Following vascular injury, adhesive ligands such as fibrinogen and von Willebrand factor engage integrin allb/?3 to effect platelet aggregation and spreading during hemostasis and thrombosis. These responses are triggered by ligand-mediated allb/?3 clustering, which initiates """"""""outside-in"""""""" signals to reorganize the actin cytoskeleton. Recent work from this project has established that outside-in signaling in platelets requires Src family tyrosine kinases (SFKs), c-Src in particular, which bind to /?3 and are activated by allb/?3 clustering in a manner dependent on PTP-1B, a protein tyrosine phosphatase. Here, three major unresolved questions will be asked concerning the molecular basis of outside-in signaling in platelets and its biological consequences. First, do direct interactions between integrins and SFKs represent a general mechanism for spatio-temporal initiation of outside-in signaling in platelets? Since platelets contain five different integrins and at least six different SFKs, this possibility will be evaluated by co-immunoprecipitation techniques, by bimolecular fluorescence complementation imaging in live cells, and by localization of Src activation in live cells using a FRET-based reporter. In addition, integrin/SFK interactions will be assessed in Drosophila cells to determine the extent to which direct activation of SFKs by integrins is an evolutionarily conserved process. Second, how does PTP-1B activate c-Src downstream of integrins? The mechanism by which PTP-1B is recruited to the allb/?3/c-Src complex, and possibly to other integrin/SFK complexes, will be evaluated in platelets and model cell systems, focusing on the possible role of adapter proteins. In addition, the effect of integrin clustering on PTP-1B catalytic activity will be determined. Third, does selective disruption of outsidein signaling affect thrombus formation in vivo? Here arterial thrombosis will be studied in novel gene-targeted mice predicted to have selective defects in the interaction of c/llb/83 with c-Src or other SFKs, or defects in downstream events required for actin reorganization. Altogether, these studies will provide molecular insights into how outside-in integrin signaling is initiated and establish the extent to which this process regulates platelet function in vivo. Thus, this line of investigation may lead to identification of new anti-thrombotic drug targets and serve as a paradigm for integrin signaling in other blood cells.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Program Projects (P01)
Project #
5P01HL057900-15
Application #
8213150
Study Section
Heart, Lung, and Blood Initial Review Group (HLBP)
Project Start
Project End
Budget Start
2011-02-01
Budget End
2012-01-31
Support Year
15
Fiscal Year
2011
Total Cost
$386,250
Indirect Cost
Name
University of California San Diego
Department
Type
DUNS #
804355790
City
La Jolla
State
CA
Country
United States
Zip Code
92093
Wilson, RaeAnna; Espinosa-Diez, Cristina; Kanner, Nathan et al. (2016) MicroRNA regulation of endothelial TREX1 reprograms the tumour microenvironment. Nat Commun 7:13597
Liao, Zhongji; Kato, Hisashi; Pandey, Manjula et al. (2015) Interaction of kindlin-2 with integrin ?3 promotes outside-in signaling responses by the ?V?3 vitronectin receptor. Blood 125:1995-2004
Desgrosellier, Jay S; Lesperance, Jacqueline; Seguin, Laetitia et al. (2014) Integrin ?v?3 drives slug activation and stemness in the pregnant and neoplastic mammary gland. Dev Cell 30:295-308
Fitzpatrick, Paul; Shattil, Sanford J; Ablooglu, Ararat J (2014) C-terminal COOH of integrin ?1 is necessary for ?1 association with the kindlin-2 adapter protein. J Biol Chem 289:11183-93
Seguin, Laetitia; Kato, Shumei; Franovic, Aleksandra et al. (2014) An integrin ??-KRAS-RalB complex drives tumour stemness and resistance to EGFR inhibition. Nat Cell Biol 16:457-68
Casar, B; Rimann, I; Kato, H et al. (2014) In vivo cleaved CDCP1 promotes early tumor dissemination via complexing with activated ?1 integrin and induction of FAK/PI3K/Akt motility signaling. Oncogene 33:255-68
Ye, Feng; Petrich, Brian G; Anekal, Praju et al. (2013) The mechanism of kindlin-mediated activation of integrin ?IIb?3. Curr Biol 23:2288-95
Cantor, Joseph M; Ginsberg, Mark H (2012) CD98 at the crossroads of adaptive immunity and cancer. J Cell Sci 125:1373-82
Scheppke, Lea; Murphy, Eric A; Zarpellon, Alessandro et al. (2012) Notch promotes vascular maturation by inducing integrin-mediated smooth muscle cell adhesion to the endothelial basement membrane. Blood 119:2149-58
Banno, Asoka; Goult, Benjamin T; Lee, HoSup et al. (2012) Subcellular localization of talin is regulated by inter-domain interactions. J Biol Chem 287:13799-812

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