Verbatim): lntegrin alpha4beta1 plays a central role in the trafficking of mononuclear leukocytes and is a potential therapeutic target in chronic inflammatory diseases such as rheumatoid arthritis. Continued support is requested for an analysis of the signaling properties of this integrin. Alpha4beta1 promotes increased cell migration and less cell spreading, stress fiber, and focal adhesion formation than other beta1 integrins. The applicant discovered that the alpha4 cytoplasmic domain binds tightly to the signaling adapter, paxillin. Furthermore, absence of paxillin or a mutation in the a4 tail that disrupts its binding reverses these biological effects. Consequently, the applicant proposes the hypothesis that the paxillin-alpha4 interaction is responsible for the unusual biological responses to alpha4 integrins. To test this, he will map the paxillin-binding site in the alpha4 tail and define mutations that disrupt the interaction. He will introduce these mutations into intact integrins, and examine their effects on cell migration, cell shape, and organization of the cytoskeleton. Conversely, he will use the mapping information to engineer the paxillin binding sites in other integrin a cytoplasmic domains. He will determine whether gain of paxillin binding causes changes in cell shape and cell migration. Furthermore, he aims to assess the consequences of the alpha4-paxillin interaction for downstream signaling events such as activation of Jun Kinase and Focal Adhesion Kinase. To further assess the biologic significance of the alpha4-paxillin interaction, the applicant proposes to generate and characterize mice bearing mutations in the a4 tail that disrupts paxillin binding.
He aims to analyze alpha4 integrin-dependent functions in mononuclear cells derived from such mice. As another test of the hypothesis, the applicant proposes to identify alpha4 cytoplasmic domain-binding sites in paxillin and assesses the effects of mutation of these sites on alpha4 specific responses. He proposes to analyze potential downstream """"""""effectors"""""""" of paxillin by mutating their binding sites in paxillin and assessing the effects of the mutations on alpha4-specific signaling. Finally, the applicant has discovered that alpha4beta1 like other integrins, undergoes active affinity modulation. He hypothesizes that the affinity changes in alpha4beta2 are important in its biological functions. To test this hypothesis he will derive cell lines that manifest defective alpha4 integrin activation or discover subtle mutations in alpha4 that disrupt the activation process. The effect of these mutations on alpha4-dependent adhesive and migratory functions will be assessed. These studies will provide important fundamental insights into the functioning of integrin alpha4beta1 and may identify novel therapeutic targets for chronic inflammatory diseases such as rheumatoid arthritis.
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