Outside-in signaling mechanisms of platelet integrin alpha-llb-beta3

血小板整合素α-IIb-β3由外而内的信号传导机制

• 批准号: 8528688
• 负责人: Xiaoping Du
• 金额: $ 37.96万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-12-15 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8528688
• 关键词: Adhesions; Adhesives; Agonist; Binding; Blood; Blood Platelets; Blood Vessels; Bone Marrow Transplantation; Calpain; Cause of Death; Chinese Hamster Ovary Cell; Clot retraction; Coagulation Process; Cytoplasmic Granules; Cytoplasmic Tail; Cytoskeleton; Data; Development; Disease; Extracellular Matrix; Family; Fibrin; Fibrinogen; Funding; GTP-Binding Proteins; Goals; In Vitro; Injury; Integrin Signaling Pathway; Integrin alpha Chains; Integrins; Ligand Binding; Ligands; Ligation; Mediating; Membrane; Modeling; Monomeric GTP-Binding Proteins; Movement; Myocardial Infarction; Names; Peptides; Pharmaceutical Preparations; Platelet Activation; Play; Protein Subunits; Publishing; Regulation; Role; SRC gene; Science; Side; Signal Pathway; Signal Transduction; Site; Stroke; Surface; Techniques; Testing; Thrombosis; Thrombus; Transgenic Mice; Tyrosine Phosphorylation; United States; Wound Healing; adhesion receptor; base; extracellular; fighting; in vivo; inhibitor/antagonist; mutant; response; rho guanine nucleotide exchange factor p115; seal; src-Family Kinases; wound

DESCRIPTION (provided by applicant): The platelet integrin, aIIb¿3, interacts with adhesive ligands such as fibrinogen and fibrin, and mediates platelet adhesion and aggregation. Integrin aIIb¿3 thus plays a critical role in the development of thrombotic diseases such as heart attack and stroke. Integrin aIIb¿3 also transmits signals bidirectionally: The ligand binding function of integrin aIIb¿3 is activated by signals from within platelets ("inside-out" signaling). Ligand binding to aIIb¿3 induces "outside-in" signals, which transmit into platelets and elicit cellular response critical in amplifying and stabilizing thrombi, in wound healing and the re-canalization of blood vessels. The goal of this application is to understand the mechanisms of integrin outside-in signaling. It is known that integrin outside-in signaling requires the activation of the Src family of kinases and regulation of small GTPases such as RhoA. However, the upstream mechanisms that initiate integrin-mediated Src activation have been unknown. Furthermore, integrin aIIb¿3 initially mediates platelet spreading, but later induces platelet-mediated clot retraction, which requires the movement of platelet membranes in the opposite direction. It has been unclear how integrin aIIb¿3 switches the direction of membrane movements. During the current funding period, we have made the following discoveries: (1) The G protein subunit, Ga13, directly interacts with the cytoplasmic domain of the integrin ¿3 subunit, and this interaction is required for the integrin-dependent c-Src activation, RhoA inhibition and platelet spreading (Gong et al, Science, 2010). (2) a calpain cleavage of ¿3 disrupts the c- Src-dependent RhoA inhibition, inducing RhoA-dependent clot retraction. (3) the small G proteins, RhoA and Rac1, are both important in integrin-dependent retractile signaling and clot retraction. Based on data from these studies, we propose following hypotheses: (1) integrin aIIb¿3 outside-in signaling is mediated by the direct binding of Ga13 to the cytoplasmic domain of ¿3. (2) Ga13 transmits integrin signal to activate c-Src and induce c-Src-dependent inhibition of RhoA, resulting in inhibition of platelet retraction and promotion of platelet spreading. (3) Calpain cleavage of ¿3 serves as a mechanism that down regulates Ga13 signaling by inactivating the Ga13/c-Src-dependent signaling pathway that inhibits RhoA. This facilitates integrin-dependent RhoA activation and stimulates clot retraction. To test these hypotheses, we propose the following specific aims: Aim 1, to determine the role of Ga13 binding to ¿3 in mediating integrin outside-in signaling. In this aim, we will determine the importance of Ga13, particularly the role of Ga13-¿3 interaction, in various integrin signaling pathways, and determine how Ga13-¿3 interaction is regulated and how this interaction activates c- Src. Aim 2, to investigate the mechanism of Ga13 and integrin-dependent dynamic regulation of RhoA in controlling platelet spreading and retraction. In this aim, we will investigate the role of Ga13-¿3 interaction in inhibiting RhoA and stimulating p190RhoGAP, competitive inhibition of Ga13-p115RhoGEF by ¿3, and the mechanisms of calpain-induced late RhoA activation and retractile signaling.

描述（由应用提供）：血小板整联蛋白AIIB课与纤维蛋白原和纤维蛋白等粘合剂配体相互作用，并介导血小板粘合剂和聚集。因此，整联蛋白AIIB课3在心脏病发作和中风等血栓性疾病的发展中起着至关重要的作用。整联蛋白AIIB€3还双向传输信号：整联蛋白AIIB课的配体结合函数被血小板内部信号激活（“内部”信号传导）。配体与AIIB课的结合3诱导“外部”信号，该信号转移到血小板中，并引起细胞反应在扩增和稳定血栓，伤口愈合和血管重新分配时至关重要。该应用的目的是了解整联蛋白外部信号传导的机制。众所周知，整联蛋白的外部信号传导需要激活SRC家族的激酶，并调节小GTPases（例如RhoA）。但是，启动整联蛋白介导的SRC激活的上游机制尚不清楚。此外，整联蛋白AIIB€3最初介导了血小板扩散，但后来诱导了血小板介导的克隆回收，这需要血小板膜在相反方向上运动。目前尚不清楚整合素AIIB课程如何切换膜运动的方向。在当前的资金期间，我们已经发现了以下发现：（1）G蛋白亚基GA13直接与整合素的细胞质结构域相互作用，3个亚基需要这种相互作用，而依赖整联蛋白的C-SRC激活需要RhoA抑制和血小板扩散（Gong et al，Science，Science，2010）。 （2）3的钙蛋白酶切割»3破坏了C- SRC依赖性的RhoA抑制作用，诱导了RhoA依赖性的凝块回缩。 （3）小的G蛋白RhoA和Rac1在整合素依赖性的伸缩信号传导和凝块回缩中都很重要。 Based on data from these studies, we propose following hypotheses: (1) integrin aIIb¿ 3 outside-in signaling is mediated by the direct binding of Ga13 to the cytoplasmic domain of ¿ 3. (2) Ga13 transmits integrin signal to activate c-Src and induce c-Src-dependent inhibition of RhoA, resulting in inhibition of platelet retraction and promotion of platelet spreading. （3）3的钙蛋白酶切割是一种机制，可通过灭活抑制RhoA的GA13/C-SRC依赖性信号通路来降低调节GA13信号传导。这有助于整联蛋白依赖性RHOA激活并刺激凝块回缩。为了检验这些假设，我们提出了以下特定目的：目标1，以确定Ga13与3在介导整联蛋白外部信号传导中的作用。在此目标中，我们将确定GA13的重要性，尤其是在各种整合素信号通路中GA13-€3相互作用的作用，并确定如何调节GA13-€3相互作用以及这种相互作用如何激活C-SRC。 AIM 2，研究RhoA在控制血小板扩散和缩回方面的GA13和整联蛋白依赖性动态调节的机理。在此目标中，我们将研究Ga13-€3相互作用在抑制RhoA和刺激P190RHOGAP，通过€3对GA13-P115RHOGEF的竞争性抑制以及CALPAIN诱导的RhoA晚期RhoA激活和缩回信号传导的作用。