A novel luminescence-based β-arrestin recruitment assay for unmodified receptors
2021; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1016/j.jbc.2021.100503
ISSN1083-351X
AutoresM. Hauge, Jennifer Pham, Helena Mancebo, Asuka Inoue, Wesley B. Asher, Jonathan A. Javitch,
Tópico(s)Photoreceptor and optogenetics research
ResumoG protein-coupled receptors (GPCRs) signal through activation of G proteins and subsequent modulation of downstream effectors. More recently, signaling mediated by β-arrestin has also been implicated in important physiological functions. This has led to great interest in the identification of biased ligands that favor either G protein or β-arrestin-signaling pathways. However, nearly all screening techniques for measuring β-arrestin recruitment have required C-terminal receptor modifications that can in principle alter protein interactions and thus signaling. Here, we have developed a novel luminescence-based assay to measure β-arrestin recruitment to the membrane or early endosomes by unmodified receptors. Our strategy uses NanoLuc, an engineered luciferase from Oplophorus gracilirostris (deep-sea shrimp) that is smaller and brighter than other well-established luciferases. Recently, several publications have explored functional NanoLuc split sites for use in complementation assays. We have identified a unique split site within NanoLuc and fused the corresponding N-terminal fragment to either a plasma membrane or early endosome tether and the C-terminal fragment to β-arrestins, which form the basis for the MeNArC and EeNArC assays, respectively. Upon receptor activation, β-arrestin is recruited to the membrane and subsequently internalized in an agonist concentration-dependent manner. This recruitment promotes complementation of the two NanoLuc fragments, thereby reconstituting functional NanoLuc, allowing for quantification of β-arrestin recruitment with a single luminescence signal. Our assay avoids potential artifacts related to C-terminal receptor modification and has promise as a new generic assay for measuring β-arrestin recruitment to diverse GPCR types in heterologous or native cells. G protein-coupled receptors (GPCRs) signal through activation of G proteins and subsequent modulation of downstream effectors. More recently, signaling mediated by β-arrestin has also been implicated in important physiological functions. This has led to great interest in the identification of biased ligands that favor either G protein or β-arrestin-signaling pathways. However, nearly all screening techniques for measuring β-arrestin recruitment have required C-terminal receptor modifications that can in principle alter protein interactions and thus signaling. Here, we have developed a novel luminescence-based assay to measure β-arrestin recruitment to the membrane or early endosomes by unmodified receptors. Our strategy uses NanoLuc, an engineered luciferase from Oplophorus gracilirostris (deep-sea shrimp) that is smaller and brighter than other well-established luciferases. Recently, several publications have explored functional NanoLuc split sites for use in complementation assays. We have identified a unique split site within NanoLuc and fused the corresponding N-terminal fragment to either a plasma membrane or early endosome tether and the C-terminal fragment to β-arrestins, which form the basis for the MeNArC and EeNArC assays, respectively. Upon receptor activation, β-arrestin is recruited to the membrane and subsequently internalized in an agonist concentration-dependent manner. This recruitment promotes complementation of the two NanoLuc fragments, thereby reconstituting functional NanoLuc, allowing for quantification of β-arrestin recruitment with a single luminescence signal. Our assay avoids potential artifacts related to C-terminal receptor modification and has promise as a new generic assay for measuring β-arrestin recruitment to diverse GPCR types in heterologous or native cells. Over the past decade, increased attention has been directed to biased signaling of G protein-coupled receptors (GPCRs) and the possibility of identifying ligands that can selectively target either G protein- or β-arrestin-mediated signaling. As an example, the β-adrenergic receptor ligand carvedilol antagonizes G protein activation but recruits β-arrestin and has been shown to increase survival rates in patients suffering from heart failure (1Wisler J.W. DeWire S.M. Whalen E.J. Violin J.D. Drake M.T. Ahn S. Shenoy S.K. Lefkowitz R.J. A unique mechanism of beta-blocker action: Carvedilol stimulates beta-arrestin signaling.Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 16657-16662Crossref PubMed Scopus (481) Google Scholar). At the μ-opioid receptor (MOR), G protein signaling has been proposed to be primarily responsible for analgesia, with side effects such as respiratory depression resulting from β-arrestin-mediated signaling (2Bohn L.M. Lefkowitz R.J. Gainetdinov R.R. Peppel K. Caron M.G. Lin F.T. Enhanced morphine analgesia in mice lacking beta-arrestin 2.Science. 1999; 286: 2495-2498Crossref PubMed Scopus (798) Google Scholar, 3Bohn L.M. Lefkowitz R.J. Caron M.G. Differential mechanisms of morphine antinociceptive tolerance revealed in (beta)arrestin-2 knock-out mice.J. Neurosci. 2002; 22: 10494-10500Crossref PubMed Google Scholar), although this paradigm has recently been challenged (4Kliewer A. Schmiedel F. Sianati S. Bailey A. Bateman J.T. Levitt E.S. Williams J.T. Christie M.J. Schulz S. Phosphorylation-deficient G-protein-biased μ-opioid receptors improve analgesia and diminish tolerance but worsen opioid side effects.Nat. Commun. 2019; 10: 367Crossref PubMed Scopus (152) Google Scholar, 5Thompson G.L. Lane J.R. Coudrat T. Sexton P.M. Christopoulos A. Canals M. Systematic analysis of factors influencing observations of biased agonism at the mu-opioid receptor.Biochem. Pharmacol. 2016; 113: 70-87Crossref PubMed Scopus (45) Google Scholar, 6Kliewer A. Gillis A. Hill R. Schmidel F. Bailey C. Kelly E. Henderson G. Christie M.J. Schulz S. Morphine-induced respiratory depression is independent of ß-arrestin2 signalling.Br. J. Pharmacol. 2020; 177: 2923-2931Crossref PubMed Scopus (117) Google Scholar). We have demonstrated that β-arrestin recruitment to the dopamine D2 receptor (D2R) in indirect pathway neurons in the ventral striatum leads to enhanced locomotion, whereas G protein signaling is necessary for incentive behavior (7Donthamsetti P. Gallo E.F. Buck D.C. Stahl E.L. Zhu Y. Lane J.R. Bohn L.M. Neve K.A. Kellendonk C. Javitch J.A. Arrestin recruitment to dopamine D2 receptor mediates locomotion but not incentive motivation.Mol. Psychiatry. 2020; 25: 2086-2100Crossref PubMed Scopus (32) Google Scholar), further emphasizing the potential importance of biased signaling in more targeted therapeutics. Agonist induced-GPCR activation leads to GPCR kinase (GRK)-mediated phosphorylation of serine and threonine residues, most notably in the C-terminal tail of receptors but also in the intracellular loops of some receptors, which are the primary receptor recognition regions for β-arrestin binding. In addition to its role in signaling, β-arrestin also regulates receptor internalization and for some receptors stays bound to endosomes where receptor-mediated signaling may continue to occur (8Irannejad R. von Zastrow M. GPCR signaling along the endocytic pathway.Curr. Opin. Cell Biol. 2014; 27: 109-116Crossref PubMed Scopus (138) Google Scholar). The most well-established techniques to investigate β-arrestin recruitment are the PathHunter, Tango, LinkLight, and bioluminescence resonance energy transfer (BRET) assays. The PathHunter assay is an enzyme complementation assay in which split enzyme fragments are fused to the receptor and to β-arrestin and complementation of the functional enzyme creates a chemiluminescence readout (9Zhao X. Jones A. Olson K.R. Peng K. Wehrman T. Park A. Mallari R. Nebalasca D. Young S.W. Xiao S.-H. A homogeneous enzyme fragment complementation-based beta-arrestin translocation assay for high-throughput screening of G-protein-coupled receptors.J. Biomol. Screen. 2008; 13: 737-747Crossref PubMed Scopus (56) Google Scholar). The Tango-GPCR assay is a reporter gene assay where a transcription factor fused to the receptor C terminus is cleaved off by a protease-tagged β-arrestin, leading to the expression of a reporter that creates a luminescence readout (10Barnea G. Strapps W. Herrada G. Berman Y. Ong J. Kloss B. Axel R. Lee K.J. The genetic design of signaling cascades to record receptor activation.Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 64-69Crossref PubMed Scopus (432) Google Scholar, 11Kroeze W.K. Sassano M.F. Huang X.-P. Lansu K. McCorvy J.D. Giguère P.M. Sciaky N. Roth B.L. PRESTO-Tango as an open-source resource for interrogation of the druggable human GPCRome.Nat. Struct. Mol. Biol. 2015; 22: 362-369Crossref PubMed Scopus (340) Google Scholar). LinkLight uses a modified luciferase attached to β-arrestin that is cleaved off by a protease fused to the C terminus of the GPCR of interest, thereby activating the luciferase and producing light (12Eishingdrelo H. Cai J. Weissensee P. Sharma P. Tocci M.J. Wright P.S. A cell-based protein-protein interaction method using a permuted luciferase reporter.Curr. Chem. Genomics. 2011; 5: 122-128Crossref PubMed Google Scholar). BRET between a receptor fused at its C terminus to a luciferase donor and β-arrestin tagged with fluorescent protein acceptor is also commonly used to measure β-arrestin recruitment (13Donthamsetti P. Quejada J.R. Javitch J.A. Gurevich V.V. Lambert N.A. Using bioluminescence resonance energy transfer (BRET) to characterize agonist-induced arrestin recruitment to modified and unmodified G protein-coupled receptors.Curr. Protoc. Pharmacol. 2015; 70: 2.14.1-2.14.14Crossref Scopus (32) Google Scholar). Notably, all of these β-arrestin assays require that fusion tags be directly attached to the C terminus of receptors of interest, which could alter GRK, arrestin, or other protein interactions with the receptors and therefore impact signaling. The only method that does not rely on modified receptors is the microscopy-based Transfluor assay where β-arrestin fused to a fluorescent protein is visualized at the membrane (14Oakley R.H. Hudson C.C. Cruickshank R.D. Meyers D.M. Payne R.E. Rhem S.M. Loomis C.R. The cellular distribution of fluorescently labeled arrestins provides a robust, sensitive, and universal assay for screening G protein-coupled receptors.Assay Drug Dev. Technol. 2002; 1: 21-30Crossref PubMed Scopus (116) Google Scholar). However, with this technique, it is difficult to quantify ligand efficacy and potency or to scale for screening ligands. In contrast, we previously introduced a modified BRET assay in which a Renilla luciferase 8 (RLuc8) donor is anchored to the membrane instead of attached to the receptor (13Donthamsetti P. Quejada J.R. Javitch J.A. Gurevich V.V. Lambert N.A. Using bioluminescence resonance energy transfer (BRET) to characterize agonist-induced arrestin recruitment to modified and unmodified G protein-coupled receptors.Curr. Protoc. Pharmacol. 2015; 70: 2.14.1-2.14.14Crossref Scopus (32) Google Scholar). In this assay, which is based on "bystander" BRET, β-arrestin fused to a venus acceptor is translocated from the cytosol to the unmodified receptor at the plasma membrane, leading to an increase in BRET by proximity between the membrane-anchored donor and acceptor attached to β-arrestin. This assay incorporated yeast-derived "helper" peptides that are attached to the donor and acceptor, thereby enhancing the affinity of the interaction between β-arrestin and the plasma-membrane anchor (15Grünberg R. Burnier J.V. Ferrar T. Beltran-Sastre V. Stricher F. van der Sloot A.M. Garcia-Olivas R. Mallabiabarrena A. Sanjuan X. Zimmermann T. Serrano L. Engineering of weak helper interactions for high-efficiency FRET probes.Nat. Methods. 2013; 10: 1021-1027Crossref PubMed Scopus (52) Google Scholar). This bystander BRET format was later adapted to a BRET2 format (16Namkung 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: 12178Crossref PubMed Scopus (152) Google Scholar). BRET assays require a specialized dual output microplate reader and filter sets compatible with BRET. The previously used BRET assays also (13Donthamsetti P. Quejada J.R. Javitch J.A. Gurevich V.V. Lambert N.A. Using bioluminescence resonance energy transfer (BRET) to characterize agonist-induced arrestin recruitment to modified and unmodified G protein-coupled receptors.Curr. Protoc. Pharmacol. 2015; 70: 2.14.1-2.14.14Crossref Scopus (32) Google Scholar) use RLuc8 instead of the more recently reported novel luciferase NanoLuc developed by Promega, which has the advantages of being smaller and significantly brighter compared with other known luciferases (17Laschet C. Dupuis N. Hanson J. A dynamic and screening-compatible nanoluciferase-based complementation assay enables profiling of individual GPCR-G protein interactions.J. Biol. Chem. 2019; 294: 4079-4090Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Recently, a split NanoLuc, known as NanoBiT, which is commercially available from Promega (Fig. S1A), has been used successfully in a direct β-arrestin recruitment assay in which a small peptide fragment, referred to as SmBiT, is attached to the receptor of interest and the larger fragment, referred to as LgBiT, is attached to β-arrestin (18Hall M.P. Unch J. Binkowski B.F. Valley M.P. Butler B.L. Wood M.G. Otto P. Zimmerman K. Vidugiris G. Machleidt T. Robers M.B. Benink H.A. Eggers C.T. Slater M.R. Meisenheimer P.L. et al.Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate.ACS Chem. Biol. 2012; 7: 1848-1857Crossref PubMed Scopus (900) Google Scholar). A slightly modified version of NanoBiT has proven to be effective for compound library screening as seen in a direct recruitment assay between D2R and Gαi1/o (19Dixon A.S. Schwinn M.K. Hall M.P. Zimmerman K. Otto P. Lubben T.H. Butler B.L. Binkowski B.F. Machleidt T. Kirkland T.A. Wood M.G. Eggers C.T. Encell L.P. Wood K.V. Nanoluc complementation reporter optimized for accurate measurement of protein interactions in cells.ACS Chem. Biol. 2016; 11: 400-408Crossref PubMed Scopus (555) Google Scholar). Thus, we expected that the NanoBiT system would readily be adapted to our bystander assay (13Donthamsetti P. Quejada J.R. Javitch J.A. Gurevich V.V. Lambert N.A. Using bioluminescence resonance energy transfer (BRET) to characterize agonist-induced arrestin recruitment to modified and unmodified G protein-coupled receptors.Curr. Protoc. Pharmacol. 2015; 70: 2.14.1-2.14.14Crossref Scopus (32) Google Scholar) by fusing SmBiT to our membrane anchor instead of directly to the receptor and LgBiT to β-arrestin, thereby substituting BRET in our assay with complemented NanoLuc detection. However, to our surprise, the NanoBiT adaptation of our assay failed to detect β-arrestin recruitment to the membrane upon receptor activation. Here, we report a novel NanoLuc split that, unlike NanoBiT, is suitable for the desired NanoLuc-complementation-based β-arrestin membrane recruitment assay where the membrane anchor is attached to the N-terminal NanoLuc fragment (referred to as MeN) and β-arrestin is attached to the C-terminal NanoLuc fragment (referred to as ArC). Our assay, which we refer to as MeNArC, does not rely on receptor modification or helper peptides and provides a simple luminescence output reading of β-arrestin recruitment as an alternative to the bystander BRET assay to investigate receptors in a more native format. We also adapted the assay to measure β-arrestin recruitment to early endosomes, referred to as EeNArC, demonstrating that the assay can detect events in specific cell compartments other than the plasma membrane. We anticipate that these novel assays will serve as general approaches to screen compounds for β-arrestin recruitment to unmodified GPCRs in vitro and potentially in vivo. As described above, we initially used the NanoBiT system (Fig. S1A) in our previously developed assay (13Donthamsetti P. Quejada J.R. Javitch J.A. Gurevich V.V. Lambert N.A. Using bioluminescence resonance energy transfer (BRET) to characterize agonist-induced arrestin recruitment to modified and unmodified G protein-coupled receptors.Curr. Protoc. Pharmacol. 2015; 70: 2.14.1-2.14.14Crossref Scopus (32) Google Scholar) to detect membrane recruitment of β-arrestin. However, the beta 2 adrenergic receptor (β2AR), D2R, and AT1R all failed to produce any increase in luminescence in this assay upon agonist stimulation (Fig. S1B). We reasoned that the nature of the NanoBiT split (Fig. S1A) might prevent proper complementation when used in our bystander membrane recruitment assay and therefore designed a new split site within NanoLuc using the crystal structure of the protein as a guide (20Tomabechi Y. Hosoya T. Ehara H. Sekine S.-I. Shirouzu M. Inouye S. Crystal structure of nanoKAZ: The mutated 19 kDa component of Oplophorus luciferase catalyzing the bioluminescent reaction with coelenterazine.Biochem. Biophys. Res. Commun. 2016; 470: 88-93Crossref PubMed Scopus (33) Google Scholar). We selected a site in a loop region that divides NanoLuc into two almost equally sized fragments (N-terminal fragment, amino acids 1–102; C-terminal fragment, amino acids 103–172), without disrupting any secondary structural element, in an effort to select fragments that would fold independently and that could efficiently compliment when brought together at the membrane (Fig. 1, A and B). This site was similar but not identical to another NanoLuc split site used previously in a direct-recruitment assay (21Yano H. Cai N.S. Javitch J.A. Ferré S. Luciferase complementation based-detection of G-protein-coupled receptor activity.BioTechniques. 2018; 65: 9-14Crossref PubMed Scopus (10) Google Scholar). To verify that our split fragments can form a fully functional protein capable of luminescence when complemented, we first attached the N- and C-terminal NanoLuc fragments to FKBP (FK506- and rapamycin-binding protein) and FRB (FKBP-rapamycin-binding domain) respectively, which can readily dimerize with high affinity upon addition of rapamycin (Fig. 1C) (22Chiu M.I. Katz H. Berlin V. RAPT1, a mammalian homolog of yeast Tor, interacts with the FKBP12/rapamycin complex.Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12574-12578Crossref PubMed Scopus (411) Google Scholar). When transfected alone, the fragments did not yield any luminescence over the baseline observed with mock transfected cells (Fig. 1D). Cotransfection of both fragments yielded a small basal luminescence signal that was increased ∼30-fold by the addition of rapamycin (Fig. 1D), suggesting only minimal spontaneous assembly of the fragments but efficient complementation when brought into proximity, demonstrating that the new fragments are suitable as a tool for complementation assays. We further validated their use for complementation-based GPCR assays in a direct recruitment assay with β-arrestin, as we have done previously with the D2R receptor in a BRET setup (13Donthamsetti P. Quejada J.R. Javitch J.A. Gurevich V.V. Lambert N.A. Using bioluminescence resonance energy transfer (BRET) to characterize agonist-induced arrestin recruitment to modified and unmodified G protein-coupled receptors.Curr. Protoc. Pharmacol. 2015; 70: 2.14.1-2.14.14Crossref Scopus (32) Google Scholar), in which the N-terminal Nanoluc fragment was attached to the C-terminal tail of D2R and the C-terminal fragment was fused to the N terminus of β-arrestin2 (Fig. 1E). In this configuration, the endogenous agonist dopamine produced a time- and dose-dependent increase in luminescence (Fig. 1F). We next incorporated the validated NanoLuc split components into our β-arrestin recruitment assay with unmodified receptors where the N-terminal fragment of NanoLuc is anchored to the plasma membrane through a double palmitoylated domain of GAP43 (MeN) and where the C-terminal fragment is fused to the N terminus of β-arrestin1 or β-arrestin2 (ArC) (Fig. 2A). The helper peptides that were used in our original BRET assay were not used in the current split Nanoluc configuration (13Donthamsetti P. Quejada J.R. Javitch J.A. Gurevich V.V. Lambert N.A. Using bioluminescence resonance energy transfer (BRET) to characterize agonist-induced arrestin recruitment to modified and unmodified G protein-coupled receptors.Curr. Protoc. Pharmacol. 2015; 70: 2.14.1-2.14.14Crossref Scopus (32) Google Scholar). To test our MeNArC assay, we used D2R and MOR, which are prototypical class A receptors based on their agonist-induced β-arrestin-binding profile after internalization (23Oakley 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: 17201-17210Abstract Full Text Full Text PDF PubMed Scopus (683) Google Scholar), as β-arrestin readily dissociates from these receptors upon receptor endocytosis. When D2R was used in this assay, in contrast to the results when the NanoBiT fragments were used (Fig. S1B), we observed a robust increase in luminescence upon agonist treatment that plateaued after ∼40 min (Fig. 2B), consistent with β-arrestin recruitment to the plasma membrane. Addition of the D2R antagonist sulpiride 18 min after receptor stimulation with the agonist quinpirole led to a strong decay in luminescence down to baseline (Fig. 2B), indicative of nearly complete reversal of complementation between the NanoLuc fragments upon receptor inhibition, suggesting that the affinity between the fragments is relatively low. Both β-arrestin isoforms in the assay gave similar fold increases of 3.1 ± 0.14 and 3.4 ± 0.20 for β-arrestin1 and 2, respectively (Fig. 2, C and D), with similar potencies (β-arrestin1 EC50 156 nM ± 0.16; β-arrestin2 EC50 74 nM ± 0.18). While the MeNArC assay also can be used with the splits in the reverse orientation, we observed a lower fold increase in luminescence upon receptor activation (Fig. S2), and therefore we used the assay in the preferred orientation described above for all subsequent experiments. For further study of the assay, we proceeded with β-arrestin2, which is the β-arrestin isoform most typically used in β-arrestin assay development. To assess the MeNArC assay's dynamic range, we tested D2R with the high-efficacy partial agonist quinpirole, medium-efficacy partial agonists bromocriptine and terguride, and the low-efficacy partial agonist (3-hydroxyphenyl)-N-propylpiperidine (PPP), giving an efficacy compared with dopamine of 78%, 46%, 24%, and 10%, respectively, similar to previously published data using the BRET direct recruitment assay (24Klewe I.V. Nielsen S.M. Tarpø L. Urizar E. Dipace C. Javitch J.A. Gether U. Egebjerg J. Christensen K.V. Recruitment of beta-arrestin2 to the dopamine D2 receptor: Insights into anti-psychotic and anti-parkinsonian drug receptor signaling.Neuropharmacology. 2008; 54: 1215-1222Crossref PubMed Scopus (61) Google Scholar). These results show that the MeNArC assay is also capable of measuring different degrees of partial agonism of unmodified receptors (Fig. 2E). To further validate the MeNArC assay, we also tested MOR compared with a previously reported MOR mutant (MOR 11S/T-A) with impaired β-arrestin binding (25Miess E. Gondin A.B. Yousuf A. Steinborn R. Mösslein N. Yang Y. Göldner M. Ruland J.G. Bünemann M. Krasel C. Christie M.J. Halls M.L. Schulz S. Canals M. Multisite phosphorylation is required for sustained interaction with GRKs and arrestins during rapid μ-opioid receptor desensitization.Sci. Signal. 2018; 11eaas9609Crossref PubMed Scopus (65) Google Scholar). In MOR 11S/T-A, 11 serine and threonine residues in the C-terminal tail of the receptor are replaced with alanine, preventing C-terminal phosphorylation and therefore inhibiting recruitment of β-arrestin. The fold change of MOR compared with MOR 11S/T-A in response to activation by DAMGO ([D-Ala2, N-MePhe4, Gly-ol]-enkephalin) was 3.7 ± 0.10 and 2.4 ± 0.11, respectively (Fig. 2F), decreasing the efficacy to 65% for MOR 11S/T-A. Previously, MOR 11S/T-A had been tested in a direct recruitment assay giving an efficacy of 42% compared with WT. In both assays the MOR 11S/T-A mutant exhibits decreased efficacy, demonstrating that our complementation assay can differentiate between receptors with known differences in β-arrestin recruitment and that the assay is sensitive to the receptor's affinity for β-arrestin. These experiments verify the system's direct link between β-arrestin recruitment and the luminescence signal. To investigate the versatility of the MeNArC assay, we further tested its use with the class B angiotensin II type 1 receptor (AT1R). Unlike class A receptors, AT1R and other class B receptors bind β-arrestin with higher affinity and form longer-lived complexes that persist after endocytosis (23Oakley 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: 17201-17210Abstract Full Text Full Text PDF PubMed Scopus (683) Google Scholar). AT1R led to a robust increase in luminescence upon stimulation with the agonist Angiotensin II (Ang II) with a fold change of 3.0 ± 0.12 (Fig. 3A). The addition of sucrose, a known inhibitor of receptor endocytosis (26Heuser J.E. Anderson R.G. Hypertonic media inhibit receptor-mediated endocytosis by blocking clathrin-coated pit formation.J. Cell Biol. 1989; 108: 389-400Crossref PubMed Scopus (771) Google Scholar), had no effect on agonist response, suggesting that the observed signal is generated at the plasma membrane (Fig. 3B). To investigate the reversibility of β-arrestin recruitment to a class B receptor, we tested AT1R with the antagonist olmesartan. Interestingly, agonist-induced β-arrestin recruitment by AT1R could not be reversed upon subsequent addition of increasing concentrations of olmesartan, either in the absence or presence of sucrose (Fig. 3B). In contrast, the class A receptor D2R was inhibited by antagonist in the same assay (Fig. 2B), showing the reversibility of activation. Notably, we were able to prevent the agonist-induced response at AT1R with olmesartan treatment when the antagonist was added before agonist (Fig. 3B). To understand whether this was due to rapid internalization of AT1R-β-arrestin complexes that might become inaccessible to inhibition by olmesartan, we inhibited internalization with sucrose, but this did not enhance olmesartan's ability to inhibit the agonist-induced response (Fig. 3B). We therefore reasoned that this irreversibility reflects the much higher affinity of β-arrestin interaction with AT1R once the receptor C tail is phosphorylated and engaged with β-arrestin (23Oakley 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: 17201-17210Abstract Full Text Full Text PDF PubMed Scopus (683) Google Scholar). Like what is reported for the beta 2 adrenergic receptor-V2R chimera, we infer that even after blocking core engagement of AT1R with β-arrestin by addition of the antagonist, the "hanging" interaction with the phosphorylated receptor C tail is sufficient to maintain the interaction (27Shukla 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: 218-222Crossref PubMed Scopus (363) Google Scholar), in contrast to MOR and D2R where core engagement and therefore continued agonist binding are essential for β-arrestin interaction. Because class B receptors form long-lived complexes with β-arrestin that can be imaged after internalization (23Oakley 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: 17201-17210Abstract Full Text Full Text PDF PubMed Scopus (683) Google Scholar), we sought to adapt our NanoLuc complementation assay to also measure β-arrestin recruitment to early endosomes containing internalized AT1R. For this assay, referred to as EeNArC, the ArC component described above is coexpressed with the N-terminal NanoLuc fragment attached to the FYVE domain of endofin (EeN), which was shown previously to selectively bind endosomes and used in a direct receptor recruitment assay (16Namkung 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: 12178Crossref PubMed Scopus (152) Google Scholar) (Fig. 3C). Stimulation of the receptors with Ang II resulted in a robust increase in luminescence over time that reached a plateau after 60 min, with an EC50 of 1.6 nM ± 0.22, consistent with β-arrestin recruitment to early endosomes (Fig. 3D). As expected, the agonist effect was dramatically blunted by sucrose for the EeNArC assay (Fig. 3, D and E), but not for the MeNArC assay (Fig. 3B), demonstrating that the former is specific to events that occur at early endosomes and thus dependent on internalization (Fig. 3, D and E). The MeNArC and EeNArC assays were also performed in human embryonic kidney (Hek)293 cells without cotransfection of any receptor, and no response was observed for dopamine, Ang II, or DAMGO, establishing the specificity of the assay for the transfected GPCRs (Fig. S3). For the experiments described above, each component of the MeNArC assay, as well as the recept
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