TARGETING ALPHA2 RECEPTORS TO RENAL BASOLATERAL MEMBRANE

本研究建议考察结构域(S)中的 α2-肾上腺素能受体(α2AR)负责靶向 和/或该受体在肾基底外侧膜上的滞留 上皮细胞。Madin-Darby犬肾(MDCKII)细胞系将 成为这些研究的主要模型系统。站点和删除 将采用突变策略来探索N-末端是否 糖基化，第三大细胞质内的二级结构 环，内吞作用信号位于预测的跨膜的基础上 螺旋7、表面芳香族残基和/或α2AR的酰化 在MDCKII细胞的α2AR极化中起关键作用。永久 MDCKII细胞表达野生型和强毒基因的转化子 突变的α2AR将被克隆，并对其表达进行鉴定。 MDCKII细胞的极化将通过在可渗透材料上生长来实现 支持(Transwell培养井)和监测的Alpha2AR分布 使用三个独立的策略，包括1) 生物素化/提取/链霉亲和素级分，2)形态 3)细胞表面酶联免疫吸附试验技术。 提出的研究将为分子基础提供新的见解。 靶向肾上皮细胞中的α2AR，可能会反映 所有GTP结合蛋白偶联蛋白所利用的结构特征 受体定位于特定的细胞域。我们会 通过检查我们在肾上皮细胞中的发现 类似的结构域以α2AR为靶点，指向基侧结构域 肠上皮细胞在培养过程中被极化。最后，未来的研究 被提议的实验所了解，有望揭示 不同Alpha2AR亚型中的基侧靶向/保持信号 确定受体递送到神经元内的离散区域，例如 躯体树突与突触终末膜。

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.