REGULATION OF PLATELET INTEGRIN FUNCTION

整合素存在于细胞表面，处于非活性构象和活性构象之间的平衡状态。因此，转移 不活跃构象的平衡会降低整合素的活性，而稳定整合素的 激活的构象会增加活性。整合素跨膜结构域在以下情况下以异构性相互作用 整合素是不活跃的，并且在激活之后是同质的。因此，生理过程 破坏异构体相互作用或稳定同聚体相互作用有望诱导整合素 激活。本申请中提出的工作继续我们对 跨膜结构域相互作用和整合素功能 和生物物理方法。研究的重点是血小板整合素α-LLB-β3。在具体目标1.我们将 表征调节α-LLB和α-LLB的异构体和均聚体相互作用的螺旋界面 β3跨膜区。我们已经证明，在α-LLB跨膜螺旋中的GxxxG基序是 对于它的同分异构体的相互作用是必不可少的。该基序还可能参与异构体的相互作用 α-LLB与β3结合，但参与这种结合的其他α-LLB残基的身份尚不清楚。这个 有关β3残基参与其异构体和同聚体相互作用的信息有限 也很矛盾。从拟议的研究中获得的数据将用于构建整合素的模型 利用计算方法确定跨膜结构域低聚物的相对贡献率 利用转基因细胞调节α-llb-beta3功能中的异构体和同聚体相互作用。最后， 细胞质结构域序列参与稳定跨膜结构域相互作用 考虑过了。特殊目的2是基于观察到的合成多肽可以被设计来调节 跨膜蛋白的组装。拟议的实验将提供更多关于 TM螺旋相互作用在α-LLB-β3活化中的作用及合成跨膜使用的原理证明 作为抗血栓药物的结构域肽。

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.