MECHANISMS AND REGULATION OF EPITHELIAL TRANSPORT

描述：肾近端小管和肠刷状缘膜 含有SGLT家族转运蛋白，其催化Na+偶联的二级转运蛋白 葡萄糖的主动运输。 两种不同的Na+/葡萄糖协同转运蛋白 同种型SGLT 1和SGLT 2在LLC-PK 1肾上皮细胞中表达， 细胞系，SGLT 1表达受细胞密度和 蛋白激酶A和C的作用。申请人已确定 调节SGLT 1 mRNA稳定性的转录后机制 可能在调节这种转运蛋白的水平方面起着关键作用， 对细胞信号传导机制的反应。 蛋白激酶A激活 促进SGLT 1消息稳定，这是一种需要 3'非翻译区（3'-UTR），与蛋白质相关 磷酸化以及增加的48 kDa蛋白（称为 SG-URBP）与SGLT 1 3' UTR中的富含尿苷的元件（URE）连接。 本 应用将进一步表征可能的顺式和反式作用因子 介导协同转运蛋白表达的转录后调节。 使用嵌合mRNA构建体，特异性目标1将检验以下假设： 位于3' UTR中的SGLT 1 URE序列调节SGLT 1信息 稳定性和努力将被用来鉴定参与 通过蛋白激酶A激活和SG-URBP结合来稳定信息。 在具体目标2中，蛋白激酶C介导的分子机制 SGLT 1信息的不稳定性将通过识别 顺式作用序列及其同源mRNA结合蛋白。 在特定 目的3、纯化SG-URBP并制备抗SG-URBP的抗体， 试剂应便于实验重点检查的影响， 蛋白激酶A介导的SG-URBP磷酸化对其mRNA结合的影响 活性，并可能有助于SG-URBP克隆和定位研究。 最后， 在具体目标4中，调节SGLT 2表达的机制也将 被定性。

DESCRIPTION: Renal proximal tubule and intestinal brush border membranes contain SGLT family transporters that catalyze the Na+ coupled secondary active transport of glucose. Two distinct Na+/glucose cotransporter isoforms, SGLT1 and SGLT2, are expressed in the LLC-PK1 renal epithelial cell line, and SGLT1 expression is regulated by cell density and by the action of protein kinases A and C. The applicant has identified a post-transcriptional mechanism for the regulation of SGLT1 mRNA stability that may play a key role in modulating the levels of this transporter in response to cell signalling mechanisms. Protein kinase A activation promotes SGLT1 message stabilization, which is an effect that requires the 3' untranslated region (3'-UTR) and is associated with protein phosphorylation as well as the increased binding of a 48 kDa protein (termed SG-URBP) to a uridine-rich element (URE) in the SGLT1 3' UTR. The present application will further characterize possible cis- and trans-acting factors that mediate post-transcriptional regulation of cotransporter expression. Using chimeric mRNA constructs, Specific Aim 1 will test the hypothesis that the SGLT1 URE sequence located in the 3' UTR regulates SGLT1 message stability and efforts will be made to identify sequence elements involved in message stabilization by protein kinase A activation and SG-URBP binding. In Specific Aim 2, the molecular mechanism of protein kinase C-mediated destabilization of the SGLT1 message will be explored by identifying cis-acting sequences and their cognate mRNA binding proteins. In Specific Aim 3, SG-URBP will be purified and antibodies generated against it. These reagents should facilitate experiments focused on examining the influence of protein kinase A-mediated SG-URBP phosphorylation on its mRNA binding activity, and may aid in SG-URBP cloning and localization studies. Finally, in Specific Aim 4, the mechanisms that regulate SGLT2 expression will also be characterized.