Verbatim): Members of the integrin family of receptors play a critical role in inducing adhesion and are involved in numerous events that require integrin-induccd changes in cell behavior. For example, signaling across integrins is involved in angiogenesis, development, metastasis, inflammation, wound healing, and hemostasis. The overall goal of our research is to understand the signaling pathways leading to integrin activation and to integrin-induced changes in cell behavior. Our work focuses on the importance of membrane skeleton proteins and associated signaling molecules in regulating the two-way signaling across integrins. Studies over the past funding period have provided evidence that a subpopulation of a11bB3 in platelets is associated with membrane skeleton complexes containing spectrin, talin, pp60c-src, and calpain. As platelets or cultured cells spread, these proteins are incorporated into integrin clusters by a mechanism that requires calpain activity. Rac is subsequently activated by a mechanism that can be inhibited by spectrin's SH3 domain. Our findings are consistent with a model in which integrin engagement causes activation of calpain. Calpain cleaves several proteins including spectrin and the beta3 integrin subunit, allowing integrin that is otherwise restrained by the cytoskeleton to cluster into focal complexes. Signaling transmitted across these complexes, by a mechanism involving spectrin, leads to activation of Rac. We have identified skelemin as a new protein that can interact with the beta3 cytoplasmic domain and shown that it is present in the integrin clusters. Experiments in the present proposal are designed to test the hypothesis that association of alIbbeta3 with the complexes of membrane skeleton proteins and signaling molecules plays a critical role in allowing two-way signaling across the integrin.
The Specific Aims are 1) to test the hypothesis that calpain is involved in Rac activation; 2) to test the hypothesis that the SH3 domain of spectrin is involved in Rac activation; 3) to test the hypothesis that interaction of skelemin with integrin cytoplasmic domains regulates two-way signaling across integrins. It is anticipated that these studies will provide information on mechanisms involved in two-way signaling across integnns. Further, the studies may lead to an increased understanding of key mechanisms involved in integrin-mediated functions such as platelet adhesion and aggregation, wound healing, angiogenesis, inflammation, metastasis, and cell migration.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL056264-07
Application #
6537260
Study Section
Hematology Subcommittee 2 (HEM)
Program Officer
Ganguly, Pankaj
Project Start
1995-08-04
Project End
2005-03-31
Budget Start
2002-04-01
Budget End
2003-03-31
Support Year
7
Fiscal Year
2002
Total Cost
$333,000
Indirect Cost
Name
Cleveland Clinic Lerner
Department
Type
DUNS #
017730458
City
Cleveland
State
OH
Country
United States
Zip Code
44195
O'Toole, Timothy E; Bialkowska, Katarzyna; Li, Xiaohong et al. (2011) Tiam1 is recruited to ?1-integrin complexes by 14-3-3? where it mediates integrin-induced Rac1 activation and motility. J Cell Physiol 226:2965-78
Bialkowska, Katarzyna; Saido, Takaomi C; Fox, Joan E B (2005) SH3 domain of spectrin participates in the activation of Rac in specialized calpain-induced integrin signaling complexes. J Cell Sci 118:381-95
Bialkowska, Katarzyna; Zaffran, Yona; Meyer, Sylvie C et al. (2003) 14-3-3 zeta mediates integrin-induced activation of Cdc42 and Rac. Platelet glycoprotein Ib-IX regulates integrin-induced signaling by sequestering 14-3-3 zeta. J Biol Chem 278:33342-50
Austin, Richard C; Fox, Joan E B; Werstuck, Geoff H et al. (2002) Identification of Dp71 isoforms in the platelet membrane cytoskeleton. Potential role in thrombin-mediated platelet adhesion. J Biol Chem 277:47106-13
Kulkarni, Sucheta; Goll, Darrel E; Fox, Joan E B (2002) Calpain cleaves RhoA generating a dominant-negative form that inhibits integrin-induced actin filament assembly and cell spreading. J Biol Chem 277:24435-41
Fox, J E (2001) Cytoskeletal proteins and platelet signaling. Thromb Haemost 86:198-213
Reddy, K B; Bialkowska, K; Fox, J E (2001) Dynamic modulation of cytoskeletal proteins linking integrins to signaling complexes in spreading cells. Role of skelemin in initial integrin-induced spreading. J Biol Chem 276:28300-8
Kulkarni, S; Fox, J E (2000) Localization of calpain by immunofluorescence in adherent cells. Methods Mol Biol 144:151-9
Bialkowska, K; Kulkarni, S; Du, X et al. (2000) Evidence that beta3 integrin-induced Rac activation involves the calpain-dependent formation of integrin clusters that are distinct from the focal complexes and focal adhesions that form as Rac and RhoA become active. J Cell Biol 151:685-96
Zaffran, Y; Meyer, S C; Negrescu, E et al. (2000) Signaling across the platelet adhesion receptor glycoprotein Ib-IX induces alpha IIbbeta 3 activation both in platelets and a transfected Chinese hamster ovary cell system. J Biol Chem 275:16779-87

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