Model for Receptor Signaling outside-in inside-out outside-in
Integrin Therapeutics: Antibodies The 24 Vertebrate Integrin   ß Heterodimers Efiluzimab Psoriasis  10 *  11 *  3  2 ...
Efficacy of Antibody to LFA-1 in Psoriasis Before treatment Efalizumab (anti-integrin LFA-1) administered for 2 months
Integrin Therapeutics: Antibodies Efiluzimab Psoriasis Abciximab Thrombosis Nataluzimab Multiple Sclerosis  10 *  11 * ...
Integrin Therapeutics: Small Molecules Epifibatide Tirofiban Thrombosis  10 *  11 *  3  2 *  4  5  6  7  8  9 ...
The cast of cell surface adhesion molecules <ul><li>Integrin   L  2, LFA-1 (lymphocyte-function associated antigen-1) </...
T lymphocytes migrating to a chemattactant-filled micropipette: Integrin   L  2-mediated migration on ICAM-1-bearing sub...
T lymphocyte migrating using integrin   L  2  on ICAM-1
C-terminal helix displacement activates high affinity of   I domain of integrin   L  2 Shimaoka, M., Xiao, T., Takagi,...
C-terminal helix displacement activates high affinity of   I domain of integrin   L  2 Shimaoka, M., Xiao, T., Takagi,...
C-terminal helix displacement activates high affinity of   I domain of integrin   L  2 Shimaoka, M., Xiao, T., Takagi,...
C-terminal helix displacement activates high affinity of   I domain of integrin   L  2 Shimaoka, M., Xiao, T., Takagi,...
Mutant I domains and a ligand-mimetic, conformation-specific Fab <ul><li>Binding of AL-57 requires Mg 2+ </li></ul><ul><li...
Migrating T lymphocytes express high affinity LFA-1 in the lamellipodium Red : non-conformation-dependent Ab to LFA-1.  Gr...
T lymphocytes recognizing antigen on dendritic cells form an immunological synapse containing high-affinity LFA-1 Red : no...
Inside-out signaling by integrin cell adhesion receptors ICAM White cell Interacting cell Foreignness recognition Activati...
L: ligand  I: resting integrin I*: high affinity integrin The equilibria for conformational change and ligand binding are ...
Integrin ectodomain crystal and EM structures in high and low affinity conformations Schematic of low affinity  V  3 cr...
Integrin ectodomain crystal structures in high and low affinity conformations  subunit    subunit Thigh Comparison of hi...
Allostery in Integrin   I and    I domains Low affinity High affinity  subunit  hybrid domain  I domain  1  7 ...
A spring pull model for I domain activation    I domain    I domain    I domain    I domain Head Upper leg Lower leg ...
Cytoplasmic and transmembrane domain separation is associated with integrin activation Head Upper legs Transmembrane / Cyt...
Conformational transitions in integrins with    I domains:   X  2  and    X  2 Leg Irons Noritaka Nishida, Can Xie, T...
Conformational transitions in integrins with    I domains:   X  2  and    X  2 Leg Irons Noritaka Nishida, Can Xie, T...
What is the effect of antibodies to activation epitopes on I-EGF modules 2 and 3 of   2? KIM127 Epitope (Activation-depen...
Effect of Fab to activation epitopes in I-EGF2 and 3 near bend in   2  leg CBR LFA-1/2 CBR LFA-1/2 Open 44% Open 52% Clos...
Arg-Gly-Asp-mimetic antagonist to   IIb  3 integrin tirofiban Allosteric antagonist to integrins   L  2  and   X  2 ...
Effect of    I-like allosteric antagonist XVA143 (Drug) CBR LFA-1/2 CBR LFA-1/2 Open 44% Open 52% Closed 48% Closed 56%...
Similar results with   L  2, different equilibria set points Leg Irons Leg Irons Cleaved
I domain displacement from the membrane
Integrin Signalling <ul><li>The conformation of integrins is regulated both by signaling/cytoskeletal molecules such as ta...
Model for Receptor Signaling Ectodomain Transmembrane Juxtamembrane Cytoplasmic domain inside-out outside-in 3. Active dim...
Collaborators <ul><li>Tsan Xiao </li></ul><ul><li>Jun Takagi - Osaka U </li></ul><ul><li>Motomu Shimaoka - Harvard Med Sch...
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Dynamic Control of Adhesion and Migration by Integrins

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  • Many of you are familiar with what I call classical, or outside-in signaling through receptors. Today I am going to be talking about adhesion molecules that can not only signal from their extracellular to their cytoplasmic domains, but can also signal from their cytoplasmic to their ectodomains, in what we call inside-out signaling. I hope to convince you that for integrins, we understand
  • Integrins are very important therapeutic targets. I already mentioned that
  • More than 2 million patients in the US have psoriasis. Both drugs are giving very significant relief to patients, and greatly improving their quality of life. Very few basic research discoveries are translated into drugs, and to have this happen two times in 2003 made this a very exciting year for me. However, it was also sobering that from basic discovery to therapy took over 20 years in each case.
  • Integrins are very important therapeutic targets. I already mentioned that
  • Integrins are very important therapeutic targets. I already mentioned that
  • In contrast to the b I domain which has two conformational states, the a I domain has three conformational states . The one shown here is the closed, low affinity conformation. A one-turn downward displacement of the C-terminal a helix results in a conformation with intermediate affinity for ligand. A two-turn displacement alters the ligand binding site to the high affinity, open conformation. These alterations adjust the position of the Mg ion and other sidechains for optimal binding to ICAM-1. Our crystal structure of the I domain in complex with ICAM-1, shown here, explains the requirement for Mg for lymphocyte adhesion to other cells that led us many years ago to start searching for and to identify adhesion molecules of the immune system.
  • 9/04/00
  • The ligand binding face of the I domain would be 230 to 240 A away from the cell surface, in contrast to 0 to 60 A distance in the bent conformation.
  • Many of you are familiar with what I call classical, or outside-in signaling through receptors. Today I am going to be talking about adhesion molecules that can not only signal from their extracellular to their cytoplasmic domains, but can also signal from their cytoplasmic to their ectodomains, in what we call inside-out signaling. I hope to convince you that for integrins, we understand
  • Dynamic Control of Adhesion and Migration by Integrins

    1. 1. Model for Receptor Signaling outside-in inside-out outside-in
    2. 2. Integrin Therapeutics: Antibodies The 24 Vertebrate Integrin  ß Heterodimers Efiluzimab Psoriasis  10 *  11 *  3  2 *  4  5  6  7  8  9  * ß1 ß  ß5 ß6 ß8  V  IIb ß   * * :   subunits that contain I domains  L*  D*  M*  X* ß2  L *  D *  M *  X * ß 
    3. 3. Efficacy of Antibody to LFA-1 in Psoriasis Before treatment Efalizumab (anti-integrin LFA-1) administered for 2 months
    4. 4. Integrin Therapeutics: Antibodies Efiluzimab Psoriasis Abciximab Thrombosis Nataluzimab Multiple Sclerosis  10 *  11 *  3  2 *  4  5  6  7  8  9  * ß1 ß  ß5 ß6 ß8  V  IIb ß   * * :   subunits that contain I domains  L*  D*  M*  X* ß2  L *  D *  M *  X * ß 
    5. 5. Integrin Therapeutics: Small Molecules Epifibatide Tirofiban Thrombosis  10 *  11 *  3  2 *  4  5  6  7  8  9  * ß1 ß  ß5 ß6 ß8  V  IIb ß   * * :   subunits that contain I domains  L*  D*  M*  X* ß2  L *  D *  M *  X * ß   I allosteric antagonists  I-like allosteric antagonists
    6. 6. The cast of cell surface adhesion molecules <ul><li>Integrin  L  2, LFA-1 (lymphocyte-function associated antigen-1) </li></ul><ul><li>Integrin  X  2 </li></ul><ul><li>Their ligand, ICAM-1 (intercellular adhesion molecule-1), contains 5 IgSF domains </li></ul><ul><li>Integrins  V  3,  IIb  3,  5  1, which lack  I domains, and bind ligands with Arg-Gly-Asp (RGD) motifs </li></ul>
    7. 7. T lymphocytes migrating to a chemattactant-filled micropipette: Integrin  L  2-mediated migration on ICAM-1-bearing substrate
    8. 8. T lymphocyte migrating using integrin  L  2 on ICAM-1
    9. 9. 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.
    10. 10. 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.
    11. 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. 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. 13. Mutant I domains and a ligand-mimetic, conformation-specific Fab <ul><li>Binding of AL-57 requires Mg 2+ </li></ul><ul><li>AL-57 blocks ligand binding </li></ul>
    14. 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. 15. 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 T cell
    16. 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. 17. L: ligand I: resting integrin I*: high affinity integrin The equilibria for conformational change and ligand binding are linked I I* Inside-out signaling IL I*L +L +L Ligand binding
    18. 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-RGD resting  V  3 Takagi et al, Cell (2002) Takagi et al, EMBO J (2003)  5  1 head  5  1 head + Fn7-10
    19. 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. 20. Allostery in Integrin  I and  I domains Low affinity High affinity  subunit  hybrid domain  I domain  1  7  I domain  1  7
    21. 21. A spring pull model for I domain activation  I domain  I domain  I domain  I domain Head Upper leg Lower leg  subunit  subunit  I  I  -propeller Second site reversion supports the model  I domain  I domain  I domain  I domain  I domain  I domain
    22. 22. Cytoplasmic and transmembrane domain separation is associated with integrin activation Head Upper legs Transmembrane / Cytoplasmic Domain 433 nm FRET mCFP mYFP 527 nm 433 nm mCFP 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 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. 23. Conformational transitions in integrins with  I domains:  X  2 and  X  2 Leg Irons Noritaka Nishida, Can Xie, Tom Walz, Tim Springer
    24. 24. Conformational transitions in integrins with  I domains:  X  2 and  X  2 Leg Irons Noritaka Nishida, Can Xie, Tom Walz, Tim Springer Negative stain EM averages of 5,000 to10,000 particles Compact 23% Extended, closed 54% Open 23% Leg Irons Cleaved Bent >95%
    25. 25. What is the effect of antibodies to activation epitopes on I-EGF modules 2 and 3 of  2? KIM127 Epitope (Activation-dependent) CBR LFA-1/2 Epitope (Activation-inducing) Beglova, Blacklow, Takagi, Springer Nat. Struct. Biol. 2002.
    26. 26. Effect of Fab to activation epitopes in I-EGF2 and 3 near bend in  2 leg CBR LFA-1/2 CBR LFA-1/2 Open 44% Open 52% Closed 48% Closed 56% Leg Irons Leg Irons Cleaved Noritaki Nishida, Can Xie, Tom Walz, Tim Springer CBR LFA-1/2 + KIM127 Open 49% Closed 51% Bent >95% Compact 23% Extended, closed 54% Open 23% CBR LFA-1/2 + KIM127 Open 49% Closed 51%
    27. 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. 28. Effect of  I-like allosteric antagonist XVA143 (Drug) CBR LFA-1/2 CBR LFA-1/2 Open 44% Open 52% Closed 48% Closed 56% Noritaki Nishida, Can Xie, Tom Walz, Tim Springer Leg Irons Leg Irons Cleaved CBR LFA-1/2 + KIM127 Open 49% Closed 51% 10  M Drug Extended, open 40% Bent 60% 10  M Drug Extended, open >95% Bent >95% Compact 23% Extended, closed 54% Open 23%
    29. 29. Similar results with  L  2, different equilibria set points Leg Irons Leg Irons Cleaved
    30. 30. I domain displacement from the membrane
    31. 31. Integrin Signalling <ul><li>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. </li></ul><ul><li>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. </li></ul><ul><li> I domain conformation and affinity for ligand is linked to  I domain conformation. </li></ul><ul><li>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. </li></ul>
    32. 32. Model for Receptor Signaling Ectodomain Transmembrane Juxtamembrane Cytoplasmic domain inside-out outside-in 3. Active dimer stabilized by bound ligand 2. Active dimer 1. Inactive dimer outside-in
    33. 33. Collaborators <ul><li>Tsan Xiao </li></ul><ul><li>Jun Takagi - Osaka U </li></ul><ul><li>Motomu Shimaoka - Harvard Med Sch </li></ul><ul><li>Jia-huai Wang - DFCI </li></ul><ul><li>Minsoo Kim - Brown Univ </li></ul><ul><li>Chris Carman </li></ul><ul><li>Bing-Hao Luo </li></ul><ul><li>Wei Yang </li></ul>http://cbr.med.harvard.edu/springer Noritaka Nishida Can Xie Tom Walz - Harvard Med Sch
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