Ankyrin-G in renal epithelia: Identification of novel isoforms and the interaction between ankyrin-G and H+/K+-ATPase alpha 2 subunit

Ankyrins are multifunctional membrane adaptors which link diverse membrane proteins to the spectrin-based membrane cytoskeleton. Ankyrin-G (Ank-G, encoded by the Ank-3 gene) is broadly expressed and exhibits alternative splicing in different tissues, giving rise to isoforms ranging in Mr from 100-480 kDa. However, little is known about the alternative promoter usage and alternative splicing patterns of the Ank-3 gene in kidney, particularly at the N-terminus. In the first study, a novel full-length Ank-G cDNA and two novel Ank-G amino termini were isolated from mouse kidney by cDNA library screening and RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE). Their distinct 5' splicing patterns and their transcription start sites were also defined. Putative promoter regions were cloned and their activities were examined by using sequential deletion analysis in different kidney cell lines. Our data demonstrate that alternative promoter usage and alternative splicing create the transcriptome complexity of Ank-G in mouse kidney.;The apical localization of H+/K+-ATPase alpha2 (HKalpha2) in renal epithelial cells is required for its role in acid-base and K+ homeostasis. There is limited information on the mechanisms contributing to the segregation of HKalpha2 to the apical membrane domain of the collecting duct epithelial cells. Ank-G is localized to both the apical and lateral domains in renal epithelia and has been reported to contribute to the specific domain localization of integral membrane proteins. In the second study, we performed overlay assays and pull-down experiments to demonstrate a direct interaction between the HKalpha2 and Ank-G. It was found that the ankyrin repeat domain, spectrin-binding domain of Ank-G, and amino-terminus, the first cytoplasmic loop (Loop1) of HK alpha2 are involved in the interaction. An interaction between Ank-G and HKalpha2 was corroborated by co-immunoprecipitation and co-localization studies. These data demonstrate a direct interaction between Ank-G and HKalpha2 which contributes to the apical localization of HKalpha2.;Our studies not only revealed that multifaceted usage of Ank-3 gene contributes to the production of Ank-G transcriptome complexity, but also demonstrated a direct interaction between Ank-G and apical membrane associated integral proteins, significantly extending our current understanding of Ank-G structural and functional diversities.