Mechanism of vascular alpha2C-adrenoceptor mobilization

血管α2C-肾上腺素受体动员机制

基本信息

批准号：
7387612
负责人：
Maqsood A. Chotani
金额：
$ 22.5万
依托单位：
OHIO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-03-04 至 2008-08-01
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7387612
关键词：
8-cyclopentyl-1,3-dimethylxanthine Ablation Actin-Binding Protein Actins Adenylate Cyclase Adrenergic Receptor Amino Acids Arginine Arteries Biological Blood Vessels Cell membrane Cell surface Cells Closure Condition Constriction procedure Cultured Cells Cutaneous Cyanosis Cyclic AMP Cytoskeleton Data Disease Expression Library F-Actin FLNC gene Family Filament Forskolin Genetic Goals Guanine Guanine Nucleotide Exchange Factors Hand Human In Vitro Individual Inflammation Inflammatory Response Injury Lead Localized Mechanics Mediating Methods Modeling Molecular Mus Norepinephrine Pallor Pathway interactions Pattern Peptides Peripheral Physiological Play Proteins Raynaud Phenomenon Regulation Rho-associated kinase Role Scleroderma Severity of illness Signal Pathway Signal Transduction Smooth Muscle Myocytes Staging Staining method Stains Stress Structure Syndrome Testing Transfection Upper arm Vascular Smooth Muscle Yeasts arteriole base cDNA Expression cold temperature digital filamin in vivo insight novel protein crosslink rap1 GTP-Binding Proteins rap1A GTP-Binding Protein receptor receptor expression research study response rho vasoconstriction vibration yeast two hybrid system

项目摘要

DESCRIPTION (provided by applicant): Vascular smooth muscle (VSM) 12C-adrenoceptors (12C-ARs) are generally retained in intracellular transGolgi compartment, and mobilized to the cell surface in response to conditions of stress including injury or cold temperature. Cell surface 12C-ARs lead to constriction of the blood vessel. These receptors have been implicated in the heightened abnormal vasoconstrictions triggered by cold temperature in individuals with Raynaud's phenomenon, secondary Raynaud's associated with scleroderma, or hand-arm vibration syndrome. Currently, there is no targeted therapy for early control of Raynaud's phenomenon. SIGNIFICANCE: The long term goal of the project is to elucidate key signaling mechanisms controlling intracellular expression and mobilization of 12C-ARs for targeted therapy in early stages of the disease. METHOD: A novel cell culture model of primary human cutaneous arteriolar VSM (microVSM) will be utilized for these studies. These cells retain high expression of 12C-ARs in culture. Preliminary results using this model implicate the cAMP-activated Ras super family small GTP-binding protein Rap1 in orchestrating 12C-AR expression and partial cell-surface mobilization at 37 oC. Rap1 mobilized 12C-ARs through RhoA-Rho kinase ROCK and the actin binding protein filamin-2, similar to receptor mobilization during cooling. The convergence of cAMP and cold-induced activation of signaling to Rho-ROCK may contribute to severity of the disease. In addition, HEK293 cells and murine microVSM with genetic ablation of Rap1 will be utilized in the studies. Two Specific Aims will be accomplished: AIM 1: Will test the HYPOTHESIS of the carboxyl terminus arginine-rich motif (RRRRR) of 12C-AR to be the key region mediating interaction with filamin 2 and receptor mobilization to filaments and cell surface. Structure-function studies are proposed that will allow testing cell-permeable decoy peptides to block interaction and receptor mobilization at 37oC and experimental condition of moderate cooling at 28 oC. AIM 2: Will test the HYPOTHESIS of Rap1-Rho-ROCK signaling in facilitating filamin 2 to mobilize 12C- ARs to the cell surface. The role of this signaling pathway in phosphorylating the conserved filamin-2 serine2113 will be examined, and the potential relevance to receptor mobilization at 370C and 280C.

描述（由申请人提供）：血管平滑肌（VSM）12C-肾上腺素受体（12C-AR）通常保留在细胞内的高尔基体区室中，并响应包括损伤或低温在内的应激条件而动员至细胞表面。细胞表面 12C-AR 导致血管收缩。这些受体与患有雷诺现象、与硬皮病相关的继发性雷诺现象或手臂振动综合征的个体因寒冷引发的异常血管收缩加剧有关。目前，尚无靶向治疗可以早期控制雷诺现象。意义：该项目的长期目标是阐明控制细胞内表达和 12C-AR 动员的关键信号机制，用于疾病早期阶段的靶向治疗。方法：这些研究将采用一种新型的原代人皮肤小动脉 VSM (microVSM) 细胞培养模型。这些细胞在培养物中保留了 12C-AR 的高表达。使用该模型的初步结果表明，cAMP 激活的 Ras 超家族小 GTP 结合蛋白 Rap1 在 37 oC 下协调 12C-AR 表达和部分细胞表面动员。 Rap1 通过 RhoA-Rho 激酶 ROCK 和肌动蛋白结合蛋白 filamin-2 动员 12C-AR，类似于冷却过程中的受体动员。 cAMP 的收敛和寒冷诱导的 Rho-ROCK 信号传导激活可能会导致疾病的严重程度。此外，研究中还将使用 HEK293 细胞和具有 Rap1 基因消除的小鼠 microVSM。将实现两个具体目标：目标 1：将测试 12C-AR 的羧基末端富含精氨酸基序 (RRRRR) 的假设，该基序是介导与细丝蛋白 2 相互作用以及受体动员至细丝和细胞表面的关键区域。提出的结构功能研究将允许测试细胞可渗透的诱饵肽，以阻断 37°C 下的相互作用和受体动员以及 28°C 适度冷却的实验条件。目标 2：将测试 Rap1-Rho-ROCK 信号传导促进细丝蛋白 2 将 12C-AR 动员至细胞表面的假设。将检查该信号传导途径在磷酸化保守的 filamin-2 serine2113 中的作用，以及与 370C 和 280C 下受体动员的潜在相关性。