MECHANISMS INVOLVED IN THE INTRACELLULAR RETENTION OF A2C ADRENERGIC RECEPTOR

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。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 表示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）的特点是增强血管收缩反应冷，情绪压力或暴露于振动。这些反应被α 2-肾上腺素能（α 2-AR）拮抗剂阻止。一些证据表明，<$2C-AR亚型介导RP血管痉挛发作，但这种作用的细胞和分子机制仍然未知。在初步研究中，我们发现α 2C-AR在37 ℃时在内质网中积累。治疗干扰受体输出贩运，但不与受体内化提高质膜α 2 C-AR水平。在暴露于低温的细胞中观察到类似的增强，表明寒冷增加了细胞表面的α 2C-AR可用性，补充了与局部儿茶酚胺相互作用的受体数量，并导致血管收缩加剧。 此外，我们鉴定了嵌入在α 2C-AR结构中的基于精氨酸的基序，其在30 ° C下充当内质网保留基序，但在37 ° C下不起作用。这些保留基序动员的机制涉及，至少在部分，温度依赖性的相互作用与<$-COP，COPI囊泡的一个亚基调节从高尔基体到内质网的逆行交通。最后，在37 ℃下用HSP 90抑制剂处理可提高α 2C-AR质膜水平，但在30 ℃下则没有。此外，受体与胞质HSP 90亚型的相互作用是温度依赖性的。基于这些初步数据，本申请的中心假设是低温导致的α 2C-AR质膜水平的增加是由于嵌入其结构中的特定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基序和辅助分子伴侣的鉴定将显着推进RP的病理机制的理解。为了实现这些目标，将在HEK 293 T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）鉴定赋予α 2C-AR运输温度敏感性的保留基序; 2）定义由这些保留基序动员的分子机制和3）表征HSP 90在温度中的作用。α 2C-AR转运至细胞表面的依赖性调节。这些特定的目标是独立的，但也相互关联的，这样的组织保证产生新的数据的细胞机制导致RP。这也将有助于识别早期发现RP的细胞生物标志物，并为设计更有效的治疗策略提供基础。

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.