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

• 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
• 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

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.

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 雷诺现象(RP)的特征是在寒冷、情绪应激或暴露于振动时血管收缩增强。这些反应可被α2-肾上腺素能(α2-AR)拮抗剂阻止。一些证据表明，2C-AR亚型介导了RP血管痉挛发作，但这种作用的细胞和分子机制尚不清楚。在初步研究中，我们发现在37℃时，α2C-AR在内质网中积聚。干扰受体输出运输的处理，但不干扰受体内化的处理，提高了质膜α2C-AR水平。在暴露在低温下的细胞中也观察到了类似的增强，表明寒冷增加了细胞表面α2C-AR的可获得性，补充了与局部儿茶酚胺相互作用的受体的数量，并导致了夸大的血管收缩。此外，我们确定了嵌入在α2C-AR结构中的精氨酸基序在30oC时作为内质网保留基序，但在37oC时不是。这些保留基序激活的机制至少部分涉及与COPI囊泡的一个亚单位-COP的依赖温度的相互作用，该亚单位调节从高尔基体到内质网的逆行交通。最后，在37℃下用HSP90抑制剂处理可提高α2C-AR质膜水平，但在30℃下不能。此外，受体与胞浆HSP90亚型的相互作用是温度依赖的。根据这些初步数据，本应用的中心假设是低温对alpha2C-AR质膜水平的增强是由于特定的RXR基序嵌入其结构中。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。识别这些RXR基序和辅助伴侣将极大地促进对RP发病机制的理解。为了实现这些目标，将在HEK293T和血管平滑肌细胞中结合使用细胞和分子生物学方法，旨在阐明以下问题：1)鉴定对α2C-AR运输具有温度敏感性的保留基序；2)确定这些保留基序动员的分子机制；3)表征HSP90在α2C-AR运输到细胞表面的依赖温度调节中的作用。这些特定的目标是独立的，但也是相互联系的，这样的组织保证了关于导致RP的细胞机制的新数据的产生。它还将有助于识别细胞生物标志物以早期发现RP，并可能为设计更有效的治疗策略提供基础。