Integrins reside on cell surfaces in an equilibrium between inactive and active conformations. Thus, shifting the equilibrium towards the inactive conformation will decrease integrin activity, whereas stabilizing the activated conformation will increase activity. Integrin transmembrane domains interact heteromerically when integrins are inactive and homomerically following activation. Accordingly, physiologic processes that destabilize heteromeric interactions or stabilize homomeric interactions would be expected to induce integrin activation. The work proposed in this application continues our examination of the relationship between transmembrane domain interactions and integrin function by integrating cell biological, molecular biological, and biophysical methods. The studies focus on the platelet integrin alpha-llb-beta3.
In Specific Aim 1. we will characterize the helical interfaces that mediate the heteromeric and homomeric interactions of the alpha-llb and beta3 transmembrane domains. We have shown that a GxxxG motif in the alpha-llb transmembrane helix is essential for its homomeric interactions. The motif also likely participates in the heteromeric interaction of alpha-llb with beta3, but the identity of other alpha-llb residues that participate in this association are not known. The information available about the beta3 residues involved in its heteromeric and homomeric interactions is limited and conflicting. The data obtained from the proposed studies will be used to construct models of integrin transmembrane domain oligomers using computational methods and to determine the relative contribution of heteromeric and homomeric interactions in regulating alpha-llb-beta3 function using transfected cells. Lastly, the participation of cytoplasmic domain sequences in stabilizing transmembrane domain interactions will be considered.
Specific Aim 2 is based on observations that synthetic peptides can be designed to modulate the assembly of transmembrane proteins. The proposed experiments will provide additional insight into the role of TM helix interactions in alpha-llb-beta3 activation and proof of principle for the use of synthetic transmembrane domain peptides as anti-thrombotic agents.
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