Cell adhesion mediated by members of the integrin superfamily plays a fundamental role in the evolution of atherosclerosis. Integrin-mediated cell adhesion is regulated, to a greater or lesser extent, by cellular metabolism, a process known as """"""""inside-out signaling"""""""". It has become apparent that inside-out signaling regulates integrin function by perturbing an equilibrium between inactive and active integrin conformations. In this regard, we have reported that enhancing the homomeric association of the beta3 transmembrane domain shifts the platelet integrin alphaIIb-beta3 to an active and clustered state, implying that inducing such associations may represent a physiologic driving force for regulating integrin activity. The function of at least two other platelet integrins, alpha-v-beta3 and alpha2beta1, are regulated by inside-out signaling. Accordingly, one objective of this proposal is to use alpha-v-beta3 and alpha2beta1 to test the hypothesis that inducing the homomeric association of integrin subunit transmembrane domains is a general mechanism for regulating integrin function.
Specific Aim 1 will test the hypothesis that homomeric interactions involving the transmembrane and membrane proximal regions of the cytoplasmic domains of alpha-v and beta3 regulate the activation state of alpha-v-beta3. Experiments will examine the functional consequences of mutations in the transmembrane and cytoplasmic domains of alpha-v and beta3 and identify sequence motifs that promote their homomeric association, alpha2beta1 is an adhesion receptor for collagen on platelets. In contrast to beta3 integrins, it binds to collagen via an inserted (I domain) located in the amino-terminal portion of alpha2. Experiments in Specific Aim 2 will use alpha2beta1 as a model to test the hypothesis that the regulation beta1 integrins follows the paradigm we have proposed for beta3 integrins. Collagen, a prominent component of the normal extracellular matrix and the matrix of atherosclerotic plaques, is an important substrate for platelet adhesion. The active conformation of the alpha2 I domain has been found to interact with a 6 residue motif in the alpha1(I)CB3 fragment of triple helical collagen. A crystal structure of this motif bound to the alpha2 I domain has been reported. Thus, collagen binding to the active conformation of alpha2beta1 may be a suitable target for the development of anti-thrombotic agents. We propose experiments in Specific Aim 3 to use available structural information to synthesize and test the efficacy of specific low molecular weight inhibitors of collagen binding to alpha2beta1.
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