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：
7578324
负责人：
Maqsood A. Chotani
金额：
$ 18万
依托单位：
RESEARCH INST NATIONWIDE CHILDREN'S HOSP
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 2012-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7578324
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 Culture Techniques Cell membrane Cell surface Cells Cutaneous Cyanosis Cyclic AMP Cytoskeleton Data Disease Early treatment 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 Mechanics Mediating Methods Modeling Molecular Mus Norepinephrine Pallor Pathway interactions Pattern Peptides Peripheral Physiological Play Process 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 constriction digital filamin in vivo insight novel novel strategies protein crosslink rap1 GTP-Binding Proteins receptor receptor expression receptor function 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-ARS）通常保留在细胞内的跨科尔基室中，并以应激条件在内，包括损伤或冷温度的压力条件，将其动员到细胞表面。细胞表面12C-ARS导致血管收缩。这些受体与雷诺现象，与硬皮病相关的次级雷诺或手臂振动综合征相关的个体触发的异常血管收缩的异常血管收缩。当前，尚无针对性的治疗雷诺现象的早期控制。意义：该项目的长期目标是阐明控制细胞内表达和动员12C-ARS在疾病早期靶向治疗中的关键信号传导机制。方法：这些研究将使用一种新的人皮肤小动脉VSM（MicroVSM）的新细胞培养模型。这些细胞在培养中保留12C-AR的高表达。使用该模型的初步结果暗示了cAMP激活的RAS超级家族小型GTP结合蛋白RAP1在策划12C-AR表达和以37 OC下的部分细胞表面动员中。 RAP1通过Rhoa-Rho激酶岩和肌动蛋白结合蛋白丝蛋白-2动员12C-ARS，类似于冷却过程中受体动员。 cAMP和冷诱导的信号传导向Rho-Rock的激活的收敛可能导致疾病的严重程度。此外，研究将在研究中使用HEK293细胞和带有RAP1遗传消融的鼠MicroVSM。将实现两个具体的目标：目标1：将测试12C-AR的羧基末端基氨酸基序（RRRRR）的假设，是介导与丝蛋白2的关键区域，以及对细丝和细胞表面的受体动员的相互作用。提出了结构功能研究，该研究将允许测试可渗透细胞的诱饵肽在37oC下阻止相互作用和受体动员，并在28 OC下进行中度冷却的实验条件。 AIM 2：将在促进Filamin 2中检验Rap1-Rho-Rock信号传导的假设，以将12C-ARS动员到细胞表面。该信号通路在磷酸化中的作用将检查保守的Filamin-2丝氨酸2113，并在370C和280C时与受体动员的潜在相关性。