NHERF1 overexpression increases functional apical expression of F508del CFTR in CFBE41o- cells. Here, we show that this occurs via the formation of the multiprotein complex NHERF1-phosphoezrin-actin, which provides a regulated linkage between F508del CFTR and the actin cytoskeleton resulting in an increased F508del CFTR stability in the membrane.
Although Cystic fibrosis transmembrane conductance regulator (CFTR) has been shown to regulate the activity of NHE3, the potential reciprocal interaction of NHE3 to modulate the protein kinase A (PKA)-dependent regulation of CFTR in epithelial cells is still unknown. In the present work, we describe experiments to define the interactions between CFTR and NHE3 with the regulatory, scaffolding protein, NHERF that organize their PKA-dependent regulation in a renal epithelial cell line that expresses endogenous CFTR. The expression of rat NHE3 significantly decreased PKAdependent activation of CFTR without altering CFTR expression, and this decrease was prevented by mutation of either of the two rat NHE3 PKA target serines to alanine (S552A or S605A). Inhibition of CFTR expression by antisense treatment resulted in an acute decrease in PKA-dependent regulation of NHE3 activity. CFTR, NHE3, and ezrin were recognized by NHERF-2 but not NHERF-1 in glutathione S-transferase pull-down experiments. Ezrin may function as a protein kinase A anchoring protein (AKAP) in this signaling complex, because blocking the binding of PKA to an AKAP by incubation with the S-Ht31 peptide inhibited the PKAdependent regulation of CFTR in the absence of NHE3. In the A6-NHE3 cells S-Ht31 blocked the PKA regulation of NHE3 whereas it now failed to affect the regulation of CFTR. We conclude that CFTR and NHE3 reciprocally interact via a shared regulatory complex comprised of NHERF-2, ezrin, and PKA.
Background information. CF (cystic fibrosis) is a disease caused by mutations within the CFTR (CF transmembrane conductance regulator) gene. The most common mutation, F508 (deletion of Phe-508), results in a protein that is defective in folding and trafficking to the cell surface but is functional if properly localized in the plasma membrane. We have recently demonstrated that overexpression of the PDZ protein NHERF1 (Na + /H + -exchanger regulatory factor 1) in CF airway cells induced both a redistribution of F508CFTR from the cytoplasm to the apical membrane and the PKA (protein kinase A)-dependent activation of F508CFTR-dependent chloride secretion. In view of the potential importance of the targeted up-regulation of NHERF1 in a therapeutic context, and since it has been demonstrated that oestrogen treatment increases endogenous NHERF1 expression, we tested the hypothesis that oestrogen treatment can increase NHERF1 expression in a human bronchiolar epithelial CF cell line, CFBE41o − , with subsequent rescue of apical F508CFTR chloride transport activity.
Results. We found that CFBE41o− cells do express ERs (oestrogen receptors) in the nuclear fraction and that β-oestradiol treatment was able to significantly rescue F508CFTR-dependent chloride secretion in CFBE41o − cell monolayers with a peak between 6 and 12 h of treatment, demonstrating that the F508CFTR translocated to the apical membrane can function as a cAMP-responsive channel, with a significant increase in chloride secretion noted at 1 nM β-oestradiol and a maximal effect observed at 10 nM. Importantly, knock-down of NHERF1 expression by transfection with siRNA (small interfering RNA) for NHERF1 inhibited the β-oestradiol-dependent increase in F508CFTR protein expression levels and completely prevented the β-oestradiol-dependent rescue of F508CFTR transport activity.Conclusions. These results demonstrate that β-oestradiol-dependent up-regulation of NHERF1 significantly increases F508CFTR functional expression in CFBE41o − cells.
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