TARGETING ALPHA2 RECEPTORS TO RENAL BASOLATERAL MEMBRANE

目前的研究建议检查什么结构域（S）内 α 2-肾上腺素能受体（α 2AR）负责靶向 和/或该受体在肾基底外侧膜上的保留 上皮细胞 Madin-Darby犬肾（MDCKII）细胞系将 是这些研究的主要模型系统。 网站和删除 将采用诱变策略来探索N-末端 糖基化，第三大细胞质内的二级结构 环，位于预测的跨膜基底的内吞信号 螺旋7、内面芳族残基和/或α 2AR的酰化 在MDCKII细胞中α 2 AR极化中起关键作用。 永久 表达编码野生型的基因的MDCKII细胞的转化体和 将克隆突变体α 2 AR并表征α 2 AR表达。 MDCKII细胞的极化将通过在可渗透的 支持物（Transwell培养威尔斯孔）和监测的α 2 AR分布 使用三个独立的策略，包括1） 生物素化/提取/链霉亲和素分级，2）形态学 定位和3）细胞表面ELISA技术。 提出的研究将提供新的见解的分子基础 在肾上皮细胞中靶向alpha 2AR， 所有GTP结合蛋白偶联的结构特征 用于定位到专门的细胞结构域的受体。 我们将 通过检测是否存在 类似的结构域将α 2 AR靶向至基底外侧域 肠上皮细胞的极化。 最后，未来的研究 通过拟议的实验，希望能揭示 不同α 2 AR亚型内的基底外侧靶向/保留信号 确定受体向神经元内离散区域的递送，例如，的 体树突与突触终膜。

The present studies propose to examine what structural domain(s) within the alpha2-adrenergic receptor (alpha2AR) are responsible for targeting and/or retention of this receptor on the basolateral membrane of renal epithelial cells. The Madin-Darby canine kidney (MDCKII) cell line will be the primary model system for these studies. Site and deletion mutagenesis strategies will be employed to explore whether N-terminal glycosylation, secondary structure within the large third cytoplasmic loop, endocytosis signals located at the base of predicted transmembrane helix 7, endofacial aromatic residues and/or acylation of the alpha2AR play a critical role in alpha2AR polarization in MDCKII cells. Permanent transformants of MDCKII cells expressing genes coding for wild-type and mutant alpha2AR will be cloned and characterized for alpha2AR expression. Polarization of the MDCKII cells will be achieved by growth on permeable supports (Transwell culture wells) and alpha2AR distribution monitored using three independent strategies, including 1) biotinylation/extraction/streptavidin fractionation, 2) morphological localization and 3) cell surface ELISA techniques. The studies proposed will provide novel insights into the molecular basis for targeting of alpha2AR in renal epithelia that likely will reflect structural features exploited by all GTP-binding protein-coupled receptors for localization to specialized cellular domains. We will extend our findings in renal epithelial cells by examining whether or not similar structural domains target the alpha2AR to the basolateral domain of intestinal epithelial polarized in culture. Finally, future studies informed by the proposed experiments hopefully will reveal whether or not basolateral targeting/retention signals within varying alpha2AR subtypes determine receptor delivery to discrete regions within neurons, e.g., the somatodendritic versus synaptic terminal membranes.