Dynamic Control of Adhesion and Migration by Integrins

transcript

Model for Receptor Signaling

outside-in

inside-out

outside-in

The 24 Vertebrate Integrin ß HeterodimersIntegrin Therapeutics: Antibodies

Efiluzimab Psoriasis

10*11*

ß1 ß

*: subunits that contain I domains

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*

ß1 ß

Epifibatide Tirofiban

Thrombosis10*11*

ß1 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

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T lymphocyte migrating using integrin L2 on ICAM-1

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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

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

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

Inside-outsignaling

IL I*L

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

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.

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

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

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

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

subunithybrid

domain

I domain

Allostery in Integrin I and I domains

Low affinity

High affinity

I domain

A spring pull model for I domain activation

Upperleg

Lower leg

subunit subunit

I-propeller I

domain I

domain

I domain

Second site reversion supports the model

I domain

Upper legs

Transmembrane /Cytoplasmic Domain

433 nm

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

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

Dynamic Control of Adhesion and Migration by Integrins

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