Inhibitory Regulation of Cystic Fibrosis Transmembrane Conductance Regulator Anion-transporting Activities by Shank2

Accumulating evidence suggests that protein-protein interactions play an important role in transepithelial ion transport. In the present study, we report on the biochemical and functional association between cystic fibrosis transmembrane conductance regulator (CFTR) and a PDZ domain-containing protein Shank2. Exploratory reverse transcription-PCR screening revealed mRNAs for several members of PDZ domain-containing proteins in epithelial cells. Shank2, one of these scaffolding proteins, showed a strong interaction with CFTR by yeast two-hybrid assays. Shank2-CFTR interaction was verified by co-immunoprecipitation experiments in mammalian cells. Notably, this interaction was abolished by mutations in the PDZ domain of Shank2. Protein phosphorylation, HCO₃^- transport and Cl^- current by CFTR were measured in NIH 3T3 cells with heterologous expression of Shank2. Of interest, expression of Shank2 suppressed cAMP-induced phosphorylation and activation of CFTR. Importantly, loss of Shank2 by stable transfection of antisense-hShank2 plasmid strongly increased CFTR currents in colonic T84 cells, in which CFTR and Shank2 were natively expressed. Our results indicate that Shank2 negatively regulates CFTR and that this may play a significant role in maintaining epithelial homeostasis under normal and diseased conditions such as those presented by secretory diarrhea.