This application focuses on integrin activation, a process important for development, cell migration and assembly of the extracellular matrix. Our previous work led to the conclusion that talin binding to integrin cytoplasmic domains is essential for activation of 1, 2, and 3 integrins and reconstituted talin-induced integrin aIb 3 activation in phospholipid nanodiscs and implicated the integrin transmembrane domains (TMD) in activation. More recent studies established the importance of the 3 TMD tilt angle in the aIIb 3 TMD interaction and showed how structural features of talin can enable it to change the topology of the 3 TMD. The present application requests continued funding for studies designed to test these paradigms and to extend them to 1 and 2 integrins with the long range goal of enabling development of new classes of agents that can modulate integrin functions.
In Aim 1 we will test the hypothesis that changes in the topology of the subunit TMD underlie integrin transmembrane allostery. We will embed 1 and 2 TMD tail-bearing environment-sensitive bimanes at the inner or outer borders of the TMD into phospholipid nanodiscs and use changes in fluorescence to assess the effects of talin head domain (THD) on TMD topology. Proline mutations will be introduced to identify sites at which flexible kinks disrupt transmissionof the topology change. Companion studies will assess the capacity of these Proline insertions to disrupt talin-induced integrin allostery in nanodiscs containing integrins a5 1, aL 2, or aIIb 3.
Aim 2 will test the hypothesis that small molecules can modulate the talin-induced change in subunit TMD topology. We will identify and optimize solvatochromic dyes, whose spectral properties are compatible with those of many pharmacophores, which can be used to assay the talin-induced change in topology of integrin TMD-tails in nanodiscs. These nanodiscs will be used as a one-step, cell-free fluorescence-based screen for inhibitors of talin-induced change in integrin TMD topology; a particular focus will be to identify integrin class-specific inhibitors. One potential such inhibitor, epigallocatechin, has been identified, and its mechanism of action and that of additional inhibitors will be tested in cells and in biochemical assays.
Aim 3 will test the hypothesis that blocking talin-induced change in subunit TMD topology will inhibit the biological functions of 2 and 3 integrins in blood and vascular cells Proline mutations that disrupt the transmission of subunit TMD topology will be introduced into 3 and 2 in cultured endothelial cells or HL60 cells, respectively. Similar mutations will also e made in mice and their effects on activation and functions of 2 integrins in leukocytes and 3 integrins in platelets and endothelial cells will be assessed. In parallel, the effects of small molecules identified in Aim 2 will be tested on the same cell types in vitro. These studies will tet and extend the paradigm that talin-induced transmembrane integrin allostery leads to integrin activation. The work will establish new methods to block talin's effects on integrins and may lead to the development of new classes of integrin antagonists.
Activation of integrins is required for platelet aggregation and for leukocyte arrest and exit from the circulation; thus; integrin activation is a potential therapeutic target in arterial thrombosis; inflammation; and other cardiovascular pathologies. These studies will probe the mechanisms of integrin activation and test and extend proposed paradigms for the mechanism of activation. In doing so; the studies will develop methods to identify antagonists of integrin signaling that can lead to new approaches to treat cardiovascular diseases.
