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Allosteric Mechanisms Underlie GPCR Signaling to SH3-Domain Proteins Through Arrestin

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Allosteric Mechanisms Underlie GPCR Signaling to SH3-Domain Proteins Through Arrestin ARTICLES https://doi.org/10.1038/s41589-018-0115-3 Allosteric mechanisms underlie GPCR signaling to SH3-domain proteins through arrestin Fan Yang1,2,3,4,9, Peng Xiao1,5,9, Chang-xiu Qu1,9, Qi Liu1,2,3,9, Liu-yang Wang6, Zhi-xin Liu1, Qing-tao He1,4, Chuan Liu4, Jian-ye Xu1, Rui-rui Li1, Meng-jing Li1, Qing Li3, Xu-zhen Guo2, Zhao-ya Yang1,4, Dong-fang He1, Fan Yi7, Ke Ruan8, Yue-mao Shen6, Xiao Yu3, Jin-peng Sun 1,4* and Jiangyun Wang2* Signals from 800 G-protein-coupled receptors (GPCRs) to many SH3 domain-containing proteins (SH3-CPs) regulate impor- tant physiological functions. These GPCRs may share a common pathway by signaling to SH3-CPs via agonist-dependent arrestin recruitment rather than through direct interactions. In the present study, 19F-NMR and cellular studies revealed that downstream of GPCR activation engagement of the receptor-phospho-tail with arrestin allosterically regulates the specific conformational states and functional outcomes of remote β -arrestin 1 proline regions (PRs). The observed NMR chemical shifts of arrestin PRs were consistent with the intrinsic efficacy and specificity of SH3 domain recruitment, which was controlled by defined propagation pathways. Moreover, in vitro reconstitution experiments and biophysical results showed that the receptor– arrestin complex promoted SRC kinase activity through an allosteric mechanism. Thus, allosteric regulation of the conforma- tional states of β -arrestin 1 PRs by GPCRs and the allosteric activation of downstream effectors by arrestin are two important mechanisms underlying GPCR-to-SH3-CP signaling. xtracellular signals received by G-protein-coupled receptors stabilization with an antibody or through extensive modification of (GPCRs) induce structural rearrangements in their cytoplasmic arrestin and its receptor26,27. Eregions, which are subsequently recognized by transducer G Alternatively, fluorescence spectroscopy and 19F-NMR have suc- proteins and in turn regulate the concentration of intracellular mes- cessfully been used to observe structural rearrangements in GPCR sengers1–6. Activated GPCRs are then phosphorylated by a group of signaling11,30–34. unnatural amino acid incorporation techniques GPCR kinases (GRKs)7–9, leading to the recruitment of a different combined with 19F-NMR, fluorescence spectroscopy and cellular type of transducer, arrestin, which then initiates another wave of approaches to study the mechanisms underlying arrestin-mediated cellular signaling10–16. In general, even a single type of GPCR can GPCR signaling to SH3 domain-containing proteins (SH3-CPs)35,36. initiate a broad range of physiological processes through arrestin Consequently, we not only determined the specificity of different engagement by scaffolding different downstream effectors11,14,17–22. PRs of arrestin in connecting different receptors to downstream Meanwhile, cellular studies suggest that conformational changes SH3-CPs and the mechanisms regulating their conformational in arrestin occur after GPCR activation, and arrestin retains its states through an allosteric mechanism, but also found that the active conformation even after dissociating from receptors11,23–25. active arrestin conformation promotes the activation of down- Consistent with these observations, recent crystallographic studies stream kinases containing SH3 domains by disrupting its autoin- have revealed that either the binding of arrestin to a phospho-recep- hibitory structural organization. tor C-tail or the direct fusion of a receptor to arrestin induces signif- icant structural rearrangements in arrestin26,27. Based on the current Results paradigm, receptor–arrestin complexes display two distinct confor- Arrestin mediates GPCR–SH3-CP coupling via three proline mations: (1) the ‘core’ or ‘snuggly’ conformation, which is impor- regions. Among the 825 GPCR sequences, 753 GPCRs (91.3%) do tant for desensitization of G-protein signaling, and (2) the ‘tail’ not contain the classic PR recognized by SH3-CPs (Supplementary or ‘hanging’ conformation, which facilitates G-protein activation Figs. 1–3; see also Supplementary Note 1). Although 72 GPCRs or G-protein-independent signaling10,15,28. Thus, conformational bearing the PR motifs were potentially able to directly interact states are an important aspect of arrestin function11,26,27,29. Despite with SH3-CPs, the remaining GPCRs might connect to SH3- this substantial research progress, the mechanisms by which con- CPs through other receptor-interacting proteins. We therefore formational states directly contribute to arrestin-mediated signal- postulated that many GPCRs might share a common pathway ing remain unclear, as high-resolution structures of active arrestin connected to a landscape of SH3-CPs via arrestin scaffolding, conformations have only been captured using crystallography via as most activated GPCRs bind to arrestin, which encodes three 1Key Laboratory Experimental Teratology of the Ministry of Education and Department of Biochemistry and Molecular Biology, Shandong University School of Medicine, Jinan, Shandong, China. 2Institute of Biophysics, Chinese Academy of Sciences, Beijing, Chaoyang district, Beijing, China. 3Key Laboratory Experimental Teratology of the Ministry of Education and Department of Physiology, Shandong University School of Medicine, Shandong, China. 4Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Peking University, Key Laboratory of Molecular Cardiovascular Science, Ministry of Education, Beijing, China. 5Key Laboratory of Chemical Biology, Ministry of Education, School of Pharmaceutical Science, Shandong University, Jinan, Shandong, China. 6Department of Molecular Genetics and Microbiology, School of Medicine, Duke University, Durham, NC, USA. 7Department of Pharmacology, Shandong University School of Medicine, Jinan, China. 8Laboratory for Physical Sciences at the Microscale and School of Life Sciences, University of Science and Technology of China, Hefei Anhui, China. 9These authors contributed equally: Fan Yang, Peng Xiao, Chang-xiu Qu, Qi Liu. *e-mail: [email protected]; [email protected] 876 NATURE CHEMIcal BIOLOgy | VOL 14 | SEPTEMBER 2018 | 876–886 | www.nature.com/naturechemicalbiology NATURE CHEMICAL BIOLOGY ARTICLES type I PRs that are potential SH3-CP docking sites (Fig. 1b and and 11a). These results suggest that the phospho-barcode in the Supplementary Fig. 4a). receptor’s C-tail plays a critical role in arrestin-mediated GPCR– To test this hypothesis, we selected a panel of GPCRs that lack a SH3-CP coupling11,37. PR but exhibit diverse arrestin recruitment and G-protein subtype coupling characteristics (Supplementary Fig. 4b) and examined The phospho-barcode encodes functional PR conformations. their ability to recruit several known SH3-CPs through arrestin Based on the crystallographic and NMR results, the phospho- after agonist stimulation. We co-transfected plasmids encoding C-tails of β 2AR and V2R lie along both the surface of the three ele- HA-β -arrestin 1, Flag-tagged GPCRs and different SH3-CPs into ments (β -strand I, α -helix I and the C terminus) and the finger-loop HEK293 cells. After agonist stimulation, the β -arrestin-1-mediated region of β -arrestin 1 in the hanging conformation of the recep- recruitment of SH3-CPs was detected by co-immunoprecipitation tor–arrestin complex10,11,15,27,28,38. Importantly, all three functional (co-IP). Importantly, regardless of the G-protein subtype coupling PRs were located between 28 Å and 36 Å away from these recep- or arrestin-binding avidity, each receptor used arrestin to recruit tor-phospho-binding sites of β -arrestin 1 (Supplementary Fig. 9a). 1–3 of the 5 SH3-CPs tested (Fig. 1a and Supplementary Fig. 5). Thus, allosteric regulation of β -arrestin 1 occurs between β -arrestin We then determined the specificity of the PR regions of β -arres- 1’s receptor-phospho-binding sites and its remote PR regions. tin 1 in mediating receptor–SH3-CP interactions using co-IP We next incorporated the unnatural amino acid 3,5-difluoroty- experiments with different PR mutations. The results showed that rosine (F2Y) in specific positions of β-arrestin 1 and used 19F-NMR all three PRs in β -arrestin 1 participated in SH3-CP recruitment to detect the conformational changes in the three PRs in response to (Supplementary Figs. 6 and 7). The PR 1 (P1) region played an different types of receptor phospho-C-tail stimuli (Supplementary important role in most of these interactions, except for the inter- Figs. 9 and 11a,b). These synthesized receptor phospho-C-tails action between SRC and SSTR2. The PR 2 (P2) region specifically induced the formation of active arrestin conformations that largely interacted with SRC by mediating its connections to β 2AR and mimicked the hanging conformation of the receptor–arrestin com- SSTR2 but not to V2R (Supplementary Fig. 7c). The PR 3 (P3) plex10,15,28. We used the F2Y technique because F2Y-incorporated region selectively bound to GRB2 (Fig. 1b and Supplementary arrestin causes minimal perturbations to the overall structure Figs. 4c, 6 and 7). In conclusion, all three PRs of β -arrestin 1 are and provides a more significant chemical shift than traditional potential SH3-CP docking sites, each with different receptor and bromo-4(trifluoromethyl)acetanilide (BTFA)-labeled cysteine- downstream effector selectivity. negative arrestin (Supplementary Fig. 10a–c). The increase in the 19F-NMR signal was due to the sensitivity of the phenolic F2Y The phospho-barcode mediates β-arrestin-1–SH3-CP coupling.
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