STRUCTURE AND FUNCTION OF THE INTEGRIN ALPHA 1 BETA 1

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Our research is focused on the structure and function of integrins, which are transmembrane heterodimeric receptors for a number of extracellular matrix molecules. These receptors are composed of alpha and beta subunits. Initially, we have used the human integrin alpha1beta1 as a model system in order to answer major questions of the structure-function relationships in alpha and beta domains. We have recently shown that both cytoplasmic domains of these receptors may be controlling integrin intracellular function. The goal of this proposal is to determine the relationship between the structure of integrin receptors and their functions. We have two primary broad goals in this project. One, we will characterize the mechanism by which post-ligand binding events in integrins occur, and isolate genes which mediate those events. Two, we will identify ligand binding domains, and then isolate genes involved in the modulation of the affinity of ligand binding. Our recent structure-function experiments have shown that the alpha cytoplasmic domain plays a regulatory role in the modulation of integrin beta cytoplasmic function. It accomplishes this via an association between these two domains, which we are studying by site- directed mutagenesis of both domains. We will use a potent dominant negative construct as a probe for the identification of beta cytoplasmic domain binding proteins, through a strategy which involves rescue of the dominant negative phenotype. Our experiments identifying ligand binding motifs within alpha1 continue, and we will pinpoint regions necessary for collagen IV binding. Lastly, we will study activation of integrins using the platelet integrin IIb/IIIa, which is then expressed in CHO cells. Using a newly developed permeabilization scheme, we will be able to identify substances required for inside out activation. These experiments will help us understand regulation of adhesion, which is central to migration in atherosclerosis. Furthermore, these experiments will also aid our understanding of the mechanism of integrin activation via inside-out signalling, which is critical in migration and in platelet adhesion.