The Na⁺/H⁺ Exchanger Cytoplasmic Tail:  Structure, Function, and Interactions with Tescalcin

Abstract

We characterized the regulatory cytoplasmic tail of the Na(+)/H(+) exchanger using a histidine-tagged protein containing the C-terminal 182 amino acids (His182). Both tescalcin and calmodulin, two Na(+)/H(+) exchanger binding proteins, bound to the His182 protein. Cascade blue was used to label the His182 protein. Calcium caused an increase in fluorescence, suggesting exposure of the label on the protein to a more hydrophilic environment. Decreasing external pH caused a transient increase in cascade blue fluorescence, followed by a decrease in fluorescence of the cascade blue labeled Na(+)/H(+) exchanger C-terminus. Tescalcin caused a decrease in fluorescence by labeled His182 protein, and calcium reversed this effect. Expression of tescalcin in vivo inhibited activity of the Na(+)/H(+) exchanger when there was an intact C-terminus of the protein. We examined the CD spectra of His182 in the presence and absence of tescalcin. The C-terminal amino acids demonstrated a very small amount of alpha-helical structure and much more beta-sheet and beta-turn. This was not greatly affected by the presence of tescalcin, but calcium caused an increase in the amount of beta-structure and a decrease in the unstructured proportion of the protein. Sedimentation equilibrium analysis demonstrated that the C-terminal 182 amino acids exist predominantly as a monomer. The results suggest that the C-terminus of the Na(+)/H(+) exchanger exists primarily as a monomeric protein that binds regulatory tescalcin and can change conformation depending on pH and calcium. Conformation changes in this region of the protein may be responsible for altering the pH sensitivity of the intact Na(+)/H(+) exchanger.

Regulation of the Na+/H+ Exchanger (NHE1) in Breast Cancer Metastasis

“…CHP2 is highly expressed in tumor cells; it is protective against serum deprivation-induced cell death by increasing pH i (10) and may play a role in the enhanced proliferation of tumor cells (11). CHP3, or tescalin (binding site uncertain), is thought to promote maturation and cell surface stability of NHE1 (12,13). Another regulatory factor of NHE1 is phosphatidylinositol 4,5-biphosphate (PIP 2 ), which binds at 2 sites between amino acids 513 and 520 and 556 and 564 (Fig.…”

Apoptosis-induced alkalinization by the Na+/H+ exchanger isoform 1 is mediated through phosphorylation of amino acids Ser726 and Ser729

“…The activity of NHE1 is regulated through the large cytosolic domain (amino acids 500 -815). This region forms a compact ␤-structure (30) containing domains for the binding of regulatory factors as well as target sites for phosphorylation. Several protein kinases phosphorylate and regulate the activity of NHE1, including Nck-interacting kinase (49), p160ROCK (45), and p90 rsk , a downstream substrate of ERK1/2.…”

Emerging roles of the single EF-hand Ca2+ sensor tescalcin in the regulation of gene expression, cell growth and differentiation

“…The structural basis and functional role of the NHE1-tescalcin interaction has been confirmed and investigated by different laboratories (Li et al, 2003(Li et al, , 2004Zaun et al, 2008). According to the detailed studies of Zaun et al, the amino acid residues of NHE1 that are necessary for tescalcin binding are also crucial for binding of CHP1 and CHP2, and their mutation has a profound effect on the 22 Na + /H + exchange activity.…”

“…To date, all studies have implicated the C-terminus of NHE1 in the interaction with tescalcin, but have identified different regions to be of functional importance, for example, either juxtamembrane sites (Zaun et al, 2008) or the distant C-tail positions (Li et al, 2003). The functional effects of the tescalcin-NHE1 interaction are also being debated, with the former report showing stimulation of NHE1 by tescalcin, whereas the latter demonstrated its suppression.…”