Laboratory of Bio-otology, Department of Otolaryngology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
J Biol Chem. 2013 Mar 8;288(10):7215-29. doi: 10.1074/jbc.M112.443226. Epub 2013 Jan 17.
Previously, we obtained evidence for a photoreceptor/olfactory type of CNGA3 transcript in a purified teleost vestibular hair cell preparation with immunolocalization of CNGA3 protein to stereocilia of teleost vestibular and mammalian cochlear hair cells. The carboxyl terminus of highly Ca(2+)-permeable CNGA3 expressed in the mammalian organ of Corti and saccular hair cells was found to interact with an intracellular domain of microfibril interface-located protein 1 (EMILIN 1), a member of the elastin superfamily, also immunolocalizd to hair cell stereocilia (Selvakumar, D., Drescher, M. J., Dowdall, J. R., Khan, K. M., Hatfield, J. S., Ramakrishnan, N. A., and Drescher, D. G. (2012) Biochem. J. 443, 463-476). Here, we provide evidence for organ of Corti proteins, of Ca(2+)-dependent binding of the amino terminus of CNGA3 specifically to the carboxyl terminus of stereocilia tip-link protein CDH23 +68 (cadherin 23 with expressed exon 68) by yeast two-hybrid mating and co-transformation protocols, pulldown assays, and surface plasmon resonance analysis. Myosin VIIa, required for adaptation of hair cell mechanotransduction (MET) channel(s), competed with CDH23 +68, with direct Ca(2+)-dependent binding to the amino terminus of CNGA3. Based upon the premise that hair cell stereocilia tip-link proteins are closely coupled with MET, these results are consistent with the possibility that CNGA3 participates in hair-cell MET. Together with the demonstration of protein-protein interaction between HCN1 and tip-link protein protocadherin 15 CD3 (Ramakrishnan, N. A., Drescher, M. J., Barretto, R. L., Beisel, K. W., Hatfield, J. S., and Drescher, D. G. (2009) J. Biol. Chem. 284, 3227-3238; Ramakrishnan, N. A., Drescher, M. J., Khan, K. M., Hatfield, J. S., and Drescher, D. G. (2012) J. Biol. Chem. 287, 37628-37646), a protein-protein interaction for CNGA3 and a second tip-link protein, CDH23 +68, further suggests possible association of two different channels with a single stereocilia tip link.
先前，我们通过免疫定位 CNGA3 蛋白在纯化的硬骨鱼类前庭毛细胞制剂中的位置，获得了感光器/嗅觉型 CNGA3 转录本的证据。在哺乳动物耳蜗毛细胞和囊斑毛细胞的 CNGA3 中，高钙通透性的羧基末端被发现与细胞内微纤维界面定位蛋白 1（EMILIN 1）的一个结构域相互作用，该蛋白属于弹性蛋白超家族，也免疫定位在毛细胞的静纤毛（Selvakumar，D.，Drescher，M. J.，Dowdall，J. R.，Khan，K. M.，Hatfield，J. S.，Ramakrishnan，N. A.，和 Drescher，D. G.（2012）Biochem. J. 443, 463-476）。在这里，我们提供了证据表明，在器官中，钙依赖性结合蛋白的氨基酸末端与静纤毛尖端连接蛋白 CDH23+68（带有表达外显子 68 的钙粘蛋白 23）的羧基末端特异性结合，通过酵母双杂交交配和共转化方案、下拉测定和表面等离子体共振分析。肌球蛋白 VIIa，是毛细胞机械转导（MET）通道适应所必需的，与 CDH23+68 竞争，与 CNGA3 的氨基酸末端直接发生钙依赖性结合。基于毛细胞静纤毛尖端连接蛋白与 MET 紧密偶联的前提，这些结果与 CNGA3 参与毛细胞 MET 的可能性一致。与 HCN1 和尖端连接蛋白原钙粘蛋白 15 CD3 之间的蛋白-蛋白相互作用的证明（Ramakrishnan，N. A.，Drescher，M. J.，Barretto，R. L.，Beisel，K. W.，Hatfield，J. S.，和 Drescher，D. G.（2009）J. Biol. Chem. 284, 3227-3238；Ramakrishnan，N. A.，Drescher，M. J.，Khan，K. M.，Hatfield，J. S.，和 Drescher，D. G.（2012）J. Biol. Chem. 287, 37628-37646），一个 CNGA3 和第二个尖端连接蛋白 CDH23+68 的蛋白-蛋白相互作用，进一步表明两个不同的通道可能与单个静纤毛尖端连接相关联。
