? ? The goal of the proposal is characterization of thrombin-mediated signaling pathways in platelets, a crucial component of the regulation of thrombosis and vascular disease. Thrombin signaling is mediated by a family of G-protein-coupled protease-activated receptors (PARs). Human platelets have been shown to express all three known thrombin receptors - PAR1, PAR3 and PAR4. Genomic analysis of the PAR cluster on chromosome 5813 identified the gene encoding IQGAP2, a putative Rho GTPase-activating protein (GAP), in close proximity to the PAR1 gene. GAPs have been shown to be required for many actin-dependent cellular processes, such as lymphocyte and fibroblast adhesion, cell motility, contraction, and cytokinesis. Preliminary data demonstrated IQGAP2 expression in human and murine platelets. Fluorescent immunostaining showed IQGAP2 and actin co-localization in filopodia of activated platelets and lamellae of COS1 cells. Thrombin/TRAP activation of human platelets was shown to induce IQGAP/actin/arp2/3 complex assembly. This complex formation appeared to require GTP-bound GTPases racl or cdc42, and was not induced by collagen or ADP. Thus, the primary hypothesis to be tested in this proposal is that IQGAP2 is a unique effector protein, possibly functioning as a scaffolding protein involved in thrombin-mediated cytoskeletal actin reorganization. Completion of Specific Aim I will characterize efficiency of racl and cdc42 at binding IQGAP2 and the effects of GTP/GDP-charging on IQGAP2 interactions and activation. Also it will identify other IQGAP2-binding proteins involved in actin cytoskeletal assembly. The goal of Specific Aim II is dissection of the molecular mechanisms regulating IQGAP2 function in thrombin-mediated signaling, including evaluation of the roles of individual thrombin receptors (PART/PAR3/PAR4). Generation and characterization of an IQGAP2-deficient (IQGAP2 -/-) knockout mouse (Specific Aim III) will define the role of platelet IQGAP2 in vivo. Should a definitive role for IQGAP2 as a scaffolding protein involved in platelet thrombin-mediated signaling be established, this will be a first example of evolutionary developed functional genomic unit of structurally unrelated clustered genes in humans. A further understanding of the mechanisms involved in signaling pathways of platelet activation and aggregation may identify potential targets for intervention in thrombosis-associated disease processes, a common long-term complications in patients with diabetes. During the award period, the candidate will develop an increased knowledge of genetics and platelet biology in a laboratory that is well-equipped for the execution of this proposal. The candidate's long-term goal is the pursuit of a career in academic medical research.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Scientist Development Award - Research & Training (K01)
Project #
5K01DK062040-03
Application #
6784643
Study Section
Diabetes, Endocrinology and Metabolic Diseases B Subcommittee (DDK)
Program Officer
Bishop, Terry Rogers
Project Start
2002-08-01
Project End
2006-06-30
Budget Start
2004-07-01
Budget End
2006-06-30
Support Year
3
Fiscal Year
2004
Total Cost
$97,200
Indirect Cost
Name
State University New York Stony Brook
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
804878247
City
Stony Brook
State
NY
Country
United States
Zip Code
11794
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Sharma, Sonia; Findlay, Gregory M; Bandukwala, Hozefa S et al. (2011) Dephosphorylation of the nuclear factor of activated T cells (NFAT) transcription factor is regulated by an RNA-protein scaffold complex. Proc Natl Acad Sci U S A 108:11381-6
Schmidt, Valentina A; Chiariello, Carmine S; Capilla, Encarnacion et al. (2008) Development of hepatocellular carcinoma in Iqgap2-deficient mice is IQGAP1 dependent. Mol Cell Biol 28:1489-502
Cupit, Lisa D; Schmidt, Valentina A; Miller, Frederick et al. (2004) Distinct PAR/IQGAP expression patterns during murine development: implications for thrombin-associated cytoskeletal reorganization. Mamm Genome 15:618-29
Cupit, Lisa D; Schmidt, Valentina A; Gnatenko, Dmitri V et al. (2004) Expression of protease activated receptor 3 (PAR3) is upregulated by induction of megakaryocyte phenotype in human erythroleukemia (HEL) cells. Exp Hematol 32:991-9
Schmidt, Valentina A; Scudder, Lesley; Devoe, Craig E et al. (2003) IQGAP2 functions as a GTP-dependent effector protein in thrombin-induced platelet cytoskeletal reorganization. Blood 101:3021-8