MECHANISMS INVOLVED IN THE INTRACELLULAR RETENTION OF A2C ADRENERGIC RECEPTOR

A2C 肾上腺素受体细胞内保留所涉及的机制

基本信息

批准号：
8360496
负责人：
Catalin Filipeanu
金额：
$ 18.66万
依托单位：
LSU HEALTH SCIENCES CENTER
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-07-01 至 2012-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8360496
关键词：
Adrenergic Antagonists Adrenergic Receptor Arginine Biological Markers Biology Blood Vessels Cardiovascular system Catecholamines Cell membrane Cell surface Cells Cellular biology Coat Protein Complex I Data Early Diagnosis Emotional Stress Endoplasmic Reticulum Exposure to Foundations Funding Generations Goals Golgi Apparatus Grant Heat-Shock Proteins 90 Investigation Mediating Mentors Molecular Molecular Biology Molecular Chaperones National Center for Research Resources Principal Investigator Protein Isoforms RXR Raynaud Phenomenon Regulation Research Research Infrastructure Resources Role Smooth Muscle Myocytes Source Structure Temperature Therapeutic United States National Institutes of Health Vesicle base cold temperature cost design inhibitor/antagonist prevent receptor receptor internalization response trafficking vasoconstriction vibration

项目摘要

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Raynaud Phenomenon (RP) is characterized by enhanced vasoconstriction in response to cold, emotional stress or exposure to vibrations. These responses are prevented by alpha2-adrenergic (alpha2-AR) antagonists. Several lines of evidence indicate that ¿2C-AR subtype mediate the RP vasospastic attacks, but the cellular and molecular mechanisms underlying this effect remain unknown. In the preliminary investigation we found that alpha2C-AR accumulates in the endoplasmic reticulum at 37oC. Treatments interfering with the receptor export trafficking, but not with receptor internalization enhanced the plasma membrane alpha2C-AR levels. A similar enhancement was observed in cells exposed to low-temperature, indicating that cold increases the alpha2C-AR availability at the cell surface, supplementing the number of receptors interacting with local catecholamines and leading to exaggerated vasoconstriction. Further, we identified arginine based motifs embedded in the alpha2C-AR structure acting as endoplasmic reticulum retention motif at 30oC, but not at 37oC. The mechanisms mobilized by these retention motifs involve, at least in part, temperature-dependent interactions with ¿-COP, a subunit of COPI vesicles regulating the retrograde traffic from Golgi to endoplasmic reticulum. Lastly, the alpha2C-AR plasma membrane levels were enhanced by treatment with HSP90 inhibitors at 37oC but not at 30oC. Further, the receptor interactions with the cytosolic HSP90 isoforms were temperature-dependent. Based on these preliminary data the central hypothesis of this application is that augmentation of the alpha2C-AR plasma membrane levels by low-temperature is due to specific RXR motifs embedded in its structure. These motifs are mediating receptor interactions with specific molecular chaperones in temperature-sensitive manner. Identification of these RXR motifs and the assisting chaperones will significantly advance the understanding of the pathological mechanisms underlying RP. To achieve these goals a combination of cell and molecular biology approaches will be used in HEK293T and vascular smooth muscle cells, aiming to elucidate the following problems: 1) identification of the retention motifs conferring temperature sensitivity to alpha2C-AR traffic; 2) defining the molecular mechanisms mobilized by these retention motifs and 3) characterization of the HSP90 roles in the temperature-dependent regulation of alpha2C-AR transport to the cell surface. These specific aims are independent, but also interconnected and such organization warrants generation of new data on the cellular mechanisms leading to RP. It will also contribute to identification of cellular biomarkers for early detection of RP and it may provide foundation for designing more effective therapeutic strategies.

该子项目是利用资源的众多研究子项目之一由 NIH/NCRR 资助的中心拨款提供该子项目的主要支持。并且子项目的主要研究者可能是由其他来源提供的，包括其他 NIH 来源的子项目可能列出的总成本。代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。雷诺现象 (RP) 的特点是对寒冷、情绪压力或振动的反应会导致血管收缩增强。多种证据表明，α2 肾上腺素能 (α2-AR) 拮抗剂可以阻止这些反应。 2C-AR 亚型介导 RP 血管痉挛发作，但这种效应背后的细胞和分子机制仍不清楚。在初步研究中，我们发现 α2C-AR 在 37°C 时在内质网中积聚，但不干扰受体输出运输。随着受体内化增强，质膜 α2C-AR 水平在暴露于低温的细胞中也观察到类似的增强，表明寒冷增加了 α2C-AR 的可用性。细胞表面，补充了与局部儿茶酚胺相互作用的受体数量，并导致过度的血管收缩。此外，我们还发现了嵌入 alpha2C-AR 结构中的精氨酸基序，在 30°C 时充当内质网保留基序，但在 37°C 时则不然。这些保留基序至少部分涉及与 ¿ -COP，COPI 囊泡的一个亚基，调节从高尔基体到内质网的逆行运输。最后，在 37°C 下使用 HSP90 抑制剂处理可增强 α2C-AR 质膜水平，但在 30°C 下则不会增强受体与胞质 HSP90 的相互作用。基于这些初步数据，本申请的中心假设是α2C-AR质膜的增强。低温水平的降低是由于其结构中嵌入了特定的 RXR 基序，这些基序以温度敏感的方式介导受体与特定分子伴侣的相互作用，这些 RXR 基序和辅助伴侣的识别将显着促进对病理机制的理解。为了实现这些目标，将在 HEK293T 和血管平滑肌细胞中使用细胞和分子生物学方法的组合，旨在阐明以下问题：1）鉴定保留基序赋予 alpha2C-AR 运输温度敏感性；2) 定义这些保留基序动员的分子机制；3) 表征 HSP90 在 alpha2C-AR 运输到细胞表面的温度依赖性调节中的作用。它们是独立的，但也是相互关联的，这样的组织保证了关于导致 RP 的细胞机制的新数据的生成，它还将有助于识别用于早期检测 RP 的细胞生物标志物，并且可能为设计更有效的治疗策略提供基础。