Model for Receptor Signaling

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Model for Receptor Signaling
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Integrin Therapeutics Antibodies
The 24 Vertebrate Integrin aß Heterodimers
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Efficacy of Antibody to LFA-1 in Psoriasis
Efalizumab (anti-integrin LFA-1) administered for
2 months
Before treatment
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Integrin Therapeutics Antibodies
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Integrin Therapeutics Small Molecules
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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

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T lymphocytes migrating to a chemattactant-filled
micropipette Integrin aLb2-mediated migration on
ICAM-1-bearing substrate
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T lymphocyte migrating using integrin aLb2 on
ICAM-1
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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.
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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.
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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.
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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.
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Mutant I domains and a ligand-mimetic,
conformation-specific Fab

• Binding of AL-57 requires Mg2
• AL-57 blocks ligand binding

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(No Transcript)
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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.
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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
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The equilibria for conformational change and
ligand binding are linked
L ligand I resting integrin I high affinity
integrin
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Integrin ectodomain crystal and EM structures in
high and low affinity conformations
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Integrin ectodomain crystal structures in high
and low affinity conformations
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Allostery in Integrin b I and a I domains
Low affinity High affinity
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A spring pull model for I domain activation
a I domain
a I domain
b I domain
b I domain
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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 3011720.
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
?
?
?
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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
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Conformational transitions in integrins with a I
domains aXb2 and aXb2
Leg Irons
Noritaka Nishida, Can Xie, Tom Walz, Tim Springer
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Conformational transitions in integrins with a I
domains aXb2 and aXb2
Negative stain EM averages of 5,000 to10,000
particles
Leg Irons
Noritaka Nishida, Can Xie, Tom Walz, Tim Springer
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What is the effect of antibodies to activation
epitopes on I-EGF modules 2 and 3 of b2?
KIM127 Epitope (Activation-dependent)
CBR LFA-1/2 Epitope (Activation-inducing)
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Effect of Fab to activation epitopes in I-EGF2
and 3 near bend in b2 leg
Leg Irons Cleaved
Leg Irons
CBR LFA-1/2
CBR LFA-1/2
Open 44
Open 52
Closed 48
Closed 56
Noritaki Nishida, Can Xie, Tom Walz, Tim Springer
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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
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Effect of a/b I-like allosteric antagonist XVA143
(Drug)
Leg Irons Cleaved
Leg Irons
CBR LFA-1/2
CBR LFA-1/2
Open 44
Open 52
Closed 48
Closed 56
Noritaki Nishida, Can Xie, Tom Walz, Tim Springer
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Similar results with aLb2, different equilibria
set points
Leg Irons
Leg Irons Cleaved
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I domain displacement from the membrane
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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.

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Model for Receptor Signaling
Ectodomain
Transmembrane
Juxtamembrane
Cytoplasmic domain
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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

Noritaka Nishida Can Xie Tom Walz - Harvard Med
Sch
http//cbr.med.harvard.edu/springer