Model for Receptor Signaling
outside-in
inside-out
outside-in
The 24 Vertebrate Integrin ß HeterodimersIntegrin Therapeutics: Antibodies
Efiluzimab Psoriasis
10*11*
3
2*
4
5
6
78
9
*
ß1 ß
ß5
ß6
ß8
V
IIb
ß*
*: subunits that contain I domains
L*
D*
M*
X*
ß2
L*
D*
M*
X*
ß
Before treatmentBefore treatmentEfalizumab (anti-integrin LFA-1) Efalizumab (anti-integrin LFA-1)
administered for 2 monthsadministered for 2 months
Efficacy of Antibody to LFA-1 in Psoriasis
Integrin Therapeutics: Antibodies
Efiluzimab Psoriasis
Abciximab Thrombosis
NataluzimabMultiple Sclerosis
10*11*
3
2*
4
5
6
78
9
*
ß1 ß
ß5
ß6
ß8
V
IIb
ß*
*: subunits that contain I domains
L*
D*
M*
X*
ß2
L*
D*
M*
X*
ß
Epifibatide Tirofiban
Thrombosis10*11*
3
2*
4
5
6
78
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
Integrin Therapeutics: Small Molecules
The cast of cell surface adhesion molecules
• Integrin L2, LFA-1 (lymphocyte-function associated antigen-1)• Integrin X2• Their ligand, ICAM-1 (intercellular adhesion molecule-1), contains 5
IgSF domains
• Integrins V3, IIb3, 51, which lack I domains, and bind ligands with Arg-Gly-Asp (RGD) motifs
T lymphocytes migrating to a chemattactant-filled micropipette:Integrin L2-mediated migration on ICAM-1-bearing substrate
QuickTime™ and aH.263 decompressor
are needed to see this picture.
T lymphocyte migrating using integrin L2 on ICAM-1
QuickTime™ and aVideo decompressor
are needed to see this picture.
QuickTime™ and aSorenson Video 3 decompressorare needed to see this picture.
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 L2
QuickTime™ and aSorenson Video 3 decompressorare needed to see this picture.
C-terminal helix displacement activates high affinity of I domain of integrin L2
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.
QuickTime™ and aSorenson Video 3 decompressorare needed to see this picture.
C-terminal helix displacement activates high affinity of I domain of integrin L2
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.
QuickTime™ and aSorenson Video 3 decompressorare needed to see this picture.
C-terminal helix displacement activates high affinity of I domain of integrin L2
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.
Mutant I domains and a ligand-mimetic, conformation-specific Fab
I domain Mutation KD, ICAM-1 KD, AL-57 Fab KD, MHM24 FabWild-type none 1.5 mM Not detected 1.9 nMIntermediate affinity I161C/V299C 3,000 nM 4,700 nM 2.0 nMHigh affinity K297C/K294C 150 nM 23 nM 6.3 nM
• Binding of AL-57 requires Mg2+
• AL-57 blocks ligand binding
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Migrating T lymphocytes express high affinity LFA-1 in the lamellipodiumRed: non-conformation-dependent Ab to LFA-1. Green: AL-57 ligand-mimetic Ab.
T lymphocytes recognizing antigen on dendritic cells form an immunological synapse containing high-affinity LFA-1
Dendritic cell T cell
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Red: non-conformation-dependent Ab to LFA-1. Green: AL-57 ligand-mimetic Ab.
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
I I*
Inside-outsignaling
IL I*L
+L +L
Lig
and
bin
din
g
L: ligand I: resting integrinI*: high affinity integrin
The equilibria for conformational change and ligand binding are linked
Integrin ectodomain crystal and EM structures in high and low affinity conformations
Schematic of low affinityV3 crystal structure
Upper legs
Lo
we
r le
gs
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
V3 + cyclo-RGDresting V3
Takagi et al, Cell (2002)
Takagi et al, EMBO J (2003)
51 head
51 head + Fn7-10
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 IIb3 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 affinityV3 crystal structure
Upper legs
Lo
we
r le
gs
Head
I
subunithybrid
domain
I domain
1
7
Allostery in Integrin I and I domains
Low affinity
High affinity
I domain
1
7
A spring pull model for I domain activation
Head
Upperleg
Lower leg
subunit subunit
I
I-propeller I
domain I
domain
I domain
I domain
Second site reversion supports the model
I domain
I domain
I domain
I domain
I domain
I domain
Head
Upper legs
Transmembrane /Cytoplasmic Domain
433 nm
FRET
mCFP mYFP527 nm 433 nm
mCFP mYFP475 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.
Conformational transitions in integrins with I domains: X2 and X2
Leg Irons
Noritaka Nishida, Can Xie, Tom Walz, Tim Springer
Compact 23% Extended, closed 54% Open 23%
Leg Irons Cleaved
Conformational transitions in integrins with I domains: X2 and X2
Bent >95%
Leg Irons
Noritaka Nishida, Can Xie, Tom Walz, Tim Springer
Negative stain EM averages of 5,000 to10,000 particles
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)
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%
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%
Arg-Gly-Asp-mimetic antagonist to IIb3integrin
tirofiban
Allosteric antagonist to integrins L2 and X2
XVA143
What is the effect of Integrin antagonists directed to the I domain MIDAS?
CBR LFA-1/2+ KIM127
Open 49%Closed 51%
Effect of I-like allosteric antagonist XVA143 (Drug)
CBR LFA-1/2 CBR LFA-1/2
Open 44%Open 52%Closed 48% Closed 56%
10M Drug
Extended, open 40%Bent 60%
10M 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
Leg Irons Leg Irons Cleaved
Similar results with L2, different equilibria set points
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 (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.
Model for Receptor Signaling
inside-out
outside-in
3. Active dimer stabilized by bound ligand
2. Active dimer
1. Inactive dimer
Ectodomain
TransmembraneJuxtamembrane
Cytoplasmic domain
outside-in
Collaborators
Tsan XiaoJun Takagi - Osaka U
Motomu Shimaoka - Harvard Med SchJia-huai Wang - DFCI
Minsoo Kim - Brown UnivChris Carman
Bing-Hao LuoWei Yang
http://cbr.med.harvard.edu/springer
Noritaka NishidaCan Xie
Tom Walz - Harvard Med Sch