Regulation of Platelet Adhesion Receptors

由整合素超家族成员介导的细胞粘附在动脉粥样硬化的演变中起着重要作用。整合素介导的细胞粘附在或多或少的程度上受到细胞代谢的调节，这一过程被称为“由内而外的信号传导”。很明显，由内而外的信号转导通过扰乱非活性和活性整合素构象之间的平衡来调节整合素功能。在这方面，我们已经报道了增强β 3跨膜结构域的同源缔合使血小板整合素α IIb-β 3转变为活性和聚集状态，这意味着诱导这种缔合 可以代表调节整联蛋白活性的生理驱动力。至少另外两种血小板整合素α-v-β 3和α 2 β 1的功能由内而外信号传导调节。因此，该建议的一个目的是使用α-v-β 3和α 2 β 1来检验诱导整联蛋白亚基跨膜结构域的同源缔合是调节整联蛋白功能的一般机制的假设。具体目标1将检验以下假设：涉及α-v和β 3胞质结构域的跨膜和近膜区域的同聚体相互作用调节α-v-β 3的活化状态。实验将检查α-v和β 3的跨膜和胞质结构域中突变的功能后果，并确定促进其同源缔合的序列基序。α 2 β 1是血小板上胶原蛋白的粘附受体。与β 3整联蛋白相反，它通过位于α 2氨基末端部分的插入（I结构域）与胶原结合。《特定目标2》中的实验将使用α 2 β 1作为模型来测试 调节β 1整联蛋白的假设遵循我们为β 3整联蛋白提出的范例。胶原蛋白是正常细胞外基质和动脉粥样硬化斑块基质的主要成分，是血小板粘附的重要底物。已发现α 2 I结构域的活性构象与三螺旋胶原的α 1（I）CB 3片段中的6个残基序相互作用。已经报道了与α 2 I结构域结合的该基序的晶体结构。因此，与α 2 β 1的活性构象结合的胶原蛋白可能是开发抗血栓形成剂的合适靶点。我们提出 具体目标3中的实验使用可用的结构信息来合成和测试与α 2 β 1结合的胶原蛋白的特定低分子量抑制剂的功效。

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.