Many important physiological processes are regulated by protein-tyrosyl phosphorylation, which is controlled by protein-tyrosyl kinases (PTKs) and protein-tyrosyl phosphatases (PTPs). Endothelial cell proliferation, migration, and differentiation are initiated, maintained, and regulated by growth factors, many of which signal through receptor PTKs (RTKs). Both endothelial cell-specific RTKs, such as VEGFRs and members of the Tie/Tek subfamily, as well as more widely expressed PTKs, such as FGFR1, the PDGFR, and the HGFR have important actions on endothelial cells. Disorders in RTK activity may play a role in human diseases; for example, Tie family RTKs are implicated in hemangioma syndromes. Interfering with tyrosyl phosphorylation pathways may yield anti-angiogenic therapies; enhancing such pathways may aid in processes such as wound healing/revascularization. Like other cells, endothelial cells respond to inhibitory signals such as cell contact. Although much has been learned about how PTKs regulate endothelial cells, almost nothing is known about the roles of specific PTPs and little is known about inhibitory signaling m endothelial cells. The goal of this research is to define the function of PTPmu and SHPTP2 in cultured endothelial cells and in vivo in mice. Work in other labs has established that PTPmu, a transmembrane PTP, participates in homophilic interactions and associates with cadherin- catenin complexes in tissue culture cells. We have found that, in vivo, HPTPmu is expressed almost exclusively in endothelial cells. Immunocytochemical studies indicate that PTPmu is located at cell-cell junctions and co-localizes with catenins in vivo. Furthermore, preliminary data indicate that antibodies against the ectodomain of HPTPmu may inhibit re-endothelialization in a cell culture "wounding" assay. SHPTP2 is a non- transmembrane PTP that is expressed widely, including in endothelial cells. Studies in fibroblast and epithelial cell lines indicate that SHPTP2 is required for proliferation in response to some, but not all RTKs and in some, but not all cell types. These RTK- and cell type-specific differences in SHPTP2 requirements indicate that SHPTP2's role in endothelial cells cannot simply be inferred from other cell types. We will test the hypotheses that: (i) PTPmu is an important negative regulator of endothelial cell proliferation, and (ii) SHPTP2 is a required positive element in endothelial RTK pathways. The effects of anti-PTPmu antibodies, soluble and particle bound ectodomain constructs, and dominant negative PTPmu mutants on aortic and capillary endothelial cell proliferation, migration, and differentiation will be determined. The mechanism by which PTPmu is removed from the cell surface will be determined, as will the nature of its association with cadherin/catenin complexes. Candidate PTPmu targets will be identified by a combined biochemical and genetic approach. Whether SHPTP2 is tyrosyl phosphorylated and/or associated with RTKs in endothelial cells will be assessed. Transient and/or stable transfections of dominant negative SHPTP2 mutants, along with microinjection of anti- SHPTP2 fusion proteins will be used to determine the function(s) and position of SHPTP2 in endothelial SHPTP2 antibodies an cell RTK signaling pathways. Finally, endothelial cell specific promoters and a novel "knock- in" strategy will be used to examine the effects of dominant negative mutants of PTPmu and SHPTP2 in vivo in transgenic mice. These studies should yield new insights into how PTPs contribute to the maintenance of vascular homeostasis and may contribute to disease states.