Dynamic Control of Adhesion and Migration by Integrins
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Model for Receptor Signaling outside-in outside-in inside-out The 24 Vertebrate Integrin aß Heterodimers Integrin Therapeutics: Antibodies Efiluzimab Psoriasis a1* a10* a11* a2* a3 a4 aL* aM* aX* ß2 aE* ß7 a5 aD* ß4 *: a subunits that contain I domains a6 a7 a8 a9 aIIb ß1 aV ß3 ß5 ß6 ß8 Efficacy of Antibody to LFA-1 in Psoriasis Before treatment Efalizumab (anti-integrin LFA-1) administered for 2 months Integrin Therapeutics: Antibodies Efiluzimab Psoriasis aL* aM* aX* Nataluzimab Multiple Sclerosis ß2 aE* ß7 a1* a10* a11* a2* a3 a4 a5 aD* ß4 *: a subunits that contain I domains a6 a7 a8 a9 aIIb Abciximab Thrombosis ß1 aV ß3 ß5 ß6 ß8 a I allosteric antagonists Integrin Therapeutics: Small Molecules a/b I-like allosteric antagonists a1* a10* a11* a2* a3 a4 aL* aM* aX* ß2 aE* ß7 a5 aD* ß4 *: a subunits that contain I domains a6 a7 a8 a9 Epifibatide Tirofiban Thrombosis ß1 aV aIIb ß3 ß5 ß6 ß8 The cast of cell surface adhesion molecules • Integrin aLb2, LFA-1 (lymphocyte-function associated antigen-1) • Integrin aXb2 • Their ligand, ICAM-1 (intercellular adhesion molecule-1), contains 5 IgSF domains • Integrins aVb3, aIIbb3, a5b1, which lack a I domains, and bind ligands with Arg-Gly-Asp (RGD) motifs T lymphocytes migrating to a chemattactant-filled micropipette: Integrin aLb2-mediated migration on ICAM-1-bearing substrate QuickTime™ and a H.263 decompressor are needed to see this picture. T lymphocyte migrating using integrin aLb2 on ICAM-1 QuickTime™ and a Video decompressor are needed to see this picture. C-terminal helix displacement activates high affinity of a I domain of integrin aLb2 QuickTime™ and a Sorenson Video 3 decompressor are needed to see this picture. Shimaoka, M., Xiao, T., Takagi, J., Wang, J, & Springer, T.A. (2003). Structures of the aL I domain and its complex with ICAM-1 reveal a shape-shifting pathway for integrin regulation. Cell 112, 99-111. C-terminal helix displacement activates high affinity of a I domain of integrin aLb2 QuickTime™ and a Sorenson Video 3 decompressor are needed to see this picture. Shimaoka, M., Xiao, T., Takagi, J., Wang, J, & Springer, T.A. (2003). Structures of the aL I domain and its complex with ICAM-1 reveal a shape-shifting pathway for integrin regulation. Cell 112, 99-111. C-terminal helix displacement activates high affinity of a I domain of integrin aLb2 QuickTime™ and a Sorenson Video 3 decompressor are needed to see this picture. Shimaoka, M., Xiao, T., Takagi, J., Wang, J, & Springer, T.A. (2003). Structures of the aL I domain and its complex with ICAM-1 reveal a shape-shifting pathway for integrin regulation. Cell 112, 99-111. C-terminal helix displacement activates high affinity of a I domain of integrin aLb2 QuickTime™ and a Sorenson Video 3 decompressor are needed to see this picture. Shimaoka, M., Xiao, T., Takagi, J., Wang, J, & Springer, T.A. (2003). Structures of the aL I domain and its complex with ICAM-1 reveal a shape-shifting pathway for integrin regulation. Cell 112, 99-111. Mutant I domains and a ligand-mimetic, conformation-specific Fab I domain Wild-type Intermedia te affi nity High affinit y Mutation none I161C/V299C K297C/K294C • Binding of AL-57 requires Mg2+ • AL-57 blocks ligand binding KD, ICAM-1 1.5 mM 3,000 nM 150 nM KD, AL-57 Fab Not detected 4,700 nM 23 nM KD, MHM24 Fab 1.9 nM 2.0 nM 6.3 nM Migrating T lymphocytes express high affinity LFA-1 in the lamellipodium Red: non-conformation-dependent Ab to LFA-1. Green: AL-57 ligand-mimetic Ab. QuickTime™ and a Animation decompressor are needed to see this picture. T lymphocytes recognizing antigen on dendritic cells form an immunological synapse containing high-affinity LFA-1 Red: non-conformation-dependent Ab to LFA-1. Green: AL-57 ligand-mimetic Ab. Dendritic cell QuickTime™ and a Animation decompressor are needed to see this picture. T cell Inside-out signaling by integrin cell adhesion receptors White cell Intracellular signals Integrin outside-in signaling talin binding Foreignness Activation signal recognition recognition Integrin inside-out signaling Binding to ligand (ICAM) ICAM Interacting cell The equilibria for conformational change and ligand binding are linked Inside-out signaling I Ligand binding +L I* +L IL I*L L: ligand I: resting integrin I*: high affinity integrin Integrin ectodomain crystal and EM structures in high and low affinity conformations resting aVb3 aVb3 + cyclo-RGD Schematic of low affinity aVb3 crystal structure Lower legs bI Takagi et al, Cell (2002) a5b1 head + Fn7-10 Xiong, J.-P., Stehle, T., Diefenbach, B., Zhang, R., Dunker, R., Scott, D. L., Joachimiak, A., Goodman, S. L., and Arnaout, M. A.. Science 294, 339-345. a5b1 head Takagi et al, EMBO J (2003) Integrin ectodomain crystal structures in high and low affinity conformations Ribbon diagram of high affinity aIIbb3 headpiece crystal structure Comparison of high and low affinity headpiece conformations Ligand b-propeller bI b-propeller Schematic of low affinity aVb3 crystal structure bI a subunit Hybrid bI Lower legs Thigh PSI b subunit Xiao, T., Takagi, J., Wang, J.-h., Coller, B. S., and Springer, T. A. Nature 432, 5967. Xiong, J.-P., Stehle, T., Diefenbach, B., Zhang, R., Dunker, R., Scott, D. L., Joachimiak, A., Goodman, S. L., and Arnaout, M. A.. Science 294, 339-345. a subunit Swung-in hybrid domain, low affinity, closed headpiece b subunit Swung-out hybrid domain, high affinity, open headpiece Allostery in Integrin b I and a I domains a I domain b I domain a1 a1 Low affinity High affinity a7 b subunit hybrid domain a7 A spring pull model for I domain activation aI Head b-propeller b I aI domain Upper leg aI domain bI domain bI domain Lower leg a subunit b subunit Second site reversion supports the model aI domain aI domain bI domain aI domain bI domain bI domain Cytoplasmic and transmembrane domain separation is associated with integrin activation Kim, M., Carman, C. V., and Springer, T. A. 2003. Bidirectional transmembrane signaling by cytoplasmic domain separation in integrins. Science 301:1720. Head Luo, B.-H., Springer, T. A., and Takagi, J. (2004). A specific interface between integrin transmembrane helices and affinity for ligand. PLoS Biol. 2, 776. Upper legs Lower legs b a 433 nm mCFP mYFP 527 nm 433 nm FRET b a mCFP Transmembrane / Cytoplasmic Domain mYFP 475 nm FRET experiments demonstrate that separation of integrin cytoplasmic domains activates the extracellular domain, and conversely, ligand binding to the extracellular domain induces cytoplasmic domain separation Conformational transitions in integrins with a I domains: aXb2 and aXb2 Leg Irons Noritaka Nishida, Can Xie, Tom Walz, Tim Springer Conformational transitions in integrins with a I domains: aXb2 and aXb2 Negative stain EM averages of 5,000 to10,000 particles Leg Irons Cleaved Leg Irons Bent >95% Noritaka Nishida, Can Xie, Tom Walz, Tim Springer Compact 23% Extended, closed 54% Open 23% What is the effect of antibodies to activation epitopes on I-EGF modules 2 and 3 of b2? KIM127 Epitope (Activation-dependent) Beglova, Blacklow, Takagi, Springer Nat. Struct. Biol. 2002. CBR LFA-1/2 Epitope (Activation-inducing) Effect of Fab to activation epitopes in I-EGF2 and 3 near bend in b2 leg Leg Irons Cleaved Leg Irons Compact 23% Bent >95% CBR LFA-1/2 Closed 48% Open 52% CBR LFA-1/2 + KIM127 Closed 51% Noritaki Nishida, Can Xie, Tom Walz, Tim Springer Open 49% Extended, closed 54% Open 23% CBR LFA-1/2 Closed 56% Open 44% What is the effect of Integrin antagonists directed to the b I domain MIDAS? Arg-Gly-Asp-mimetic antagonist to aIIbb3integrin tirofiban Allosteric antagonist to integrins aLb2 and aXb2 XVA143 Effect of a/b I-like allosteric antagonist XVA143 (Drug) Leg Irons Cleaved Leg Irons Compact 23% Bent >95% CBR LFA-1/2 + KIM127 CBR LFA-1/2 Closed 48% Open 52% Closed 51% 10mM Drug Bent 60% Extended, open 40% Noritaki Nishida, Can Xie, Tom Walz, Tim Springer Open 49% Extended, closed 54% Open 23% CBR LFA-1/2 Closed 56% 10mM Drug Extended, open >95% Open 44% Similar results with aLb2, different equilibria set points Leg Irons Leg Irons Cleaved I domain displacement from the membrane Integrin Signalling • The conformation of integrins is regulated both by signaling/cytoskeletal molecules such as talin inside the cell (insideout signaling) and binding to ligands outside the cell. • Work with the same antibodies/Fab on live cells and EM definitively establishes that integrin extension is sufficient for activation, and occurs in vivo when integrin adhesiveness is activated. • a I domain conformation and affinity for ligand is linked to b I domain conformation. • Small changes in b I domain conformation are linked to very large conformational changes in the integrin ectodomain by hybrid domain swing-out, facilitating communication of allostery across the cell membrane by separation of the a and b subunit TM and cytoplasmic domains. Model for Receptor Signaling outside-in outside-in Ectodomain inside-out 1. Inactive dimer 2. Active dimer Transmembrane Juxtamembrane Cytoplasmic domain 3. Active dimer stabilized by bound ligand Collaborators Tsan Xiao Jun Takagi - Osaka U Motomu Shimaoka - Harvard Med Sch Jia-huai Wang - DFCI Noritaka Nishida Can Xie Tom Walz - Harvard Med Sch Minsoo Kim - Brown Univ Chris Carman Bing-Hao Luo Wei Yang http://cbr.med.harvard.edu/springer
