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News and Publications
The Molecular Basis of Integrin Signaling in Hematopoietic and
Vascular Cells
S.J. Shattil, A. Bertoni, C. Buensuceso, K. Eto, M. de Virgilio,
E. Garcia Arias-Salgado, A. Kasirer-Friede, B. Moran, R. Murphy,
A. Obergfell, S. Tadokoro
Integrin adhesion receptors transfer information between the extracellular
and intracellular environments of cells at sites on the plasma membrane
referred to as focal complexes and focal adhesions. The general
term "integrin signaling" is often used to refer to this process.
The term encompasses "inside-out" signals that regulate integrin
affinity and avidity for matrix ligands and "outside-in" signals
that regulate anchorage-dependent cellular responses, such as motility
and gene expression.
Our goal is to unravel the molecular basis of integrin signaling
as it pertains to hematopoietic and vascular cells, particularly
(1) platelets and their precursor megakaryocytes and (2) endothelial
cells. Indeed, integrins are required for proliferation and differentiation
of stem cells and hematopoietic progenitors into megakaryocytes,
for platelet function in hemostasis and thrombosis, and for the
participation of endothelial cells in wound healing and angiogenesis.
One current challenge is to integrate the wealth of information
obtained from studies of human blood and vascular cells with that
obtained from studies of gene-targeted or mutant mice to yield a
complete understanding of the protein-protein interactions involved
in integrin signaling.
An interesting form of inside-out signaling in platelets is related
to the relationship between integrin aIIbb3
and the nonintegrin adhesion receptor glycoprotein Ib-IX (GPIb-IX).
Von Willebrand factor (VWF) is a modular adhesive protein with specific
binding sites for both GPIb-IX and aIIbb3.
The binding of VWF to GPIb-IX under conditions of blood flow mediates
initial adhesion of platelets to the walls of damaged blood vessels.
In addition, binding of GPIb-IX by VWF appears to increase the adhesive
function of aIIbb3. Because GPIb-IX
responses are promoted most effectively by large VWF multimers,
we hypothesized that the function of GPIb-IX is modulated by clustering
of this receptor within the plane of the plasma membrane.
To test this hypothesis, GPIX was fused at its cytoplasmic tail
to tandem repeats of the dimerization motif FKBP, and GPIb-IX(FKBP)2
and aIIbb3 were expressed
in CHO cells, long used as a model system to study aIIbb3.
The behavior of these cells under conditions of flow was examined
in collaborative studies with Z. Ruggeri and J. Ware, Department
of Molecular and Experimental Medicine. At wall shear rates up to
2000 s-1, GPIb-IX(FKBP)2 mediated cell tethering
on immobilized VWF, just as in platelets. Conditional oligomerization
of GPIb-IX(FKBP)2 by AP20187, a cell-permeable FKBP dimerizer,
caused a decrease in cell translocation velocities on VWF. Moreover,
clustering of GPIb-IX(FKBP)2 by AP20187 led to an increase
in the function of aIIbb3, manifested under
static conditions by increased cell adhesion to the aIIbb3-specific
ligand fibrinogen and under flow by increased stable cell adhesion
to VWF. Conditional clustering of GPIb-IX(FKBP)2 also
stimulated rapid tyrosine phosphorylation of ectopically expressed
Syk tyrosine kinase, a putative downstream effector of GPIb-IX in
platelets.
These studies established that GPIb-IX oligomerization per se
affects the interaction of this receptor with VWF and the ability
of GPIb-IX to influence the adhesive function of aIIbb3.
By extrapolation, GPIb-IX clustering in platelets may promote formation
of thrombus.
Integrins regulate cell adhesion and motility through nonreceptor
tyrosine kinases, but initiation of this process is poorly understood.
In particular, the precise mechanisms whereby integrin ligation
activates Src family kinases remains a mystery. We found that Src
associates constitutively with integrin aIIbb3
in human and murine platelets. Platelet adhesion to fibrinogen or
binding of soluble fibrinogen to platelets via aIIbb3
caused a rapid increase in aIIbb3-associated
Src activity, and active Src localized to the filopodia and edges
of adherent platelets. Another tyrosine kinase, Csk, which can negatively
regulate Src by phosphorylating Tyr529, was also constitutively
associated with aIIbb3. However, fibrinogen
binding caused Csk to dissociate from aIIbb3,
concomitant with dephosphorylation of Src Tyr529 and phosphorylation
of Src activation loop Tyr418.
In contrast to the behavior of Src and Csk, the tyrosine kinase
Syk was associated with aIIbb3 only after fibrinogen
binding. Murine platelets multiply deficient in Src, Hck, Fgr, and
Lyn or normal human platelets treated with Src kinase inhibitors
did not spread on fibrinogen. Furthermore, inhibition of Src kinases
blocked Syk activation and inhibited phosphorylation of Syk substrates
implicated in cytoskeletal regulation. Syk-deficient murine platelets
had Src activation upon adhesion to fibrinogen but no spreading
or phosphorylation of Syk substrates. These studies establish that
platelet spreading on fibrinogen requires sequential activation
of Src and Syk in proximity to aIIbb3,
thus providing a model for Src activation by integrins and initiation
of integrin signaling to the actin cytoskeleton.
PUBLICATIONS
Baron, W., Shattil, S.J., ffrench-Constant, C. The oligodendrocyte
precursor mitogen PDGF stimulates proliferation by activation of
avb3 integrins. EMBO
J. 21:1957, 2002.
Derrick, J.M., Shattil, S.J., Poncz, M., Gruppo, R.A., Gartner,
T.K. Distinct domains of aIIbb3
support different aspects of outside-in signal transduction and
platelet activation induced by LSARLAF, an aIIbb3
interacting peptide. Thromb. Haemost. 86:894, 2001.
Hato, T., Ginsberg, M.H., Shattil, S.J. Integrin aIIbb3 and platelet aggregation. In: Platelets.
Michelson, A.D. (Ed.). Academic Press, San Diego, in press.
Judd, B.A., Myung, P.S., Obergfell, A., Myers, E.E., Chung,
A.M., Watson, S.P., Pear, W.S., Allman, D., Shattil, S.J., Koretzky,
G.A. Differential requirement for LAT and SLP-76 in GPVI versus
T cell receptor signaling. J. Exp. Med. 195:705, 2002.
Kasirer-Friede, A., Ware, J., Leng, L., Marchese, P., Ruggeri,
Z., Shattil, S.J. Lateral clustering of GP Ib-IX complexes leads
to up-regulation of the adhesive function of integrin aIIbb3.
J. Biol. Chem. 277:11949, 2002.
Obergfell, A., Eto, K., Mocsai, A., Buensuceso, C., Moores,
S.L., Brugge, J.S., Lowell, C.A., Shattil, S.J. Coordinate interactions
of Csk, Src and Syk kinases with aIIbb3
initiate integrin signaling to the cytoskeleton. J. Cell Biol. 157:265,
2002.
Tomiyama, Y., Shiraga, M., Shattil, S.J. Platelet membrane
proteins as adhesion receptors. In: Platelets in Thrombotic
and Nonthrombotic Disorders: Pathophysiology, Pharmacology and Therapeutics.
Gresele, P., et al. (Eds.). Cambridge University Press, New York,
in press.
Tzima, E., del Pozo, M.A., Shattil, S.J., Chien, S., Schwartz,
M.A. Activation of integrins in endothelial cells by fluid shear
stress mediates Rho-dependent cytoskeletal alignment. EMBO J. 20:4639,
2001.
Wonerow, P., Obergfell, A., Wilde, J., Bobe, R., Asazuma, N.,
Brdicka, T., Leo, A., Schraven, B., Horeji, V., Shattil, S.J., Watson,
S. Differential role of glycolipid-enriched membrane domains
in glycoprotein VI- and integrin-mediated phospholipase Cg2 activation in platelets. Biochem.
J. 364:755, 2002.
Woodside, D.G., Obergfell, A., Leng, L., Wilsbacher, J.L.,
Miranti, C.K., Brugge, J.S., Shattil, S.J., Ginsberg, M.H. Activation
of Syk protein tyrosine kinase through interaction with integrin
b cytoplasmic domains.
Curr. Biol. 11:1799, 2001.
Woodside, D.G., Shattil, S.J., Ginsberg, M.H. The T cell
receptor SLAPS integrins together. Nat. Immunol. 2:904, 2001.
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