The endothelial cell (EC)-EC adherens junction or the zonula adherens (ZA) and the pulmonary vascular endothelial paracellular pathway are regulated, in part, through protein tyrosine phosphorylation. Several protein tyrosine phosphatases (PTPs) that associate with and/or dephosphorylate components of the ZA multiprotein complex appear to regulate the state of ZA protein tyrosine phosphorylation and assembly. One such PTP, PTP-mu, is highly expressed in lung tissue, almost exclusively expressed in EC, binds to the cytoplasmic domain of vascular endothelial (VE)-cadherin and/or to one or more of the catenins, and through its own ectodomain participates in homophilic adhesion. We will use an immortalized human microvascular EC line together with a tetracycline-responsive, retrovirus-mediated gene transfer system to stably overexpress wild-type PTP-mu, a dominant-negative catalytically-inactive PTP-mu and antisense PTP-mu together with coimmunoprecipitation, in vitro GST-fusion protein binding, and gel-overlay assays, fluorescence microscopy, phosphotyrosine immunoblotting, and actin pool measurements to address the following Specific Aims: 1) To determine whether PTP-mu catalytic function regulates the microvascular endothelial paracellular pathway; 2) To define the PTP-mu structural requirements for its association with VE-cadherin and/or other junctional proteins in human lung microvascular EC; 3) To identify the junctional and/or actin-binding protein substrates for PTP-mu in human microvascular EC. 4) To determine whether PTP-mu catalytic dysfunction disrupts the ZA-actin cytoskeletal linkage and/or induces actin reorganization; and 5) To determine whether PTP-mu overexpression protects against agonist-driven endothelial barrier disruption and/or enhances barrier recovery. These studies will test whether PTP-mu serves a counter-regulatory role in the maintenance of basal pulmonary microvascular endothelial barrier function and/or participates in the opening of the paracellular pathway in response to established mediators of permeability.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Special Emphasis Panel (ZRG1-RESP (03))
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Gail, Dorothy
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University of Maryland Baltimore
Internal Medicine/Medicine
Schools of Medicine
United States
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