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

2. The 24 Vertebrate Integrin  ß Heterodimers Integrin Therapeutics: Antibodies Efiluzimab Psoriasis  10*  11* 33 2*2* 44 55 66 77 88 99  * ß1 ßß ß5 ß6 ß8 VV  IIb ßß  * * :   subunits that contain I domains  L*  D*  M*  X* ß2 L*L* D*D* M*M* X*X* ßß

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

4. Integrin Therapeutics: Antibodies Efiluzimab Psoriasis Abciximab Thrombosis Nataluzimab Multiple Sclerosis  10*  11* 33 2*2* 44 55 66 77 88 99  * ß1 ßß ß5 ß6 ß8 VV  IIb ßß  * * :   subunits that contain I domains  L*  D*  M*  X* ß2 L*L* D*D* M*M* X*X* ßß

5. Epifibatide Tirofiban Thrombosis  10*  11* 33 2*2* 44 55 66 77 88 99  * ß1 ßß ß5 ß6 ß8 VV  IIb ßß  * * :   subunits that contain I domains  L*  D*  M*  X* ß2 L*L* D*D* M*M* X*X* ßß  I allosteric antagonists  I-like allosteric antagonists Integrin Therapeutics: Small Molecules

6. The cast of cell surface adhesion molecules Integrin  L  2, LFA-1 (lymphocyte-function associated antigen-1) Integrin  X  2 Their ligand, ICAM-1 (intercellular adhesion molecule-1), contains 5 IgSF domains Integrins  V  3,  IIb  3,  5  1, which lack  I domains, and bind ligands with Arg-Gly-Asp (RGD) motifs

7. T lymphocytes migrating to a chemattactant-filled micropipette: Integrin  L  2-mediated migration on ICAM-1-bearing substrate

8. T lymphocyte migrating using integrin  L  2 on ICAM-1

9. 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. C-terminal helix displacement activates high affinity of  I domain of integrin  L  2

10. 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  I domain of integrin  L  2 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  I domain of integrin  L  2 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 Mg 2+ 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 ICAM White cell Interacting cell Foreignness recognition Activation signal recognition Integrin inside-out signaling Binding to ligand (ICAM) Intracellular signals talin binding Integrin outside-in signaling

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

18. Integrin ectodomain crystal and EM structures in high and low affinity conformations Schematic of low affinity  V  3 crystal structure Upper legs Lower legs Head 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.  I  V  3 + cyclo-RGDresting  V  3 Takagi et al, Cell (2002) Takagi et al, EMBO J (2003)  5  1 head  5  1 head + Fn7-10

19. Integrin ectodomain crystal structures in high and low affinity conformations  subunit  subunit Thigh Comparison of high and low affinity headpiece conformations Swung-in hybrid domain, low affinity, closed headpiece Swung-out hybrid domain, high affinity, open headpiece  -propeller  I 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. Ribbon diagram of high affinity  IIb  3 headpiece crystal structure  -propeller  I Hybrid PSI  subunit  subunit Ligand Xiao, T., Takagi, J., Wang, J.-h., Coller, B. S., and Springer, T. A. Nature 432, 59- 67. Schematic of low affinity  V  3 crystal structure Upper legs Lower legs Head  I

20.  subunit  hybrid domain  I domain 11 77 Allostery in Integrin  I and  I domains Low affinity High affinity  I domain 11 77

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

22. Head Upper legs Transmembrane / Cytoplasmic Domain 433 nm FRET mCFPmYFP 527 nm433 nm mCFPmYFP 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 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. Lower legs 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.

23. Conformational transitions in integrins with  I domains:  X  2 and  X  2 Leg Irons Noritaka Nishida, Can Xie, Tom Walz, Tim Springer

24. Compact 23% Extended, closed 54%Open 23% Leg Irons Cleaved Conformational transitions in integrins with  I domains:  X  2 and  X  2 Bent >95% Leg Irons Noritaka Nishida, Can Xie, Tom Walz, Tim Springer Negative stain EM averages of 5,000 to10,000 particles

25. What is the effect of antibodies to activation epitopes on I-EGF modules 2 and 3 of  2? 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  2 leg CBR LFA-1/2 Open 44% Open 52%Closed 48% CBR LFA-1/2 + KIM127 Open 49% Closed 51% Closed 56% Bent >95% Compact 23% Extended, closed 54%Open 23% Leg Irons Leg Irons Cleaved Noritaki Nishida, Can Xie, Tom Walz, Tim Springer CBR LFA-1/2 + KIM127 Open 49% Closed 51%

27. Arg-Gly-Asp-mimetic antagonist to  IIb  3 integrin tirofiban Allosteric antagonist to integrins  L  2 and  X  2 XVA143 What is the effect of Integrin antagonists directed to the  I domain MIDAS?

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

29. Leg IronsLeg Irons Cleaved Similar results with  L  2, different equilibria set points

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.  I domain conformation and affinity for ligand is linked to  I domain conformation. Small changes in  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  and  subunit TM and cytoplasmic domains.

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

33. Collaborators 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 Noritaka Nishida Can Xie Tom Walz - Harvard Med Sch