The purpose of this exploratory and developmental research project application is to identify proteins involved in the regulation of ?1 integrin activation. Integrin adhesion receptors are heterodimers formed from ? and ? subunits - each of which is a type I transmembrane protein with a large extracellular portion, a single transmembrane domain and a short cytoplasmic tail. The ability of integrin adhesion receptors to undergo activation (rapid regulated changes in affinity for their extracellular ligands) is essential for the development and functioning of the cardiovascular system. Inappropriate integrin activation contributes to thrombosis, atherosclerosis, myocardial infarction, stroke and reperfusion injury, and integrins are targets for therapeutic regulation of these conditions and for both inhibition and stimulation of angiogenesis. Detailed insight into the mechanisms controlling integrin activation is therefore directly relevant to strategies aimed at understanding and controlling cardiovascular disease and stroke. Our long-term aim is to understand the molecular mechanisms regulating integrin activation. We previously showed that binding of the cytoskeletal protein talin to the ?3 subunit cytoplasmic tail is necessary and sufficient for activation of the platelet integrin ?IIb?3. Our new data show that talin is also required but not sufficient for ?1 integrin activation and indicate that other ?1 tail binding factors are also required.
We aim to identify and characterize those additional factors. Specifically we aim to: 1) Use expression cloning to identify proteins that cooperate with talin to activate ?1 integrins: 2) Find proteins required for ?1 integrin activation using a siRNA screen: 3) Identify proteins whose binding to ?1 cytoplasmic tails is altered by mutations which we suggest impair binding of a 2nd activating factor: and 4) Characterize identified proteins. To achieve these aims we will rely on our ability to assess integrin activation in live cells in FACS based assays. We will transfect cells with talin and a cDNA expression library and collect cells where ?1 integrins have become activated; the transfected library cDNA will be recovered, re- screened and sequenced to identify genes that cooperate with talin to activate ?1 integrins. Target cell populations will also be transduced with siRNA libraries and cells expressing inactivated integrins collected; siRNA sequences will be recovered and sequences enriched in the inactivated cell population identified by microarray analysis. This should reveal genes, and so proteins, essential to maintain integrin activation. We have identified point mutations in the ?1 tail which seem to inhibit binding of a ?1 activating factor. We will apply protein profiling and identification techniques to identify proteins ca[able of binding to the wild-type but not mutant ? tails. Finally, we will characterize identified proteins biochemically and attempt to place them in integrin activation pathways. Tight control of the adhesive interactions between cells and between cells and the extracellular matrix which surrounds them is essential for the development and functioning of the cardiovascular system and inappropriate cell adhesion contributes to cardiovascular disease. Cell surface adhesion receptors called integrins mediate many of the cell's adhesive interactions and their activity is normally tightly regulated.
We aim to identify and characterize the proteins that regulate integrin activity with a view towards understanding the molecular mechanisms regulating integrin function. ? ? ?
