Usher综合征相关基因SLC4A7在视网膜中的作用机制

The electroneutral Na+/HCO3- cotransporter NBCn1 (SLC4A7), a member of solute carrier 4 (SLC4) family, is one of the major players in the regulation of intracellular pH with demonstrated broad physiological and pathological significance. NBCn1 is highly expressed in the central nervous system including the retina, and is also expressed in the cochlea. In the photoreceptor cells, NBCn1 is present in the synaptic region. By knock-out study with mouse and genetic studies with human, NBCn1 has been associated with Usher syndrome-a hereditary sensory disorder characteristic of blindness and deafness, due to sensory neuron degeneration. In addition, NBCn1 is also shown to be involved in breast cancer and hypertension..In contrast to the extremely important significance of NBCn1, litter is known about the mechanisms underlying the functional regulation of NBCn1. The SLC4A7 gene is able to produce as many as 48 NBCn1 variants, differing in the extreme Nt and the presence/absence of four major alternative cassettes: cassette I through IV. The full-length NBCn1 have a large cytosolic amino-terminus, a transmembrane domain, and a short cytosolic carboxyl terminus (Ct) which contains the alternative cassette IV. Our preliminary study has shown that cassette IV plays stimulatory effect on the transport activity of NBCn1, suggesting that cassette IV play a key role in the functional regulation of NBCn1. We then performed yeast-two-hybrid with NBCn1 Ct as bait, and identified Dab2 which is a phosphotyrosine-binding-domain-containing protein, and ARIP2 (activin receptor 2) which is a PDZ-domain containing protein. Others have shown that both Dab2 and ARIP2 are involved in the functional regulation of transmembrane proteins via endocytosis. By RT-PCR, we found that both Dab2 and ARP2 are expressed at high abundance in mouse eyes. Taking together, we hypothesize that Dab2 and ARIP2 form protein complex with NBCn1 and play an important role in the functional regulation of NBCn1 in retina..In the present planned study, I will: (1) demonstrate the molecular mechanism of protein interaction between NBCn1 and Dab2 as well as ARIP2; (2) examine whether Dab2 and ARIP2 colocalize with and form complex with NBCn1 in the retina; (3) examine the effect of Dab2 and ARIP2 on the physiological activity as well as the subcellular localization of NBCn1 in cells. This study will provide key insights into our understanding of the physiological as well as pathological roles of NBCn1 in the retina.

碳酸氢根转运体SLC4A7（简称A7）是一个重要的酸碱平衡调控蛋白。基因敲除及人类遗传学研究证实A7是一个Usher综合征（视网膜色素变性及听觉感音性耳聋）致病基因。然而，人们对于A7的功能调控机制还所知甚少。在此，我拟通过蛋白相互作用研究，探讨A7在视网膜神经细胞中的作用机理。在前期研究中，我们以A7的羧基端为诱饵，通过酵母双杂交筛选获得了相互作用蛋白Dab2和ARIP2（可通过参与胞吞作用，动态调控膜蛋白在质膜上的生物学功能）。我推测这两个蛋白与A7形成蛋白复合体，调控其在质膜上的表达水平，从而对胞内的酸碱平衡进行态调控。在此，我拟研究：（1）这两个蛋白与A7相互作用的分子机制（确定相互作用位点）；（2）它们与A7在视网膜中的表达及精细共定位；（3）它们对A7在细胞膜上的动态分布及其生物学功能的影响。本研究将为认识A7在视网膜中的功能调控机制及其与视网膜色素变性的关系提供科学依据。

NBCn1是一个Na+-HCO3–共转运体，在机体酸碱平衡调控中具有重要作用。编码NBCn1的SLC4A7是一个重要的疾病相关基因，与Usher综合征、乳腺癌、高血压、心肌肥大等多种疾病有关。SLC4A7基因可以表达多种不同的NBCn1异构体，这些异构体有两种不同的氨基端（Nt），有三个已知的结构可变区：cassette I，II，III。这些cassette由外显子的选择性剪接产生。NBCn1的这些结构可变区（不同Nt，以及cassette）的功能相关性仍然未知。其次，关于NBCn1的功能调控机制在很大程度上依然不清楚。.在本项目的研究中，我们在以下几个方面取得了一些积极进展：.(1).SLC4A7基因结构（表达机制）。我们阐明了SLC4A7基因具有两个不同的转录启动子，分别控制四类不同Nt NBCn1的表达（其中两类是我们首次发现）。此外，我们揭示了SLC4A7基因中存在的三个新的选择性剪接外显子，一个新的选择性结构单元，即cassette IV，克隆了一系列新的NBCn1异构体，使总数从已知的6种增加到18种，大大扩展了对SLC4A7基因表达产物多样性的认识。.(2).NBCn1结构与功能分析。利用生物化学及生物物理学手段，完成了NBCn1不同异构体在模式细胞（非洲爪蟾卵母细胞）中的表达及活性分析，发现cassette II，III，IV是NBCn1中重要的功能结构域，具有增强NBCn1离子转运活性的作用。.(3).ARIP2与NBCn1相互作用机制及对NBCn1功能的影响：通过pull-down技术进一步验证了NBCn1和ARIP2的相互作用，通过酵母双杂交技术阐明了NBCn1与ARIP2相互作用的结构基础，通过模式细胞外源表达并结合免疫学手段，研究了ARIP2对NBCn1蛋白表达的影响，初步探讨了ARIP2对NBCn1功能潜在的影响。.(4).NBCn1与Dab2的相互作用机制：通过酵母双杂交研究了NBCn1与不同Dab2异构体的相互作用。.以上的研究成果对于进一步深入研究SLC4A7基因的生理学作用，以及NBCn1结构与功能的关系都具有积极意义，也为我们进一步研究NBCn1的功能调控就机制打下了良好的基础。.在项目资助下，我们共发表SCI论文2篇，参加国际会议2次，培养研究生6人。