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Model for Receptor Signaling

Model for Receptor Signaling. outside-in. outside-in. inside-out. The 24 Vertebrate Integrin a ß Heterodimers. Integrin Therapeutics: Antibodies. Efiluzimab Psoriasis. a1 *. a IIb. a 10 *. a 11 *. a L*. a L *. aE *. ß 7. a 2 *. a M*. a M *. ß2. a 3. a 4. ß 3. a X*. a X *.

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Model for Receptor Signaling

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  1. Model for Receptor Signaling outside-in outside-in inside-out

  2. The 24 Vertebrate Integrin aß Heterodimers Integrin Therapeutics: Antibodies Efiluzimab Psoriasis a1* aIIb a10* a11* aL* aL* aE* ß7 a2* aM* aM* ß2 a3 a4 ß3 aX* aX* ß1 aV a5 ß5 aD* aD* a6 ß4 a7 ß6 a8 ß8 a9 *: asubunits that contain I domains

  3. Efficacy of Antibody to LFA-1 in Psoriasis Efalizumab (anti-integrin LFA-1) administered for 2 months Before treatment

  4. Integrin Therapeutics: Antibodies Efiluzimab Psoriasis a1* aIIb a10* Nataluzimab Multiple Sclerosis a11* aL* aL* aE* ß7 a2* Abciximab Thrombosis aM* aM* ß2 a3 a4 ß3 aX* aX* ß1 aV a5 ß5 aD* aD* a6 ß4 a7 ß6 a8 ß8 a9 *: asubunits that contain I domains

  5. a I allosteric antagonists a/b I-like allosteric antagonists a1* Epifibatide Tirofiban Thrombosis aIIb a10* a11* aL* aL* aE* ß7 a2* aM* aM* ß2 a3 a4 ß3 aX* aX* ß1 aV a5 ß5 aD* aD* a6 ß4 a7 ß6 a8 ß8 a9 *: asubunits that contain I domains Integrin Therapeutics: Small Molecules

  6. 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

  7. T lymphocytes migrating to a chemattactant-filled micropipette: Integrin aLb2-mediated migration on ICAM-1-bearing substrate

  8. T lymphocyte migrating using integrin aLb2 on ICAM-1

  9. C-terminal helix displacement activates high affinity of a I domain of integrin aLb2 Shimaoka, M., Xiao, T., Takagi, J., Wang, J, & Springer, T.A. (2003). Structures of the L I domain and its complex with ICAM-1 reveal a shape-shifting pathway for integrin regulation. Cell 112, 99-111.

  10. C-terminal helix displacement activates high affinity of a I domain of integrin aLb2 Shimaoka, M., Xiao, T., Takagi, J., Wang, J, & Springer, T.A. (2003). Structures of the L I domain and its complex with ICAM-1 reveal a shape-shifting pathway for integrin regulation. Cell 112, 99-111.

  11. C-terminal helix displacement activates high affinity of a I domain of integrin aLb2 Shimaoka, M., Xiao, T., Takagi, J., Wang, J, & Springer, T.A. (2003). Structures of the L I domain and its complex with ICAM-1 reveal a shape-shifting pathway for integrin regulation. Cell 112, 99-111.

  12. C-terminal helix displacement activates high affinity of a I domain of integrin aLb2 Shimaoka, M., Xiao, T., Takagi, J., Wang, J, & Springer, T.A. (2003). Structures of the L I domain and its complex with ICAM-1 reveal a shape-shifting pathway for integrin regulation. Cell 112, 99-111.

  13. Mutant I domains and a ligand-mimetic, conformation-specific Fab • Binding of AL-57 requires Mg2+ • AL-57 blocks ligand binding

  14. 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.

  15. T lymphocytes recognizing antigen on dendritic cells form an immunological synapse containing high-affinity LFA-1 Dendritic cell T cell Red: non-conformation-dependent Ab to LFA-1. Green: AL-57 ligand-mimetic Ab.

  16. Inside-out signaling by integrin cell adhesion receptors White cell Intracellular signals Integrin outside-in signaling talin binding Integrin inside-out signaling Activation signal recognition Foreignness recognition Binding to ligand (ICAM) ICAM Interacting cell

  17. Inside-out signaling I I* Ligand binding +L +L IL I*L The equilibria for conformational change and ligand binding are linked L: ligand I: resting integrin I*: high affinity integrin

  18. resting aVb3 aVb3 + cyclo-RGD Head Upper legs Takagi et al, Cell (2002) Schematic of low affinity aVb3 crystal structure a5b1 head + Fn7-10 Lower legs a5b1 head b I Takagi et al, EMBO J (2003) 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. Integrin ectodomain crystal and EM structures in high and low affinity conformations

  19. Ribbon diagram of high affinity aIIbb3 headpiece crystal structure Comparison of high and low affinity headpiece conformations Ligand Head Upper legs b-propeller b I b I b-propeller a subunit Hybrid Schematic of low affinity aVb3 crystal structure Thigh PSI Lower legs b subunit a subunit b subunit b I Xiao, T., Takagi, J., Wang, J.-h., Coller, B. S., and Springer, T. A. Nature 432, 59-67. Swung-in hybrid domain, low affinity, closed headpiece Swung-out hybrid domain, high affinity, open headpiece 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. Integrin ectodomain crystal structures in high and low affinity conformations

  20. a I domain b I domain a1 a1 a7 a7 b subunithybrid domain Allostery in Integrin b I and a I domains Low affinity High affinity

  21. A spring pull model for I domain activation a I Head b-propeller b I Second site reversion supports the model Upper leg a I domain a I domain a I domain Lower leg b I domain b I domain b I domain a subunit b subunit a I domain a I domain b I domain b I domain

  22. Kim, M., Carman, C. V., and Springer, T. A. 2003. Bidirectional transmembrane signaling by cytoplasmic domain separation in integrins. Science 301:1720. Cytoplasmic and transmembrane domain separation is associated with integrin activation 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     Transmembrane / Cytoplasmic Domain mCFP mYFP mCFP mYFP 433 nm 527 nm 433 nm 475 nm FRET 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

  23. Conformational transitions in integrins with a I domains: aXb2 andaXb2 Leg Irons Noritaka Nishida, Can Xie, Tom Walz, Tim Springer

  24. Conformational transitions in integrins with a I domains: aXb2 andaXb2 Leg Irons Cleaved Extended, closed 54% Open 23% Compact 23% Bent >95% Negative stain EM averages of 5,000 to10,000 particles Leg Irons Noritaka Nishida, Can Xie, Tom Walz, Tim Springer

  25. What is the effect of antibodies to activation epitopes on I-EGF modules 2 and 3 of b2? Beglova, Blacklow, Takagi, Springer Nat. Struct. Biol. 2002. KIM127 Epitope (Activation-dependent) CBR LFA-1/2 Epitope (Activation-inducing)

  26. Effect of Fab to activation epitopes in I-EGF2 and 3 near bend in b2 leg Extended, closed 54% Open 23% Compact 23% Bent >95% CBR LFA-1/2 + KIM127 CBR LFA-1/2 + KIM127 Open 49% Open 49% Closed 51% Closed 51% Leg Irons Cleaved Leg Irons CBR LFA-1/2 CBR LFA-1/2 Open 44% Closed 56% Closed 48% Open 52% Noritaki Nishida, Can Xie, Tom Walz, Tim Springer

  27. Arg-Gly-Asp-mimetic antagonist to aIIbb3integrin What is the effect of Integrin antagonists directed to the b I domain MIDAS? tirofiban Allosteric antagonist to integrins aLb2 and aXb2 XVA143

  28. Effect of a/b I-like allosteric antagonist XVA143 (Drug) Extended, closed 54% Open 23% Compact 23% Bent >95% CBR LFA-1/2 + KIM127 Open 49% Closed 51% 10mM Drug 10mM Drug Bent 60% Extended, open 40% Extended, open >95% Leg Irons Cleaved Leg Irons CBR LFA-1/2 CBR LFA-1/2 Open 44% Closed 56% Closed 48% Open 52% Noritaki Nishida, Can Xie, Tom Walz, Tim Springer

  29. Similar results with aLb2, different equilibria set points Leg Irons Leg Irons Cleaved

  30. I domain displacement from the membrane

  31. Integrin Signalling • The conformation of integrins is regulated both by signaling/cytoskeletal molecules such as talin inside the cell (inside-out 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.

  32. Model for Receptor Signaling outside-in outside-in inside-out 3. Active dimer stabilized by bound ligand 2. Active dimer 1. Inactive dimer Ectodomain Transmembrane Juxtamembrane Cytoplasmic domain

  33. Collaborators Noritaka Nishida Can Xie Tom Walz - Harvard Med Sch Tsan Xiao Jun Takagi - Osaka U Motomu Shimaoka - Harvard Med Sch Jia-huai Wang - DFCI Minsoo Kim - Brown Univ Chris Carman Bing-Hao Luo Wei Yang http://cbr.med.harvard.edu/springer

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