Kizhatil Krishnakumar, Yoon Woohyun, Mohler Peter J, Davis Lydia H, Hoffman Janis A, Bennett Vann
Howard Hughes Medical Institute and Departments of Cell Biology, Biochemistry, and Neurobiology, Duke University Medical Center, Durham, North Carolina 27710, USA.
J Biol Chem. 2007 Jan 19;282(3):2029-37. doi: 10.1074/jbc.M608921200. Epub 2006 Oct 29.
Ankyrins are a family of adapter proteins required for localization of membrane proteins to diverse specialized membrane domains including axon initial segments, specialized sites at the transverse tubule/sarcoplasmic reticulum in cardiomyocytes, and lateral membrane domains of epithelial cells. Little is currently known regarding the molecular basis for specific roles of different ankyrin isoforms. In this study, we systematically generated alanine mutants of clusters of charged residues in the spectrin-binding domains of both ankyrin-B and -G. The corresponding mutants were evaluated for activity in either restoration of abnormal localization of the inositol trisphosphate receptor in the sarcoplasmic reticulum in mutant mouse cardiomyocytes deficient in ankyrin-B or in prevention of loss of lateral membrane in human bronchial epithelial cells depleted of ankyrin-G by small interfering RNA. Interestingly, ankyrin-B and -G share two homologous sites that result in loss of function in both systems, suggesting that common molecular interactions underlie diverse roles of these isoforms. Ankyrins G and B also exhibit differences; mutations affecting spectrin binding had no effect on ankyrin-B function but did abolish activity of ankyrin-G in restoring lateral membrane biogenesis. Depletion of beta(2)-spectrin by small interfering RNA phenocopied depletion of ankyrin-G and resulted in a failure to form new lateral membrane in interphase and mitotic cells. These results demonstrate that ankyrin-G and beta(2)-spectrin are functional partners in biogenesis of the lateral membrane of epithelial cells.
锚蛋白是一类衔接蛋白，对于膜蛋白定位于多种特殊膜结构域至关重要，这些膜结构域包括轴突起始段、心肌细胞横管/肌浆网的特化位点以及上皮细胞的侧膜结构域。目前对于不同锚蛋白亚型发挥特定作用的分子基础了解甚少。在本研究中，我们系统地构建了锚蛋白-B和 -G的血影蛋白结合结构域中带电荷残基簇的丙氨酸突变体。通过小干扰RNA使锚蛋白-B缺陷的突变小鼠心肌细胞中肌醇三磷酸受体在肌浆网中的异常定位得以恢复，或者使锚蛋白-G缺失的人支气管上皮细胞中侧膜得以维持，以此来评估相应突变体的活性。有趣的是，锚蛋白-B和 -G有两个同源位点，在这两个系统中均导致功能丧失，这表明这些亚型的不同作用有着共同的分子相互作用基础。锚蛋白G和B也存在差异；影响血影蛋白结合的突变对锚蛋白-B的功能没有影响，但确实消除了锚蛋白-G在恢复侧膜生物发生方面的活性。通过小干扰RNA使β(2)-血影蛋白缺失可模拟锚蛋白-G缺失的情况，并导致间期细胞和有丝分裂细胞无法形成新的侧膜。这些结果表明，锚蛋白-G和β(2)-血影蛋白是上皮细胞侧膜生物发生中的功能伙伴。
