Platelet aggregation and leukocyte transmigration depend on regulated changes in integrin affinity ('activation'). New studies by the applicant established the H-Ras and its downstream effector, cRaf-1, suppress the activation of certain integrins. Consequently, the applicant hypothesizes the existence of signaling pathways that suppress integrin function. He also proposes that malignant transformation can produce a sustained activation of these suppressor pathways and that integrin occupancy and/or clustering may stimulate a suppressor pathway resulting in trans-dominant inhibition. To test these hypotheses, he will dissect the mechanisms by which Ras and Raf suppress integrin activation using chimeric integrins in which the extracellular and transmembrane domain of platelet alphaIIbeta3 joined to various integrin cytoplasmic domains. The binding of PAC1, an activation- specific antibody, will be used to asess the affinity state of the alphaIIbeta3 reporter. Requirements for phosphorylatable residues in integrin cytoplasmic domain and integrin specificity will be evaluated. A conditional Raf mutant or microinjected of activated Ras will be used to activate the suppressor pathway. These tools will provide the means to assess the kinetics of suppression and requirements for protein and mRNA synthesis. The role of this suppressor pathway will be tested in three processes controlled by integrin activation: platelet aggregation, fibronectin matrix assembly, and cell migration. Downstream elements of the suppressive pathway triggered by H-Ras and activated Raf-1 will be traced by use of activated and dominant negative comoponents of the MAP kinase cascade. The applicant hypothesis that there are cell-type specific elements in integrin suppressor pathways. To identify these elements, an expression cloning system will be developed isolate suppressive cDNAs from normal and transformed hematoopoietic cells. In addition, if Ras - Raf induced genes are integrin suppressors, then expression cloning will be used to isolate their cDNAs. Integrin occupancy or isolated integrin cytoplasmic domains inhibit functions of other integrins (trans-dominant inhibition). The applicant will test the hypothesis that trans-dominant inhibition is mediated via these suppressor mechanisms by examining the effects of blockers of suppressor pathway(s) on trans-dominant inhibition. These studies will provide new insights into the regulation of integrin affinity, an event of central importance in the functioning of platelets and leukocytes.
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