Abstract
Disorganized ion transport caused by hypo- or hyperfunctioning of the cystic fibrosis transmembrane conductance regulator (CFTR) can be detrimental and may result in life-threatening diseases such as cystic fibrosis or secretory diarrhea. Thus, CFTR is controlled by elaborate positive and negative regulations for an efficient homeostasis. It has been shown that expression and activity of CFTR can be regulated either positively or negatively by PDZ (PSD-95/discs large/ZO-1) domain-based adaptors. Although a positive regulation by PDZ domain-based adaptors such as EBP50/NHERF1 is established, the mechanisms for negative regulation of the CFTR by Shank2, as well as the effects of multiple adaptor interactions, are not known. Here we demonstrate a physical and physiological competition between EBP50-CFTR and Shank2-CFTR associations and the dynamic regulation of CFTR activity by these positive and negative interactions using the surface plasmon resonance assays and consecutive patch clamp experiments. Furthermore whereas EBP50 recruits a cAMP-dependent protein kinase (PKA) complex to CFTR, Shank2 was found to be physically and functionally associated with the cyclic nucleotide phosphodiesterase PDE4D that precludes cAMP/PKA signals in epithelial cells and mouse brains. These findings strongly suggest that balanced interactions between the membrane transporter and multiple PDZ-based adaptors play a critical role in the homeostatic regulation of epithelial transport and possibly the membrane transport in other tissues.
Highlights
Research Foundation, Ministry of Education and Human Resources Development, Korea and Grant 03-PJ10-PG13-GD01-0002 from the Korea Health 21 R&D Project, Ministry of Health and Welfare, Korea
Recent evidence demonstrates that native CFTR and EBP50 are co-immunoprecipitated in human airway epithelial cells [15] and that CFTRdependent anion current is reduced in the intestine of EBP50 knock-out mice [6]
His-tagged PDZ domains were captured on NTA chips, and their binding to the GST-tagged C terminus of CFTR was sensored by surface plasmon resonance (SPR)
Summary
Research Foundation, Ministry of Education and Human Resources Development, Korea and Grant 03-PJ10-PG13-GD01-0002 from the Korea Health 21 R&D Project, Ministry of Health and Welfare, Korea We reported functional and physical associations between the PDZ domain-containing protein Shank2 and two epithelial transporters, CFTR and the Naϩ/Hϩ exchanger 3 (NHE3) [11, 12]. Consecutive patch clamp studies revealed that CFTR ClϪ channel activity was dynamically regulated by the competition of Shank2 and EBP50 binding.
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