Role of Carboxyterminal Hypervariable Region in Rap1b Function

羧基末端高变区在 Rap1b 功能中的作用

• 批准号: 7851206
• 负责人: GILBERT C. WHITE, II
• 金额: $ 59.32万
• 依托单位: VERSITI WISCONSIN, INC.
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7851206
• 关键词: Abnormal Platelet; Adhesions; Affect; Agonist; Amino Acids; Binding; Binding Proteins; Biochemical; Biochemical Genetics; Biological Assay; Blood Clot; Blood Platelets; Blood Vessels; Blood coagulation; C-terminal; Cell Aggregation; Cells; Charge; Chemicals; Complementarity Determining Regions; Complex; Confocal Microscopy; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cytoskeletal Proteins; Cytoskeleton; Dominant-Negative Mutation; Dot Immunoblotting; Electrostatics; Family; Fluorescence Resonance Energy Transfer; G-Protein-Coupled Receptors; GTP-Binding Proteins; Glutamine; Goals; Guanine Nucleotides; Guanosine Triphosphate Phosphohydrolases; Healed; Hemorrhage; Immunoprecipitation; In Vitro; Injury; Integrins; Lead; Length; Lipids; Mediating; Megakaryocytes; Membrane; Membrane Microdomains; Methods; Methylation; Micelles; Molecular Weight; Mus; Mutate; Mutation; PH Domain; Peptides; Phosphatidylinositols; Phospholipids; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphorylation Site; Plasma; Platelet aggregation; Process; Protein Binding; Protein Kinase; Proteins; Regulation; Role; Serine; Signal Pathway; Signal Transduction; Signaling Molecule; Site; Structure; Tertiary Protein Structure; Testing; Triton; Work; Yeasts; attachment protein G; carboxymethylation; emergency service responder; genetic regulatory protein; healing; improved; in vitro testing; in vivo; insight; member; mutant; phosphoinositide-3,4,5-triphosphate; prenyl; prenylation; protein protein interaction; public health relevance; response

DESCRIPTION (provided by applicant): Rap1b is a ubiquitous, membrane associated low molecular weight GTPase that has been shown to be important for integrin-mediated platelet aggregation and adhesion. The mechanism of rap1b action appears to be through a signaling cascade that leads to activation of integrins. Mice deficient in rap1b have abnormal platelet responses to all agonists tested. As with all low molecular weight GTPases, the membrane association of rap1b is important for its function. The purpose of the work in this proposal is to explore the membrane interactions of rap1b in more detail in order to better understand its mechanism of action. The attachment of rap1b to the membrane is through its C-terminal hypervariable region, a region that contains polybasic sequences believed to interact with membrane phospholipids, a post-translationally added prenyl group that associates with membranes, and a hydrophobic C-terminal carboxymethyl group that also associates with the membrane. The hypervariable region is also the site of phosphorylation by cyclic AMP (cAMP)-dependent protein kinase (PKA) when platelets are inhibited by agents that increase cAMP levels. Using various hypervariable region mutations that affect the structure of the polybasic cluster and the sites of carboxymethylation, prenylation, and phosphorylation, we will examine the role of these various moeities on the subcellular localization and function of rap1b, the interaction of the hypervariable region with specific phoshoplipids, and the interaction of the hypervariable region with other proteins. The results of these studies will lead to an improved understand of how GTPases work in general, how rap1b functions in platelets to regulate integrin activation, and may lead to new methods for inhibiting platelet function. PUBLIC HEALTH RELEVANCE: Platelets are the first responders when there is an injury to a blood vessel. The overall goal of this application is to understand how platelets respond to such an injury, as well as the mechanisms involved with their response. These findings will provide insight into how the body heals bleeding injuries, as well as the underlying cause of blood clotting conditions.

描述（由申请人提供）：Rap1b 是一种普遍存在的膜相关低分子量 GTP 酶，已被证明对整合素介导的血小板聚集和粘附很重要。 rap1b 的作用机制似乎是通过信号级联导致整合素激活。 rap1b 缺陷的小鼠对所有测试的激动剂都有异常的血小板反应。与所有低分子量 GTP 酶一样，rap1b 的膜缔合对其功能很重要。本提案的工作目的是更详细地探索 rap1b 的膜相互作用，以便更好地理解其作用机制。 rap1b 通过其 C 端高变区与膜连接，该区域包含被认为与膜磷脂相互作用的多元序列、与膜结合的翻译后添加的异戊二烯基团，以及也与膜结合的疏水性 C 端羧甲基基团。当血小板受到增加 cAMP 水平的药物抑制时，高变区也是环 AMP (cAMP) 依赖性蛋白激酶 (PKA) 磷酸化的位点。利用影响多碱基簇结构以及羧甲基化、异戊二烯化和磷酸化位点的各种高变区突变，我们将研究这些不同部分对 rap1b 的亚细胞定位和功能的作用、高变区与特定磷脂的相互作用以及高变区与其他蛋白质的相互作用。这些研究的结果将有助于更好地理解 GTPases 的一般工作原理、rap1b 在血小板中如何发挥作用来调节整合素激活，并可能带来抑制血小板功能的新方法。公共卫生相关性：当血管受伤时，血小板是第一反应者。该应用的总体目标是了解血小板如何对这种损伤做出反应，以及与其反应相关的机制。这些发现将有助于深入了解身体如何治愈出血损伤，以及血液凝固的根本原因。