Exploring GPCR‐arrestin interfaces with genetically encoded crosslinkers
2020; Springer Nature; Volume: 21; Issue: 11 Linguagem: Inglês
10.15252/embr.202050437
ISSN1469-3178
AutoresThore Böttke, Stefan Ernicke, Robert Serfling, Christian Ihling, Edyta Burda, Vsevolod V. Gurevich, Andrea Sinz, Irene Coin,
Tópico(s)Neuropeptides and Animal Physiology
ResumoArticle14 September 2020Open Access Transparent process Exploring GPCR-arrestin interfaces with genetically encoded crosslinkers Thore Böttke Thore Böttke orcid.org/0000-0003-4817-9487 Institute of Biochemistry, Faculty of Life Sciences, University of Leipzig, Leipzig, Germany Search for more papers by this author Stefan Ernicke Stefan Ernicke Institute of Biochemistry, Faculty of Life Sciences, University of Leipzig, Leipzig, Germany Search for more papers by this author Robert Serfling Robert Serfling Institute of Biochemistry, Faculty of Life Sciences, University of Leipzig, Leipzig, Germany Search for more papers by this author Christian Ihling Christian Ihling Institute of Pharmacy, Department of Pharmaceutical Chemistry and Bioanalytics, Charles Tanford Protein Center, Martin Luther University Halle-Wittenberg, Halle/Saale, Germany Search for more papers by this author Edyta Burda Edyta Burda Institute of Pharmacy, Faculty of Medicine, University of Leipzig, Leipzig, Germany Search for more papers by this author Vsevolod V Gurevich Vsevolod V Gurevich orcid.org/0000-0002-3950-5351 Department of Pharmacology, Vanderbilt University, Nashville, TN, USA Search for more papers by this author Andrea Sinz Andrea Sinz Institute of Pharmacy, Department of Pharmaceutical Chemistry and Bioanalytics, Charles Tanford Protein Center, Martin Luther University Halle-Wittenberg, Halle/Saale, Germany Search for more papers by this author Irene Coin Corresponding Author Irene Coin [email protected] orcid.org/0000-0002-7722-004X Institute of Biochemistry, Faculty of Life Sciences, University of Leipzig, Leipzig, Germany Search for more papers by this author Thore Böttke Thore Böttke orcid.org/0000-0003-4817-9487 Institute of Biochemistry, Faculty of Life Sciences, University of Leipzig, Leipzig, Germany Search for more papers by this author Stefan Ernicke Stefan Ernicke Institute of Biochemistry, Faculty of Life Sciences, University of Leipzig, Leipzig, Germany Search for more papers by this author Robert Serfling Robert Serfling Institute of Biochemistry, Faculty of Life Sciences, University of Leipzig, Leipzig, Germany Search for more papers by this author Christian Ihling Christian Ihling Institute of Pharmacy, Department of Pharmaceutical Chemistry and Bioanalytics, Charles Tanford Protein Center, Martin Luther University Halle-Wittenberg, Halle/Saale, Germany Search for more papers by this author Edyta Burda Edyta Burda Institute of Pharmacy, Faculty of Medicine, University of Leipzig, Leipzig, Germany Search for more papers by this author Vsevolod V Gurevich Vsevolod V Gurevich orcid.org/0000-0002-3950-5351 Department of Pharmacology, Vanderbilt University, Nashville, TN, USA Search for more papers by this author Andrea Sinz Andrea Sinz Institute of Pharmacy, Department of Pharmaceutical Chemistry and Bioanalytics, Charles Tanford Protein Center, Martin Luther University Halle-Wittenberg, Halle/Saale, Germany Search for more papers by this author Irene Coin Corresponding Author Irene Coin [email protected] orcid.org/0000-0002-7722-004X Institute of Biochemistry, Faculty of Life Sciences, University of Leipzig, Leipzig, Germany Search for more papers by this author Author Information Thore Böttke1, Stefan Ernicke1, Robert Serfling1, Christian Ihling2, Edyta Burda3, Vsevolod V Gurevich4, Andrea Sinz2 and Irene Coin *,1 1Institute of Biochemistry, Faculty of Life Sciences, University of Leipzig, Leipzig, Germany 2Institute of Pharmacy, Department of Pharmaceutical Chemistry and Bioanalytics, Charles Tanford Protein Center, Martin Luther University Halle-Wittenberg, Halle/Saale, Germany 3Institute of Pharmacy, Faculty of Medicine, University of Leipzig, Leipzig, Germany 4Department of Pharmacology, Vanderbilt University, Nashville, TN, USA *Corresponding author. Tel: +49 (0)341 9736996; Fax: +49 (0)341 9736909; E-mail: [email protected] EMBO Reports (2020)21:e50437https://doi.org/10.15252/embr.202050437 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract β-arrestins (βarr1 and βarr2) are ubiquitous regulators of G protein-coupled receptor (GPCR) signaling. Available data suggest that β-arrestins dock to different receptors in different ways. However, the structural characterization of GPCR-arrestin complexes is challenging and alternative approaches to study GPCR-arrestin complexes are needed. Here, starting from the finger loop as a major site for the interaction of arrestins with GPCRs, we genetically incorporate non-canonical amino acids for photo- and chemical crosslinking into βarr1 and βarr2 and explore binding topologies to GPCRs forming either stable or transient complexes with arrestins: the vasopressin receptor 2 (rhodopsin-like), the corticotropin-releasing factor receptor 1, and the parathyroid hormone receptor 1 (both secretin-like). We show that each receptor leaves a unique footprint on arrestins, whereas the two β-arrestins yield quite similar crosslinking patterns. Furthermore, we show that the method allows defining the orientation of arrestin with respect to the GPCR. Finally, we provide direct evidence for the formation of arrestin oligomers in the cell. Synopsis Chemical and photo-crosslinkers genetically incorporated into β-arrestins allow the mapping of footprints of different receptors on the arrestin surface in living cells, and to identify intermolecular pairs of proximal amino acids in GPCR-arrestin complexes. Genetically encoded crosslinkers can capture arrestin-GPCR interactions with high spatial precision. Each G protein-coupled receptor leaves a unique footprint on arrestins. Arrestins βarr1 and βarr2 may bind to a GPCR in a similar way. Introduction Arrestins are a small family of highly homologous cytosolic proteins that dock to activated and phosphorylated G protein-coupled receptors (GPCRs) to desensitize G protein-mediated signaling (Gurevich, 2014; Shukla & Dwivedi-Agnihotri, 2020). Four arrestin isoforms are expressed in vertebrates: Two quench the signaling by rhodopsin (Rho) and cone opsins in the retina (arr1 and arr4, also called "visual" arrestins), whereas the other two serve ubiquitously the hundreds of non-visual GPCRs encoded in the human genome (arr2 and arr3, a.k.a. βarr1 and βarr2, called "non-visual" or β-arrestins). All arrestins in their basal state consist of β-sheets organized in two cup-like domains (N- and C-domain) with four exposed loops in the central crest of the receptor binding side (Fig 1A). The finger loop shares a consensus motif with the C-terminal helix of Gα from Gi/transducin and competes with it for the cavity that opens in the transmembrane (TM) core of active GPCRs (Szczepek et al, 2014). This loop is indispensable for the formation of a fully engaged arrestin–receptor complex (core conformation). Arrestin variants lacking the finger loop only bind to the phosphorylated GPCR C-terminal tail via their N-domain (tail conformation) (Shukla et al, 2014; Cahill et al, 2017; Nguyen et al, 2019). Moreover, the finger loop determines the binding preferences of arrestin to receptors (Vishnivetskiy et al, 2004, 2011; Chen et al, 2017). Figure 1. Genetic incorporation of Bpa into β-arrestins Ribbon representation of bovine βarr1 (PDB 1G4M) (Han et al, 2001). The finger loop is highlighted in blue and β-strand VI in pale green. Sequence alignment of the finger loop and N-terminal part of β-strand VI in bovine β-arrestins, color coded as in (A). Divergent positions are shown in bold. Western blots of the lysates of HEK293T cells, resolved on SDS–PAGE and stained with a pan-arrestin antibody. mt, mock transfected. Bpa: benzoyl-phenylalanine. Arrestins were equipped with a 3xHA tag at the C-terminus, which increases their size of about 3 kDa compared to the endogenous proteins (βarr1 47 kDa, βarr2 46 kDa). βarr1 and βarr2 were transfected at 1/3 DNA compared to the corresponding TAG-mutants. Download figure Download PowerPoint G protein-coupled receptors are divided into two classes with respect to arrestin binding (Oakley et al, 1999, 2000; Luttrell & Lefkowitz, 2002). Class A receptors form transient and rapidly dissociating complexes with arrestin, and resensitize rapidly. These receptors interact with both β-arrestins, but show a bias toward βarr2. Besides the prototypical β2-adrenergic, class A receptors include, among others, the μ opioid, endothelin A, and dopamine D1A receptors (all rhodopsin-like GPCRs), as well as the corticotropin-releasing factor receptor (CRF1R, secretin-like) (Oakley et al, 2007; Grammatopoulos, 2012). Class B receptors form long-lived complexes with arrestin that remain stable through the internalization via clathrin-coated pits, and resensitize slowly. Class B receptors bind with high affinity either β-arrestin. The prototypic class B arrestin binder is the vasopressin 2 receptor (V2R). Other receptors forming stable complexes with arrestin include the angiotensin II type 1 receptor (AT1R), the oxytocin receptor, the neurotensin 1 receptor (NTS1R), and the secretin-like parathyroid hormone receptor (PTH1R) (Oakley et al, 2001; Vilardaga et al, 2002). The latter has been shown to form GPCR–arrestin–G protein megaplexes that mediate prolonged signaling after internalization in endosomes (Wehbi et al, 2013; Thomsen et al, 2016). A major determinant for the stability of GPCR-arrestin complexes is the C-terminal tail of the receptor. In general, GPCRs carrying clusters of Set/Thr in the C-terminal tail show a class B behavior (Oakley et al, 1999). Overall, GPCR-arrestin complexes are highly dynamic and the biophysical determination of their structure is technically very challenging. To date, only a few 3D structures of arrestin–GPCR complexes have been solved: the crystal structure of the rhodopsin (Rho)-bound arr1 (Kang et al, 2015; Zhou et al, 2017), and the three very recent cryo-electron microscopy (cryo-EM) structures of βarr1 in complex with the class B NTS1R (Yin et al, 2019; Huang et al, 2020), the muscarinic acetylcholine-2-receptor (M2R) (Staus et al, 2020), and the β1-adrenoceptor (β1-AR) (Lee et al, 2020). The β1-AR belongs to class A arrestin binders (Shiina et al, 2000, Eichel et al, 2016), whereas contradictory findings have been reported for the M2R (Gurevich et al, 1995, Jones et al, 2006). As expected on the basis of numerous biochemical studies (reviewed in (Gurevich & Gurevich, 2006)), the existing structures confirm that βarr1 can assume distinct orientations depending on which receptor it binds to. Likewise, position and conformation of the finger loop differ between the complexes. Moreover, the binding mode of arrestin to the same GPCR can be affected by specific phosphorylation patterns, which lead to different downstream responses (barcode hypothesis) (Tobin et al, 2008; Nobles et al, 2011; Yang et al, 2015; Zhou et al, 2017; Mayer et al, 2019; Kaya et al, 2020). These facts, together with the observation that there are no conserved motifs in the intracellular part of the GPCRs, suggest that homology models based on the few available structures likely have limited predictive value. In addition, the lipid environment has been shown to affect the conformation of isolated GPCR-arrestin complexes (Staus et al, 2020). It is also worth mentioning that the structures of M2R and β1-AR could only be achieved by equipping the receptors with the C-terminal tail of the V2R, a modification that stabilizes the GPCR-arrestin complexes by transforming class A arrestin binders into class B ones (Oakley et al, 2000). Methods are needed both to address complexes that elude direct structural characterization and to validate biophysical data in the living cell. We have a long experience in the application of genetically encoded chemical tools to map interactions of GPCRs with their ligands (Coin et al, 2011, 2013; Seidel et al, 2017), as well as interactions of nuclear receptors (Schwarz et al, 2013, 2016). Here we show that the incorporation of crosslinking non-canonical amino acids (ncAAs) into arrestins allows the comparison of the topology of GPCR-arrestin complexes in intact cells. Results and Discussion We selected 24 positions in βarr1 and βarr2 covering the finger loop italic and the N-terminal segment of the following β-strand (Fig 1B). The codons of each amino acid along this stretch were substituted with the amber stop codon TAG for the incorporation of the photo-activatable ncAA benzoyl-Phe (Bpa). A preliminary test on a pair of homologous βarr1/βarr2 mutants showed that Bpa was smoothly incorporated into both arrestins via amber suppression (Wang, 2017a; Lang et al, 2018), with minimal read-through occurring in the absence of the ncAA (Fig 1C). Transfected arrestins were expressed in very large excess compared to the endogenous counterparts. βarr1 is known to express at a higher level than βarr2 in native cells and different cell lines (Gurevich et al, 2004; Coffa et al, 2011). This can be observed also here, both in the case of endogenous arrestins and the Bpa-mutants, which expressed at a homogenous yield (Fig 2). The Bpa-mutation did not hamper the recruitment of either arrestin to the PTH1R receptor, suggesting that the overall functionality of the arrestins is preserved (Appendix Fig S1). Figure 2. Photo-crosslinking of Bpa-arrestins with three different GPCRs with and without agonist stimulationEach panel represents immunoblots of whole cell lysates stained with an anti-HA antibody detecting arrestin. In the left panels, GPCRs were combined with βarr1 variants and in the right panels with βarr2 variants. Residues replaced by Bpa are indicated above each lane. Covalent arrestin–receptor complexes are expected between 100–200 kDa, considering the size of the receptors (V2R 41.5 kDa, PTH1R 63.8 kDa, CRF1R, 46.3 kDa) and their glycosylation. PTH1R, agonist PTH(1–34). CRF1R, agonist Urocortin 1 (Ucn1). V2R, agonist arginine-vasopressin (AVP). No receptor. Download figure Download PowerPoint The two sets of Bpa-βarr1 and Bpa-βarr2 were combined with GPCRs forming either stable (class B) or transient (class A) complexes with arrestins. We selected two class B receptors belonging to two distinct phylogenetic GPCR families, the V2R (rhodopsin-like) and the PTH1R (secretin-like), as well as the class A receptor CRF1R (secretin-like). The latter was preferred to other class A GPCRs because the CRF system is well established in our laboratory. Photo-crosslinking experiments were carried out in live HEK293T cells expressing one receptor and one arrestin variant, for a total of 144 combinations, with or without agonist stimulation (Fig EV1). Cell lysates were resolved on SDS–PAGE and immunoblotted with antibodies detecting either arrestin (Fig 2) or the GPCR (Fig EV2). For a subset of arrestin–receptor combinations, high molecular weight (MW) bands were observed (Fig 2A–C upper panels). Most bands appeared only when arrestin recruitment was triggered by the agonist treatment, but were not visible when the receptor was not activated (Fig 2A–C, lower panels) or absent (Fig 2D). The apparent MW was receptor-dependent and was reduced by deglycosylation (Fig 3). These activation-dependent bands were therefore attributed to covalently crosslinked receptor–arrestin complexes. The crosslinking patterns differed substantially between the different receptors, whereas the strongest bands with the same receptor appeared at homologous Bpa positions in the two arrestins. The most prominent bands were obtained with Bpa63/64-βarr1/2 (crosslinker at the beginning of the finger loop) with the PTH1R, Bpa75/76-βarr1/2 (crosslinker at the C-terminal portion of the finger loop) with the CRF1R and Bpa81/82-βarr1/2 (crosslinker in β-strand VI) with the V2R. Overall, these results suggest specific binding modes of arrestins at different receptors, whereas the two arrestins appear to bind to each receptor in a similar fashion. Click here to expand this figure. Figure EV1. βarr-GPCR photo-crosslinking 1: Cultured HEK293T cells are supplemented with 250 μM Bpa and transfected with three plasmids. The first plasmid carries the ORF for a GPCR, the second plasmid carries the ORF for one βarr TAG-mutant, and the third plasmid encodes for the tRNA/synthetase pair that incorporates Bpa. 2: The GPCR and the βarr-xxxBpa variant are expressed for 48 h. 3: The GPCR is activated with an agonist for 15 min. 4: The cells are irradiated with UV light (365 nm) for 15 min 5: Samples are lysed, resolved on SDS–PAGE, and the crosslinked arrestin–GPCR complex is detected by immunostaining. Mechanism of photo-activation of Bpa. If the diradical species is in close proximity to the GPCR, crosslinking can occur. In general, Bpa inserts into C-H bonds (Dorman & Prestwich, 1994). A covalent arrestin–GPCR complex is formed. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Photo-crosslinking of Bpa-arrestins with three different GPCRsThe same samples described in Fig 2 of the main text were immunostained using an antibody detecting the receptor. Overall, crosslinking signals are easier to detect when looking from the side of arrestin with the anti-HA antibody (Fig 2 in the main text). PTH1R was detected with a commercial anti-PTH1R antibody, which is very reliable, although it shows some background at about the same size were the crosslinking band is expected. CRF1R was equipped with a FLAG tag at the N-terminus, which was inserted right after the natural signal peptide. V2R was equipped with the 1D4 epitope at its C-terminus. Download figure Download PowerPoint Figure 3. Response of photo-crosslinked samples to deglycosylationAliquots from agonist-treated samples shown in Fig 2 were deglycosylated with PNGaseF, run on SDS–PAGE and immunoblotted with antibodies detecting arrestin (upper panels, α-HA) and the GPCRs (as indicated, CRF1R is equipped with a N-terminal FLAG, V2R carries a C-terminal 1D4 epitope). Download figure Download PowerPoint These findings confirm the observation from the 3D structures, which show distinct binding modes of βarr1 to the different receptors. In a recent report, photo-crosslinkers genetically incorporated into the intracellular domains of the AT1R have revealed distinct binding modalities of AT1R to βarr1 depending on the type of the agonist used for its activation (natural angiotensin vs. arrestin-biased AT1R ligands) (Gagnon et al, 2019). Collectively, these results demonstrate that genetically encoded photo-crosslinkers incorporated either into a GPCR or into arrestin allow elucidating with a good sensitivity differences in the arrangement of arrestin–GPCR complexes. Other crosslinking bands observed in our experiments appeared independently from activation of a GPCR. The most prominent activation-independent bands were observed with Bpa78- and Bpa81-βarr1 at apparent MW ~ 120 kDa. They appeared in all analyses (Fig 2A–C) even in the absence of a co-transfected receptor (Fig 2D) and were not affected by deglycosylation (Fig 3). Only very faint bands were observed with the Bpa-βarr2 set. To identify the nature of these crosslinking products, Bpa78-βarr1 was equipped with a 2xStrep tag at the C-terminus, expressed in large scale in HEK293T cells, and isolated via the Strep-Tactin® XT system either before or after UV irradiation (Appendix Fig S2A). As shown in Fig 4, the crosslinking product obtained by in vitro irradiation of the isolated Bap78-βarr1 runs exactly as the crosslinking product obtained when irradiating the intact cells (Fig 4A). Furthermore, the crosslinked samples were subjected to size exclusion chromatography (SEC), yielding two partially resolved major peaks (Fig 4B). Eluates were analyzed by immunoblotting (Fig 4C) and by MALDI mass spectrometry (MS, Fig 4D). The peak at higher retention volume (VR3) yielded a single signal in Western blot and a whole-protein mass spectrum compatible with a βarr1 monomer (50.4 kDa). The other eluted fraction (VR2) contained a mixture of βarr1 with the crosslinking product, and featured in MS a further signal at double mass. Furthermore, SEC fractions were enzymatically digested and applied to nano-HPLC/nano-ESI-Orbitrap-MS/MS. In the SEC fraction containing the crosslinked product, βarr1 was identified as the by far most abundant protein with sequence coverage of 90% (Appendix Fig S2B) and 1,951 PSMs (peptide spectral matches) compared to only 22 PSMs for the second most abundant protein. Collectively, the results identify this crosslinking product as a covalent βarr1 dimer. Figure 4. Analysis of activation-independent crosslinking band at D78Bpa-βarr1 SDS–PAGE and immunoblots of immunoprecipitated samples. Sample 1 was immunoprecipitated without previous UV treatment. Sample 2 is the product of irradiation of sample 1. Sample 3 was treated with UV light before immunoprecipitation. All samples were run in parallel on the same gel. Size exclusion chromatography (SEC) of Sample 3. Western blot of the fractions eluted by SEC. MALDI-TOF analysis of the SEC fractions VR2 and VR3. The spectrum of sample VR2 shows a [M + H]+ peak featuring twice the mass (˜ 108,000 Da) of the [M + H]+ peak of the βarr1 monomer (sample VR3, ˜ 50,400 Da). Download figure Download PowerPoint β-arrestins are known to self-associate in solution (Hanson et al, 2008; Chen et al, 2014), as well as in crystals and in cells (Han et al, 2001; Milano et al, 2006; Boularan et al, 2007; Zhan et al, 2011). Our results support the notion that β-strand VI participates in the βarr1 association interface, which is in line with the current oligomerization model of this arrestin. On the other hand, it cannot be excluded that at least some of the weak activation-independent crosslinking signals at high MW belong to complexes of arrestin with endogenous proteins. Both arrestins are known to function as scaffolds for a wide variety of proteins, with the most prominent examples being kinases like ERKs, JNK3, or other MAPKs (Xiao et al, 2007; Song et al, 2009), as well as proteins involved in GPCR trafficking, such as clathrin and AP2 (Goodman et al, 1996; Laporte et al, 1999). Elucidation of the nature of all receptor-independent crosslinking signals awaits further experiments. Other receptor-independent bands were visible below the apparent MW of arrestin itself (e.g., Bpa75/76-βarr1/2; Fig 2). Position 75/76 is quite intriguing, because Bpa75/76-βarr1/2 give both a low-MW band and an activation-dependent crosslinking band (especially with CRF1R, Fig 2). To test whether low-MW bands are due to intra-molecular crosslinking, we applied proximity-induced pair-wise crosslinking (Fig 5A) (Xiang et al, 2013; Wang, 2017b; Coin, 2018; Seidel et al, 2019). The mildly electrophilic ncAA O-(2-bromoethyl)-tyrosine (BrEtY) (Xiang et al, 2014) was genetically incorporated at position F75 of βarr1. Simultaneously, F244, which lies in its close proximity in the inactive βarr1 (Fig 5B, left), was mutated to Cys. Indeed, BrEtY75-Cys244-βarr1, but not BrEtY75-βarr1, yielded on Western blot a major band at the exact position of the receptor-independent product generated by UV irradiation of Bpa75-βarr1 (Fig 5C). When arrestin is activated, the finger loop moves away from F244 to insert into the receptor TM cavity (Fig 5B, right), so that Bpa at position 75 can capture the receptor. Thus, the photo-crosslinking data reveal the co-existence of two distinct populations of arrestins in our experiments: a receptor-engaged population and an inactive population in basal conformation. The result is not surprising, as especially βarr1 is largely overexpressed. Figure 5. Proximity-induced pair-wise crosslinking A. Mechanism of the nucleophilic substitution reaction between BrEtY and Cys. The product is a thioether, which is stable in reducing SDS–PAGE. B. Ribbon representation of inactive (PDB 1G4M (Han et al, 2001)) and active (PDB 6U1N (Staus et al, 2020)) βarr1. Arrestin is in gray and M2R in pale green. C. Western blot of whole cell lysates from HEK293T cells expressing βarr1 mutated as indicated in the top lines. All samples were run in parallel on the same gel. D, E. Pair-wise crosslinking between BrEtY-βarr1-3xHA and wt PTH1R. (D) Ribbon representation of active PTH1R as seen from the intracellular side (PDB 6NBF) (Zhao et al, 2019). Cys residues are shown in red. ECL3 (not resolved in the structure) is represented as a yellow dashed line. The approximate position of the Cys residue in this loop is marked with a fuzzy red circle. (E) Western blot of whole cells lysates stained with an α-HA antibody. Residues of βArr1 exchanged with BrEtY are indicated in the upper row, and mutations at PTH1R are indicated on the left side. The red arrows indicate the two most prominent signals. Red squares indicate signals that vanish upon removal of specific Cys residues in PTH1R. Download figure Download PowerPoint We further explored whether chemical crosslinking can be applied to determine inter-molecular receptor-arrestin proximity points, as it was shown to capture protein–protein interactions in live cells (Yang et al, 2017). The PTH1R contains four Cys residues in the juxtamembrane region at the cytosolic side (Fig 5D). To investigate whether any of these residues lie close to the finger loop of the associated arrestin, we incorporated BrEtY in the same 24 positions of βarr1 previously substituted with Bpa. BrEtY-βarr1 mutants were co-expressed with PTH1R in HEK293T cells, and arrestin recruitment was triggered by agonist activation. Strong arrestin–receptor crosslinking was detected when BrEtY was incorporated at position E66 and D78, along with some minor signals (Fig 5E). To investigate which Cys reacted with which BrEtY, the four Cys in PTH1R where mutated one by one into Ser and Ser-PTH1R mutants were combined with the same BrEtY-βarr1 set. Crosslinking obtained with BrEtY66- and BrEtY78-βarr1 selectively disappeared upon removal of C217 and C397, respectively (Fig 5E). Overall, the results of chemical crosslinking suggest that in the PTH1R-βarr1 complex, the N-terminal portion of the finger loop (E66) points toward ICL1 (C217) of the receptor, whereas the C-terminal segment (T74-K77, weaker crosslinking signals) up to the beginning of β-strand VI (D78) is proximal to ICL3 (C397). This is compatible with the orientation observed in the arr1-Rho and the βarr1-M2R complex, but not the βarr1-NTS1R (Fig EV3). This result clearly confirms the observation from the existing structures that the orientation of arrestin on the receptor does not determine whether the GPCR–arrestin complexes are stable or transient. Click here to expand this figure. Figure EV3. M2R, β1AR, and NTS1R in complex with βarr1Ribbon representation of βarr1 in complex with the M2R (gold), β1AR (violet), and NTS1R (coral) from recent cryo-EM structures (A, D: PDB 6U1N (Staus et al, 2020); B, E: PDB 6TKO (Lee et al, 2020); C, F: PDB 6PWC (Huang et al, 2020)). βarr1 is shown in gray with the finger loop in blue and the β-strand VI in pale green as in Fig 1 of the main text. The two main hits of chemical crosslinking with the PTH1R (Fig 5 in the main text), position E66 and D78, are marked in red. βarr1 binds to the M2R and β1AR in a similar pose as arr1 to Rho (not shown here), whereas it is rotated by about 90° when docked to the NTS1R. A–C. These panels focus on the position of the ICL1 in the GPCR–arrestin complex. For the sake of clarity, only the region TM1-ICL1-TM2 in the foreground is fully colored, whereas the rest of the GPCRs is transparent. In all three cases, E66 in the finger loop points toward ICL1 of the GPCR, which is compatible with the chemical crosslinking results at the PTH1R. D–F. These panels focus on the position of the ICL3, which is not resolved in any of the structures. For the sake of clarity, only the region TM5-ICL3-TM6 in the foreground is fully colored, whereas the rest of the GPCRs is transparent. The ICL3 of the M2R and β1AR is oriented toward β-strand VI, which is compatible with the chemical crosslinking results at the PTH1R, whereas the ICL3 of NTS1R points toward the arrestin N-terminus (cyan in panel F), away from β-strand VI. Download figure Download PowerPoint In conclusion, we have devised a biochemical method to investigate topologies of arrestin binding to GPCRs in intact cells, which exploits the genetic incorporation of photo-activatable and mildly electrophilic ncAAs into arrestin for photo- and chemical crosslinking experiments. In the first instance, photo-crosslinking enables distinguishing footprints of different receptors on the same arrestin. We show that different footprints are not related to the phylogenetic class of the investigated GPCRs (rhodopsin-like vs. secretin-like) nor to the type of interaction with arrestin (transient or stable), but are indeed specific for individual GPCRs. The similar footprints obtained on βarr1 and βarr2 suggest a common binding mode for the two arrestins. In the second instance, chemical crosslinking reveals inter-molecular pairs of amino acids coming into proximity in the GPCR-arrestin complex. By exploiting the presence of natural Cys residues in PTH1R, we have defined the orientation of the finger loop in the intracellular crevice of the active receptor. We anticipate that the incorporation of BrEtY into arrestins combined with Cys-mutagenesis of the GPCR will allow the detailed investigation of whole GPCR–arrestin interface
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