Integrins alphaIIbeta3 and alphavbeta3 play important roles in hemostasis and in blood vessel development respectively. Furthermore, these receptors are promising therapeutic targets in cardiovascular disease. Continued support is requested for studies directed at the hypothesis that vascular integrin signaling involves interactions of the and subunits' cytoplasmic tails with each other and with other intracellular proteins. In addition, CD98 was identified as a potential integrin regulatory protein in a complementation of dominant suppression (CODS) expression cloning scheme. The applicant hypothesizes that CD98 controls integrin function by interacting with cytoplasmic domains. To test these hypotheses, he will use affinity chromatography to examine interactions of CD98 with integrin and to map interactive sites in both the CD98 and integrin cytoplasmic domains. He will examine the effects of mutations that perturb these interactions on CODS and will analyze the phenotype of mice rendered CD98 null by homologous recombination. To gain further insight into the structure-relationships of beta3 integrin tails, the applicant will subject that region of alphaIIbbeta3 to saturation mutagenesis. He will select and characterize mutants that result in constitutive signaling or that disrupt signaling. To gain structural insight into the mutants that result in constitutive signaling or that disrupt signaling. To gain structural insight into the cytoplasmic domain of beta3 integrins the applicant will construct and characterize model protein structural mimics of the alphaIIbbeta3 and Vbeta3 cytoplasmic face. He will use immunochemistry, protease susceptibility, and interactions with cytoplasmic proteins to test the hypothesis that the alpha and beta tails interact to form a discrete domain. Mutational analysis will be used to assess the functional significance of integrin cytoplasmic domain interactions for bi-directional integrin signaling. Finally, the applicant will seek platelet and endothelial cell proteins that bind to alphaIIbbeta3 and alphaVbeta3 integrin cytoplasmic domains. he will use the model protein structural mimics to identify integrin cytoplasmic domain binding proteins by affinity chromatography or by cDNA expression cloning. As alternative strategy, cDNA libraries derived from endothelial cells, bone marrow or differentiated megakaryocytes will be screened for proteins that complement dominant suppression of integrin activation initiated by over-expression of free beta3 tails. The studies will provide new insights into vascular cell function and may suggest novel therapeutic strategies for cardiovascular disease.
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