Identification of IQGAP1 as a SLC26A4 (Pendrin)-Binding Protein in the Kidney.
Identification of IQGAP1 as a SLC26A4 (Pendrin)-Binding Protein in the Kidney.
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DOI：
10.3389/fmolb.2022.874186
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发表时间：
2022
影响因子：
5
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中科院分区：
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背景：SLC26a转运蛋白家族中的几个成员，包括SLC26A3(DRA)、SLC26A5(Prestin)、SLC26A6(PAT-1；CFEX)和SLC26A9，与许多分子(如细胞骨架蛋白、锚定或连接蛋白、囊性纤维化跨膜电导调节因子和蛋白激酶)形成多蛋白复合体。这些相互作用为这些分子提供了调节信号。然而，与Cl−/HCO3−交换器SLC26A4(Pendrin)相互作用的蛋白质的同一性尚未确定。本研究的目的是确定与垂垂蛋白相互作用的蛋白质(S)。方法：采用酵母双杂交(Y2H)系统，以SLC26A4的C端片段为诱饵，筛选小鼠肾脏cDNA文库。用亲和纯化的抗体和肾蛋白提取物进行肾切片免疫荧光显微镜检查和免疫共沉淀实验，以证实Pendrin与已鉴定的结合伙伴的共定位和相互作用。在培养的细胞中进行了共表达研究，以检测结合伙伴对悬垂蛋白运输和活性的影响。结果：Y2H研究证实含有IQ基序的GTP酶激活蛋白1(IQGAP1)是一种与SLC26A4‘S C末端结合的蛋白。用亲和纯化的抗IQGAP1抗体进行免疫共沉淀实验，然后用Pendrin特异性抗体对肾脏蛋白洗脱液进行免疫印迹分析，证实了Pendrin和IQGAP1的相互作用。免疫荧光显微镜研究表明，IQGAP1与Pendrin共定位于B细胞的顶膜，而在皮质集合管的A细胞中，IQGAP1呈基底侧向表达。在HEK293细胞中的功能和共聚焦研究，以及在mDCK细胞中的共聚焦研究表明，侧翼蛋白和IQGAP1的共转染显示出这两个分子在质膜上的强烈共定位，并增强了Cl−/HCO3−交换活性。结论：在B细胞中，IQGAP1是一种与膜蛋白C末端结合的蛋白。IQGAP1与Pendrin共定位于B细胞顶膜。IQGAP1和Pendrin的共表达导致两个分子的强烈共定位，并增加了Pendrin在培养细胞质膜中的活性。我们认为，Pendrin与IQGAP1的相互作用可能在CCDs的功能和生理调节中发挥关键作用，这种相互作用的破坏可能有助于改变Pendrin在病理生理状态下的运输和/或活性。
Background: Several members of the SLC26A family of transporters, including SLC26A3 (DRA), SLC26A5 (prestin), SLC26A6 (PAT-1; CFEX) and SLC26A9, form multi-protein complexes with a number of molecules (e.g., cytoskeletal proteins, anchoring or adaptor proteins, cystic fibrosis transmembrane conductance regulator, and protein kinases). These interactions provide regulatory signals for these molecules. However, the identity of proteins that interact with the Cl−/HCO3 − exchanger, SLC26A4 (pendrin), have yet to be determined. The purpose of this study is to identify the protein(s) that interact with pendrin. Methods: A yeast two hybrid (Y2H) system was employed to screen a mouse kidney cDNA library using the C-terminal fragment of SLC26A4 as bait. Immunofluorescence microscopic examination of kidney sections, as well as co-immunoprecipitation assays, were performed using affinity purified antibodies and kidney protein extracts to confirm the co-localization and interaction of pendrin and the identified binding partners. Co-expression studies were carried out in cultured cells to examine the effect of binding partners on pendrin trafficking and activity. Results: The Y2H studies identified IQ motif-containing GTPase-activating protein 1 (IQGAP1) as a protein that binds to SLC26A4’s C-terminus. Co-immunoprecipitation experiments using affinity purified anti-IQGAP1 antibodies followed by western blot analysis of kidney protein eluates using pendrin-specific antibodies confirmed the interaction of pendrin and IQGAP1. Immunofluorescence microscopy studies demonstrated that IQGAP1 co-localizes with pendrin on the apical membrane of B-intercalated cells, whereas it shows basolateral expression in A-intercalated cells in the cortical collecting duct (CCD). Functional and confocal studies in HEK-293 cells, as well as confocal studies in MDCK cells, demonstrated that the co-transfection of pendrin and IQGAP1 shows strong co-localization of the two molecules on the plasma membrane along with enhanced Cl−/HCO3 − exchanger activity. Conclusion: IQGAP1 was identified as a protein that binds to the C-terminus of pendrin in B-intercalated cells. IQGAP1 co-localized with pendrin on the apical membrane of B-intercalated cells. Co-expression of IQGAP1 with pendrin resulted in strong co-localization of the two molecules and increased the activity of pendrin in the plasma membrane in cultured cells. We propose that pendrin’s interaction with IQGAP1 may play a critical role in the regulation of CCD function and physiology, and that disruption of this interaction could contribute to altered pendrin trafficking and/or activity in pathophysiologic states.