A non–GPCR-binding partner interacts with a novel surface on β-arrestin1 to mediate GPCR signaling
2020; Elsevier BV; Volume: 295; Issue: 41 Linguagem: Inglês
10.1074/jbc.ra120.015074
ISSN1083-351X
AutoresYa Zhuo, Vsevolod V. Gurevich, Sergey A. Vishnivetskiy, Candice S. Klug, Adriano Marchese,
Tópico(s)Monoclonal and Polyclonal Antibodies Research
ResumoThe multifaceted adaptor protein β-arr1 (β-arrestin1) promotes activation of focal adhesion kinase (FAK) by the chemokine receptor CXCR4, facilitating chemotaxis. This function of β-arr1 requires the assistance of the adaptor protein STAM1 (signal-transducing adaptor molecule 1) because disruption of the interaction between STAM1 and β-arr1 reduces CXCR4-mediated activation of FAK and chemotaxis. To begin to understand the mechanism by which β-arr1 together with STAM1 activates FAK, we used site-directed spin-labeling EPR spectroscopy-based studies coupled with bioluminescence resonance energy transfer–based cellular studies to show that STAM1 is recruited to activated β-arr1 by binding to a novel surface on β-arr1 at the base of the finger loop, at a site that is distinct from the receptor-binding site. Expression of a STAM1-deficient binding β-arr1 mutant that is still able to bind to CXCR4 significantly reduced CXCL12-induced activation of FAK but had no impact on ERK-1/2 activation. We provide evidence of a novel surface at the base of the finger loop that dictates non-GPCR interactions specifying β-arrestin–dependent signaling by a GPCR. This surface might represent a previously unidentified switch region that engages with effector molecules to drive β-arrestin signaling. The multifaceted adaptor protein β-arr1 (β-arrestin1) promotes activation of focal adhesion kinase (FAK) by the chemokine receptor CXCR4, facilitating chemotaxis. This function of β-arr1 requires the assistance of the adaptor protein STAM1 (signal-transducing adaptor molecule 1) because disruption of the interaction between STAM1 and β-arr1 reduces CXCR4-mediated activation of FAK and chemotaxis. To begin to understand the mechanism by which β-arr1 together with STAM1 activates FAK, we used site-directed spin-labeling EPR spectroscopy-based studies coupled with bioluminescence resonance energy transfer–based cellular studies to show that STAM1 is recruited to activated β-arr1 by binding to a novel surface on β-arr1 at the base of the finger loop, at a site that is distinct from the receptor-binding site. Expression of a STAM1-deficient binding β-arr1 mutant that is still able to bind to CXCR4 significantly reduced CXCL12-induced activation of FAK but had no impact on ERK-1/2 activation. We provide evidence of a novel surface at the base of the finger loop that dictates non-GPCR interactions specifying β-arrestin–dependent signaling by a GPCR. This surface might represent a previously unidentified switch region that engages with effector molecules to drive β-arrestin signaling. β-Arrestins (β-arrestin1 and β-arrestin2, also called arrestin-2 and arrestin-3, respectively) are multifaceted adaptor proteins that bind to ligand-activated and G protein–coupled receptor kinase (GRK) phosphorylated G protein–coupled receptors (GPCRs), thereby limiting the magnitude and duration of G protein–mediated signaling, leading to homologous receptor desensitization (1Carman C.V. Benovic J.L. G-protein–coupled receptors: turn-ons and turn-offs.Curr. Opin. Neurobiol. 1998; 8 (9687355): 335-34410.1016/S0959-4388(98)80058-5Crossref PubMed Scopus (218) Google Scholar, 2Gurevich 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). Once bound to a GPCR, β-arrestins can also promote distinct branches of signaling by interacting with various signaling molecules (3Shenoy S.K. Lefkowitz R.J. Multifaceted roles of β-arrestins in the regulation of seven-membrane-spanning receptor trafficking and signalling.Biochem. J. 2003; 375 (12959637): 503-51510.1042/BJ20031076Crossref PubMed Scopus (325) Google Scholar, 4DeWire S.M. Ahn S. Lefkowitz R.J. Shenoy S.K. β-Arrestins and cell signaling.Annu. Rev. Physiol. 2007; 69 (17305471): 483-51010.1146/annurev.physiol.69.022405.154749Crossref PubMed Scopus (1080) Google Scholar). β-Arrestins are members of a family of four proteins that also includes the two visual arrestins (arrestin-1 and arrestin-4). Arrestins share a conserved fold characterized by two elongated domains, termed the N and C domains, that are connected by a hinge region (5Hirsch J.A. Schubert C. Gurevich V.V. Sigler P.B. A Model for arrestin's regulation: the 2.8 Å crystal structure of visual arrestin.Cell. 1999; 97 (10219246): 257-26910.1016/S0092-8674(00)80735-7Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar, 6Han M. Gurevich V.V. Vishnivetskiy S.A. Sigler P.B. Schubert C. Crystal structure of β-arrestin at 1.9 A: possible mechanism of receptor binding and membrane translocation.Structure. 2001; 9 (11566136): 869-88010.1016/S0969-2126(01)00644-XAbstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar, 7Milano S.K. Pace H.C. Kim Y.-M. Brenner C. Benovic J.L. Scaffolding functions of arrestin-2 revealed by crystal structure and mutagenesis.Biochemistry. 2002; 41 (11876640): 3321-332810.1021/bi015905jCrossref PubMed Scopus (159) Google Scholar, 8Zhan X. Gimenez L.E. Gurevich V.V. Spiller B.W. Crystal structure of arrestin-3 reveals the basis of the difference in receptor binding between two non-visual subtypes.J. Mol. Biol. 2011; 406 (21215759): 467-47810.1016/j.jmb.2010.12.034Crossref PubMed Scopus (134) Google Scholar, 9Sutton R.B. Vishnivetskiy S.A. Robert J. Hanson S.M. Raman D. Knox B.E. Kono M. Navarro J. Gurevich V.V. Crystal structure of cone arrestin at 2.3Å: evolution of receptor specificity.J. Mol. Biol. 2005; 354 (16289201): 1069-108010.1016/j.jmb.2005.10.023Crossref PubMed Scopus (143) Google Scholar). Αrrestins are mainly cytosolic proteins where they are maintained in an inactive state by several intramolecular interaction networks (5Hirsch J.A. Schubert C. Gurevich V.V. Sigler P.B. A Model for arrestin's regulation: the 2.8 Å crystal structure of visual arrestin.Cell. 1999; 97 (10219246): 257-26910.1016/S0092-8674(00)80735-7Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar, 10Gurevich V.V. Benovic J.L. Visual arrestin interaction with rhodopsin: sequential multisite binding ensures strict selectivity toward light-activated phosphorylated rhodopsin.J. Biol. Chem. 1993; 268 (8505295): 11628-11638Abstract Full Text PDF PubMed Google Scholar). Agonist activation and phosphorylation of GPCRs promotes high-affinity arrestin binding characterized by a global conformational change leading to a fully activated arrestin (11Charest 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, 12Nuber S. Zabel U. Lorenz K. Nuber A. Milligan G. Tobin A.B. Lohse M.J. Hoffmann C. β-Arrestin biosensors reveal a rapid, receptor-dependent activation/deactivation cycle.Nature. 2016; 531 (27007855): 661-66410.1038/nature17198Crossref PubMed Scopus (127) Google Scholar, 13Kim M. Vishnivetskiy S.A. Van Eps N. Alexander N.S. Cleghorn W.M. Zhan X. Hanson S.M. Morizumi T. Ernst O.P. Meiler J. Gurevich V.V. Hubbell W.L. Conformation of receptor-bound visual arrestin.Proc. Natl. Acad. Sci. U.S.A. 2012; 109 (23091036): 18407-1841210.1073/pnas.1216304109Crossref PubMed Scopus (86) Google Scholar, 14Kim Y.J. Hofmann K.P. Ernst O.P. Scheerer P. Choe H.W. Sommer M.E. Crystal structure of pre-activated arrestin p44.Nature. 2013; 497 (23604253): 142-14610.1038/nature12133Crossref PubMed Scopus (127) Google Scholar, 15Zhuo Y. Vishnivetskiy S.A. Zhan X. Gurevich V.V. Klug C.S. Identification of receptor binding-induced conformational changes in non-visual arrestins.J. Biol. Chem. 2014; 289 (24867953): 20991-2100210.1074/jbc.M114.560680Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 16Shukla A.K. Manglik A. Kruse A.C. Xiao K. Reis R.I. Tseng W.C. Staus D.P. Hilger D. Uysal S. Huang L.Y. Paduch M. Tripathi-Shukla P. Koide A. Koide S. Weis W.I. et al.Structure of active β-arrestin-1 bound to a G-protein–coupled receptor phosphopeptide.Nature. 2013; 497 (23604254): 137-14110.1038/nature12120Crossref PubMed Scopus (280) Google Scholar). β-Arrestins might also assume receptor-specific conformations when bound to GPCRs, possibly endowing them with the ability to engage with discrete effector molecules (7Milano S.K. Pace H.C. Kim Y.-M. Brenner C. Benovic J.L. Scaffolding functions of arrestin-2 revealed by crystal structure and mutagenesis.Biochemistry. 2002; 41 (11876640): 3321-332810.1021/bi015905jCrossref PubMed Scopus (159) Google Scholar, 16Shukla A.K. Manglik A. Kruse A.C. Xiao K. Reis R.I. Tseng W.C. Staus D.P. Hilger D. Uysal S. Huang L.Y. Paduch M. Tripathi-Shukla P. Koide A. Koide S. Weis W.I. et al.Structure of active β-arrestin-1 bound to a G-protein–coupled receptor phosphopeptide.Nature. 2013; 497 (23604254): 137-14110.1038/nature12120Crossref PubMed Scopus (280) Google Scholar, 17Gurevich V.V. Gurevich E.V. Synthetic biology with surgical precision: targeted reengineering of signaling proteins.Cell Signal. 2012; 24 (22664341): 1899-190810.1016/j.cellsig.2012.05.012Crossref PubMed Scopus (24) Google Scholar, 18Zhan X. Perez A. Gimenez L.E. Vishnivetskiy S.A. Gurevich V.V. Arrestin-3 binds the MAP kinase JNK3α2 via multiple sites on both domains.Cell Signal. 2014; 26 (24412749): 766-77610.1016/j.cellsig.2014.01.001Crossref PubMed Scopus (29) Google Scholar). However, this remains poorly understood. The GPCR CXC motif chemokine receptor 4 (CXCR4) and its cognate chemokine CXCL12 (also called stromal cell-derived factor 1α) mediate chemotaxis during organ development, immune responses, stem cell mobilization, and metastatic cancer (19Peled A. Petit I. Kollet O. Magid M. Ponomaryov T. Byk T. Nagler A. Ben-Hur H. Many A. Shultz L. Lider O. Alon R. Zipori D. Lapidot T. Dependence of human stem cell engraftment and repopulation of NOD/SCID mice on CXCR4.Science. 1999; 283 (9933168): 845-84810.1126/science.283.5403.845Crossref PubMed Scopus (1415) Google Scholar, 20Tachibana K. Hirota S. Iizasa H. Yoshida H. Kawabata K. Kataoka Y. Kitamura Y. Matsushima K. Yoshida N. Nishikawa S. Kishimoto T. Nagasawa T. The chemokine receptor CXCR4 is essential for vascularization of the gastrointestinal tract.Nature. 1998; 393 (9634237): 591-59410.1038/31261Crossref PubMed Scopus (1285) Google Scholar, 21Nagasawa T. Hirota S. Tachibana K. Takakura N. Nishikawa S. Kitamura Y. Yoshida N. Kikutani H. Kishimoto T. Defects of B-cell lymphopoiesis and bone-marrow myelopoiesis in mice lacking the CXC chemokine PBSF/SDF-1.Nature. 1996; 382 (8757135): 635-63810.1038/382635a0Crossref PubMed Scopus (1940) Google Scholar, 22Müller A. Homey B. Soto H. Ge N. Catron D. Buchanan M.E. McClanahan T. Murphy E. Yuan W. Wagner S.N. Barrera J.L. Mohar A. Verástegui E. Zlotnik A. Involvement of chemokine receptors in breast cancer metastasis.Nature. 2001; 410 (11242036): 50-5610.1038/35065016Crossref PubMed Scopus (4258) Google Scholar, 23Hurley J.H. Stenmark H. Molecular mechanisms of ubiquitin-dependent membrane traffic.Annu. Rev. Biophys. 2011; 40 (21332354): 119-14210.1146/annurev-biophys-042910-155404Crossref PubMed Scopus (72) Google Scholar). CXCR4 is widely expressed on multiple cell types including lymphocytes, hematopoietic stem cells, epithelial cells, and cancer cells (24Teicher B.A. Fricker S.P. CXCL12 (SDF-1)/CXCR4 pathway in cancer.Clin. Cancer Res. 2010; 16 (20484021): 2927-293110.1158/1078-0432.CCR-09-2329Crossref PubMed Scopus (898) Google Scholar). CXCR4 is overexpressed in more than 23 human cancers, and its expression is associated with overall poor survival (22Müller A. Homey B. Soto H. Ge N. Catron D. Buchanan M.E. McClanahan T. Murphy E. Yuan W. Wagner S.N. Barrera J.L. Mohar A. Verástegui E. Zlotnik A. Involvement of chemokine receptors in breast cancer metastasis.Nature. 2001; 410 (11242036): 50-5610.1038/35065016Crossref PubMed Scopus (4258) Google Scholar, 25Li Y.M. Pan Y. Wei Y. Cheng X. Zhou B.P. Tan M. Zhou X. Xia W. Hortobagyi G.N. Yu D. Hung M.C. Upregulation of CXCR4 is essential for HER2-mediated tumor metastasis.Cancer Cell. 2004; 6 (15542430): 459-46910.1016/j.ccr.2004.09.027Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar, 26Orimo A. Gupta P.B. Sgroi D.C. Arenzana-Seisdedos F. Delaunay T. Naeem R. Carey V.J. Richardson A.L. Weinberg R.A. Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion.Cell. 2005; 121 (15882617): 335-34810.1016/j.cell.2005.02.034Abstract Full Text Full Text PDF PubMed Scopus (2670) Google Scholar, 27Zhang X.H. Wang Q. Gerald W. Hudis C.A. Norton L. Smid M. Foekens J.A. Massagué J. Latent bone metastasis in breast cancer tied to Src-dependent survival signals.Cancer Cell. 2009; 16 (19573813): 67-7810.1016/j.ccr.2009.05.017Abstract Full Text Full Text PDF PubMed Scopus (490) Google Scholar, 28Balkwill F. The significance of cancer cell expression of the chemokine receptor CXCR4.Semin. Cancer Biol. 2004; 14 (15246052): 171-17910.1016/j.semcancer.2003.10.003Crossref PubMed Scopus (610) Google Scholar, 29Hung C.S. Su H.Y. Liang H.H. Lai C.W. Chang Y.C. Ho Y.S. Wu C.H. Ho J.D. Wei P.L. Chang Y.J. High-level expression of CXCR4 in breast cancer is associated with early distant and bone metastases.Tumour Biol. 2014; 35 (24101191): 1581-158810.1007/s13277-013-1218-9Crossref PubMed Scopus (21) Google Scholar). CXCR4 signaling is involved in several aspects of tumor progression and metastases (30Shi J. Wei Y. Xia J. Wang S. Wu J. Chen F. Huang G. Chen J. CXCL12-CXCR4 contributes to the implication of bone marrow in cancer metastasis.Future Oncol. 2014; 10 (24799056): 749-75910.2217/fon.13.193Crossref PubMed Scopus (22) Google Scholar, 31Balkwill F. Cancer and the chemokine network.Nat. Rev. Cancer. 2004; 4 (15229479): 540-55010.1038/nrc1388Crossref PubMed Scopus (1777) Google Scholar), yet the mechanisms governing CXCR4 signaling remain poorly understood. Although GRKs and β-arrestins negatively regulate CXCR4 by canonical homologous desensitization (32Busillo J.M. Benovic J.L. Regulation of CXCR4 signaling.Biochim. Biophys. Acta. 2007; 1768 (17169327): 952-96310.1016/j.bbamem.2006.11.002Crossref PubMed Scopus (393) Google Scholar), β-arrestins can also promote CXCR4 signaling. In particular, β-arr1 (β-arrestin 1) is necessary for the activation of focal adhesion kinase (FAK), but not Akt or ERK-1/2, by CXCR4 (33). A unique feature of FAK activation by β-arr1 is that it requires the assistance of endocytic adaptor protein STAM1 (signal-transducing adaptor molecule 1) (33Alekhina O. Marchese A. β-Arrestin1 and signal-transducing adaptor Molecule 1 (STAM1) cooperate to promote focal adhesion kinase autophosphorylation and chemotaxis via the chemokine receptor CXCR4.J. Biol. Chem. 2016; 291 (27789711): 26083-2609710.1074/jbc.M116.757138Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). β-arr1, and to a lesser extent β-arr2, interacts directly with STAM1, and disruption of the interaction between them reduces FAK activation and chemotaxis by CXCR4 (33). Because binding to β-arr1 is enhanced by CXCR4 activation (34Malik R. Marchese A. Arrestin-2 interacts with the endosomal sorting complex required for transport machinery to modulate endosomal sorting of CXCR4.Mol. Biol. Cell. 2010; 21 (20505072): 2529-254110.1091/mbc.e10-02-0169Crossref PubMed Scopus (70) Google Scholar), it is likely that STAM1 prefers to bind to the receptor-bound, fully activated conformation of β-arr1. This is likely required to interact with and activate FAK, because a physical complex of β-arr1, STAM1, and FAK is formed following receptor activation, and they also co-localize at or near the cell periphery (33Alekhina O. Marchese A. β-Arrestin1 and signal-transducing adaptor Molecule 1 (STAM1) cooperate to promote focal adhesion kinase autophosphorylation and chemotaxis via the chemokine receptor CXCR4.J. Biol. Chem. 2016; 291 (27789711): 26083-2609710.1074/jbc.M116.757138Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Why β-arr1 requires STAM1 to activate FAK remains to be determined. Once bound to GPCRs, β-arrestins undergo a global conformational change that enhances their ability to bind to effector molecules (15Zhuo Y. Vishnivetskiy S.A. Zhan X. Gurevich V.V. Klug C.S. Identification of receptor binding-induced conformational changes in non-visual arrestins.J. Biol. Chem. 2014; 289 (24867953): 20991-2100210.1074/jbc.M114.560680Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 35Coffa S. Breitman M. Hanson S.M. Callaway K. Kook S. Dalby K.N. Gurevich V.V. The effect of arrestin conformation on the recruitment of c-Raf1, MEK1, and ERK1/2 activation.PLoS One. 2011; 6 (22174878): e2872310.1371/journal.pone.0028723Crossref PubMed Scopus (59) Google Scholar, 36Kumari P. Srivastava A. Banerjee R. Ghosh E. Gupta P. Ranjan R. Chen X. Gupta B. Gupta C. Jaiman D. Shukla A.K. Functional competence of a partially engaged GPCR–β-arrestin complex.Nat. Commun. 2016; 7 (27827372): 1341610.1038/ncomms13416Crossref PubMed Scopus (86) Google Scholar, 37Lee M.H. Appleton K.M. Strungs E.G. Kwon J.Y. Morinelli T.A. Peterson Y.K. Laporte S.A. Luttrell L.M. The conformational signature of β-arrestin2 predicts its trafficking and signalling functions.Nature. 2016; 531 (27007854): 665-66810.1038/nature17154Crossref PubMed Scopus (128) Google Scholar). Because β-arr1 binding to activated and phosphorylated CXCR4 changes its ability to bind to STAM1, the interaction with STAM1 is likely driven by conformational changes in β-arrestin. STAM1 mainly binds to the N domain of β-arr1 between residues 25 and 161 (33Alekhina O. Marchese A. β-Arrestin1 and signal-transducing adaptor Molecule 1 (STAM1) cooperate to promote focal adhesion kinase autophosphorylation and chemotaxis via the chemokine receptor CXCR4.J. Biol. Chem. 2016; 291 (27789711): 26083-2609710.1074/jbc.M116.757138Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar), which also contains key contact sites for ligand-activated and phosphorylated GPCRs (10Gurevich V.V. Benovic J.L. Visual arrestin interaction with rhodopsin: sequential multisite binding ensures strict selectivity toward light-activated phosphorylated rhodopsin.J. Biol. Chem. 1993; 268 (8505295): 11628-11638Abstract Full Text PDF PubMed Google Scholar, 38Vishnivetskiy S.A. Gimenez L.E. Francis D.J. Hanson S.M. Hubbell W.L. Klug C.S. Gurevich V.V. Few residues within an extensive binding interface drive receptor interaction and determine the specificity of arrestin proteins.J. Biol. Chem. 2011; 286 (21471193): 24288-2429910.1074/jbc.M110.213835Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). However, the precise structural or conformational determinants that dictate STAM1 binding remain unknown. Arrestins have several regions that become available for binding to effector molecules once activated by receptor binding. Importantly, the region between amino acid residues 89 and 97 of the N domain in β-arr2 referred to as arrestin switch region I (39Chen Q. Perry N.A. Vishnivetskiy S.A. Berndt S. Gilbert N.C. Zhuo Y. Singh P.K. Tholen J. Ohi M.D. Gurevich E.V. Brautigam C.A. Klug K.S. Gurevich V.V. Iverson T.M. Structural basis of arrestin-3 activation and signaling.Nat. Commun. 2017; 8 (29127291): 142710.1038/s41467-017-01218-8Crossref PubMed Scopus (46) Google Scholar) is an element that undergoes considerable movement from the basal state to the active state and might mediate agonist-dependent interactions with effector molecules (40Chen Q. Iverson T.M. Gurevich V.V. Structural basis of arrestin-dependent signal transduction.Trends Biochem. Sci. 2018; 43 (29636212): 412-42310.1016/j.tibs.2018.03.005Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). This region in β-arr2 contains two PXXP motifs, which are recognition sites for SH3 domains and likely mediate agonist-dependent binding to c-Src by GPCRs (39Chen Q. Perry N.A. Vishnivetskiy S.A. Berndt S. Gilbert N.C. Zhuo Y. Singh P.K. Tholen J. Ohi M.D. Gurevich E.V. Brautigam C.A. Klug K.S. Gurevich V.V. Iverson T.M. Structural basis of arrestin-3 activation and signaling.Nat. Commun. 2017; 8 (29127291): 142710.1038/s41467-017-01218-8Crossref PubMed Scopus (46) Google Scholar). The analogous region in β-arr1 contains only one PXXP motif, but it is unclear whether it can interact with SH3 domains. Notably, although STAM1 encodes an SH3 domain, it mainly interacts with β-arr1 via its coiled-coil region (34Malik R. Marchese A. Arrestin-2 interacts with the endosomal sorting complex required for transport machinery to modulate endosomal sorting of CXCR4.Mol. Biol. Cell. 2010; 21 (20505072): 2529-254110.1091/mbc.e10-02-0169Crossref PubMed Scopus (70) Google Scholar). Therefore, whether arrestin switch region I or a previously unidentified switch region binds to STAM1 remains to be determined. Here, we investigated the structural and biophysical properties of the interaction between β-arr1 and STAM1 by EPR spectroscopy. Using continuous wave (CW) EPR, we mapped the STAM1-binding site to a discrete region at the base of the finger loop. To determine whether binding to this region causes a global or localized conformational change in β-arr1, we used another EPR approach known as double electron–electron resonance (DEER) spectroscopy that measures distances between two intramolecular spin labels attached at specific sites. The DEER data show that STAM1 binding induces movement of the finger loop, similar to GPCRs, but does not induce movement of two other regions known to undergo large movements upon GPCR binding (15Zhuo Y. Vishnivetskiy S.A. Zhan X. Gurevich V.V. Klug C.S. Identification of receptor binding-induced conformational changes in non-visual arrestins.J. Biol. Chem. 2014; 289 (24867953): 20991-2100210.1074/jbc.M114.560680Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). The finger loop is located between β-strands 5 and 6 and is considered to represent a major binding region for activated GPCRs (39Chen Q. Perry N.A. Vishnivetskiy S.A. Berndt S. Gilbert N.C. Zhuo Y. Singh P.K. Tholen J. Ohi M.D. Gurevich E.V. Brautigam C.A. Klug K.S. Gurevich V.V. Iverson T.M. Structural basis of arrestin-3 activation and signaling.Nat. Commun. 2017; 8 (29127291): 142710.1038/s41467-017-01218-8Crossref PubMed Scopus (46) Google Scholar, 40Chen Q. Iverson T.M. Gurevich V.V. Structural basis of arrestin-dependent signal transduction.Trends Biochem. Sci. 2018; 43 (29636212): 412-42310.1016/j.tibs.2018.03.005Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Mutation of residues within the STAM1-binding site reduced agonist-dependent STAM1 binding but not CXCR4 binding, indicating that the STAM1-binding site does not overlap with the GPCR-binding site. Expression of a STAM1-deficient β-arr1–binding mutant that could still bind to CXCR4 reduced FAK activation, but not ERK-1/2, by CXCR4. We provide evidence of a novel region of β-arr1 specifying β-arrestin–dependent signaling. We used DEER spectroscopy to determine whether STAM1 binding induces conformational changes in β-arr1 by introducing spin labels at two reporter sites within β-arr1. The position of the spin labels was selected based on previous studies that revealed large conformational changes upon GPCR binding, including C-tail displacement, finger-loop extension, and middle-loop movement (15Zhuo Y. Vishnivetskiy S.A. Zhan X. Gurevich V.V. Klug C.S. Identification of receptor binding-induced conformational changes in non-visual arrestins.J. Biol. Chem. 2014; 289 (24867953): 20991-2100210.1074/jbc.M114.560680Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) (Fig. 1a). To report on C-tail displacement, the spin labels were placed on the C-tail (A392C) and the N domain (A12C). DEER-derived distance distributions are shown as overlays for the free (black) and STAM1-bound (blue) states (Fig. 1b). In the basal state, the C-tail is folded back onto the N domain, which helps to stabilize the inactive conformation of β-arr1 and is consistent with a small interspin distance between positions 12 and 392 (Fig. 1b). Upon GPCR binding, there is a large increase in the interspin distance, consistent with C-tail release from the N domain, as previously reported (15Zhuo Y. Vishnivetskiy S.A. Zhan X. Gurevich V.V. Klug C.S. Identification of receptor binding-induced conformational changes in non-visual arrestins.J. Biol. Chem. 2014; 289 (24867953): 20991-2100210.1074/jbc.M114.560680Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). However, STAM1 did not induce distance changes between spin labels at 12 and 392, indicating that the C-tail position is not affected by STAM1 binding (Fig. 1b). Middle-loop movement was assessed by placing both spin labels on the N domain at positions 136 and 167 (T136C and V167C) (Fig. 1a). STAM1 did not induce conformational changes in the middle loop, because no distance changes were observed between spin labels at positions 136 and 167 (Fig. 1c). This is in contrast to GPCR binding, which induced distance changes between spin labels in these positions, indicating the N domain moving away from the C domain (15Zhuo Y. Vishnivetskiy S.A. Zhan X. Gurevich V.V. Klug C.S. Identification of receptor binding-induced conformational changes in non-visual arrestins.J. Biol. Chem. 2014; 289 (24867953): 20991-2100210.1074/jbc.M114.560680Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 16Shukla A.K. Manglik A. Kruse A.C. Xiao K. Reis R.I. Tseng W.C. Staus D.P. Hilger D. Uysal S. Huang L.Y. Paduch M. Tripathi-Shukla P. Koide A. Koide S. Weis W.I. et al.Structure of active β-arrestin-1 bound to a G-protein–coupled receptor phosphopeptide.Nature. 2013; 497 (23604254): 137-14110.1038/nature12120Crossref PubMed Scopus (280) Google Scholar). By placing one spin label at site 167 (V167C) and the other at 68 (L68C), we were able to assess finger-loop movement (Fig. 1a). In contrast to C-tail displacement or middle-loop movement, STAM1 binding induced finger-loop extension (Fig. 1d). STAM1 induced a large distance change between these spin labels from 21 to 25 Å (Fig. 1d). This is similar to GPCR-binding, which also induces a distance change between positions 68 and 167, as previously shown (15Zhuo Y. Vishnivetskiy S.A. Zhan X. Gurevich V.V. Klug C.S. Identification of receptor binding-induced conformational changes in non-visual arrestins.J. Biol. Chem. 2014; 289 (24867953): 20991-2100210.1074/jbc.M114.560680Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Shown in Fig. 1 (e–g) are the fits to the free (black) and the STAM1-bound (blue) background-corrected dipolar evolution data (gray dots) to indicate data quality to support the distance distribution data (41Klug C.S. Feix J.B. Methods and applications of site-directed spin labeling EPR spectroscopy.Methods Cell Biol. 2008; 84 (17964945): 617-65810.1016/S0091-679X(07)84020-9Crossref PubMed Scopus (130) Google Scholar, 42Jeschke G. DEER distance measurements on proteins.Annu. Rev. Phys. Chem. 2012; 63 (22404592): 419-44610.1146/annurev-physchem-032511-143716Crossref PubMed Scopus (624) Google Scholar). The attached spin labels on the β-arr1 double-cysteine mutants did not impact STAM1 binding, as assessed by pulldown assay (Fig. 2a), similar to what was previously observed with binding to GPCR (15Zhuo Y. Vishnivetskiy S.A. Zhan X. Gurevich V.V. Klug C.S. Identification of receptor binding-induced conformational changes in non-visual arrestins.J. Biol. Chem. 2014; 289 (24867953): 20991-2100210.1074/jbc.M114.560680Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). These data provide evidence that unlike GPCRs, STAM1 does not promote global conformational change in β-arr1, suggesting that STAM1 and GPCRs interact with the N domain differently. To precisely map the STAM1-binding site on β-arr1, we employed site-directed spin-labeling CW EPR spectroscopy. EPR spectroscopy takes advantage of the fact that the spin labels have a paramagnetic center and when the label is attached to a unique cysteine on the surface of a protein, the spectrum reflects its mobility. Spin labels that are mobile display spectra that are narrow with sharp peaks; however, when a protein partner binds at or near the spin label or a conformational change constrains its mobility, the spectral lines broaden and decrease in amplitude (43Hubbell W.L. Gross A. Langen R. Lietzow M.A. Recent advances in site-directed spin labeling of proteins.Curr. Opin. Struct. Biol. 1998; 8 (9818271): 649-65610.1016/S0959-440X(98)80158-9Crossref PubMed Scopus (480) Google Scholar, 44Hubbell W.L. Cafiso D.S. Altenbach C. Identifying conformational changes with site-directed spin labeling.Nat. Struct. Biol. 2000; 7 (10966640): 735-73910.1038/78956Crossref PubMed Scopus (697) Google Scholar). This method has been previously used to successfully map the rhodopsin footprint on β-arr1 (38Vishnivetskiy S.A. Gimenez L.E. Francis D.J. Hanson S.M. Hubbell W.L. Klug C.S. Gurevich V.V. Few residues within an extensive binding interface drive receptor interaction and determine the specificity of arrestin proteins.J. Biol. Chem. 2011; 286 (21471193): 24288-2429910.1074/jbc.M110.213835Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 45Hanson S.M. Francis D.J. Vishnivetskiy S.A. Kolobova E.A. Hubbell W.L. Klug C.S. Gurevich V.V. Differential interaction of spin-labeled arrestin with inactive and active phosphorhodopsin.Proc. Natl. Acad. Sci. U.S.A. 2006; 103 (16547131): 4900-490510.1073/pnas.0600733103Crossref PubMed Scopus (154) Google Scholar). Because the N domain has previously been shown to bind STAM1 (34Malik R. Marchese A. Arrestin-2 interacts with the endosomal sorting complex required for tra
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