REGULATION OF PLATELET INTEGRIN FUNCTION

整合素以非活性构象和活性构象之间的平衡存在于细胞表面。因此， 朝向无活性构象的平衡将降低整联蛋白活性，而稳定整联蛋白活性， 活化构象将增加活性。整合素跨膜结构域异聚体相互作用时， 整联蛋白是无活性的，并且在活化后是同聚的。因此， 使异聚体相互作用不稳定或使同聚体相互作用稳定预期会诱导整联蛋白 activation.在本申请中提出的工作继续我们的研究之间的关系， 通过整合细胞生物学，分子生物学， 和生物物理学方法。这些研究集中在血小板整合素α-IIb-β 3。具体目标1。我们将 表征介导α-IIb的异聚和同聚相互作用的螺旋界面， β 3跨膜结构域。我们已经表明，在α-IIb跨膜螺旋中的GxxxG基序是 对于其同质异构体相互作用至关重要。该基序也可能参与了 α-IIb与β 3结合，但参与这种结合的其他α-IIb残基的身份尚不清楚。的 关于β 3残基参与其异聚体和同聚体相互作用的信息有限 和冲突。从拟议的研究中获得的数据将用于构建整合素模型 跨膜结构域寡聚体，并确定相对贡献 使用转染细胞调节α-IIb-β 3功能的异聚体和同聚体相互作用。最后 胞质结构域序列在稳定跨膜结构域相互作用中的参与将是 考虑了具体目标2是基于这样的观察，即合成肽可以被设计成调节 跨膜蛋白的组装。拟议的实验将提供更多的见解， TM螺旋相互作用在α-IIb-β 3活化中的作用和使用合成跨膜 结构域肽作为抗血栓形成剂。

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.