This competitive renewal application is to continue on-going studies of novel functions of the integrin, alpha9beta1. Based on analysis of alpha9 knockout mice, alpha9beta1 plays a critical role in development of the thoracic duct and other lymphatic vessels. One clue to the mechanism by which this integrin might contribute to lymphatic development comes from our identification of the lymphangiogenic growth factors, VEGFC and VEGFD as putative alpha9beta1 ligands. During the current funding period a unique mechanism by which alpha9beta1 enhances cell migration was also identified, and this effect was shown to be mediated by specific sequences in the alpha9 cytoplasmic domain. A single protein, the enzyme spermine/spermidine acetyltransferase, was found to bind to the alpha9 cytoplasmic domain and to specifically modulate alpha9-dependent enhancement of cell migration. This application proposes to evaluate each of these clues in more detail. The significance of binding of the extracellular domains of alpha9beta1 to VEGFC and D will be examined by assessing cell migration, proliferation, and early steps in integrin and growth factor receptor signaling in response to recombinant forms of each growth factor in mock- and alpha9-transfected cells. The importance of co-ligation of the canonical receptor for these growth factors, VEGFR3 will be assessed by performing all of these studies in the presence or absence of co-expression of VEGFR3. The alpha9 expressing cells critical for lymphatic development will be evaluated in mice homozygous for a conditional alpha9 null allele that will be used to inactivate this gene in specific cell types that might contribute to developing lymphatics. Stable cell lines co-expressing wild type and mutant forms of alpha9beta1 along with wild type or mutant forms of SSAT will be used to map the interaction sites in each protein and determine the importance of SSAT binding and enzymatic activity for alpha9beta1-mediated enhancement of cell migration. In vitro binding of recombinant versions of each protein, co-immunoprecipitation and double staining immunofluorescence will be used to determine whether this interaction is direct, occurs in living mammalian cells and results in co-localization to informative cellular compartments. Finally, the in vivo significance of alpha9-mediated migration and interactions with SSAT will be determined utilizing mice expressing knock-in mutations of the alpha9 cytoplasmic domain specifically designed to eliminate enhanced migration and/or SSAT binding. The proposed studies should provide important information about the role this widely expressed integrin plays in lymphatic development and cell migration and could ultimately lead to the design of novel interventions in diseases affected by each of these processes.
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