with of vasopressin for and or signals on a with the and for and and for The as the of the by the energy the by energy are in 7 and in by the in for the and of is in The and using and the details of and are in the are available in the We the of for of the G-protein–coupled receptor β-arrestin antibody bioluminescence resonance energy transfer kinase of intrabody receptor receptor receptor receptor receptor single-chain variable

Topics & Concepts