Amino Acids 563–566 of the Na+/H+ Exchanger Isoform 1 C-Terminal Cytosolic Tail Prevent Protein Degradation and Stabilize Protein Expression and Activity

Abstract

Isoform one of the mammalian Na+/H+ exchanger is a plasma membrane protein that is ubiquitously present in humans. It regulates intracellular pH through the removal of one intracellular proton in exchange for a single extracellular sodium. It consists of a 500 amino acid membrane domain plus a 315 amino acid, C-terminal tail. We examined amino acids of the C-terminal tail that are important in the targeting and activity of the protein. A previous study demonstrated that stop codon polymorphisms can result in decreased activity, expression, targeting and enhanced protein degradation. Here, we determine elements that are critical in these anomalies. A series of progressive deletions of the C-terminal tail demonstrated a progressive decrease in activity and targeting, though these remained until a final drop off with the deletion of amino acids 563–566. The deletion of the 562LIAGERS568 sequence or the alteration to the 562LAAAARS568 sequence caused the decreased protein expression, aberrant targeting, reduced activity and enhanced degradation of the Na+/H+ exchanger (NHE1) protein. The 562LIAGERS568 sequence bound to other regions of the C-terminal cytosolic domain. We suggest this region is necessary for the activity, targeting, stability, and expression of the NHE1 protein. The results define a new sequence that is important in maintenance of NHE1 protein levels and activity.

Rainbow Trout (Oncorhynchus mykiss) Na+/H+ Exchangers tNhe3a and tNhe3b Display Unique Inhibitory Profiles Dissimilar from Mammalian NHE Isoforms

“…The likely explanation is that the protein was not well targeted to the cell surface, as shown by our localization experiments. The mammalian NHE1 C-terminal, tail has several regions that are important in targeting the protein to the cell surface and when these are absent, targeting to the cell surface is reduced [ 51 ]. These targeting sequences are not present in the tNhe3a and tNhe3b isoforms.…”

Roles of the Na+/H+ Exchanger Isoform 1 and Urokinase in Prostate Cancer Cell Migration and Invasion

“…The larger band corresponds to the full-length glycosylated protein, while the smaller band corresponds to the partial or deglycosylated NHE1 protein [28]. A smaller nonspecific immunoreactive band was present both in the wild-type and knockout cells, as well as in other cell types [29], and for unknown reasons was more abundant in the knockout cells. Re-probing the blot with anti-tubulin antibodies confirmed that the cell extract from knockout cells was present in equivalent amounts.…”

“…The secondary antibody used was peroxidase-conjugated goat anti-mouse antibody (Bio-Can, Mississauga, ON, Canada). To examine the reintroduction of HA-tagged NHE1, we used anti-HA monoclonal antibody [29].…”

“…Intracellular pH was measured using 2 ,7-bis(2-carboxyethyl)-5(6) carboxyfluorescein acetoxymethyl ester (BCECF-AM), in a similar manner to that described in [29]. DU 145 cells were grown to ~80-90% confluence on glass coverslips.…”

“…To return the human NHE1 protein to the knockout cells, we transfected DU-145knockout cells with the plasmid pYN4+ that contains human NHE1 cDNA with a HA (hemagglutinin) tag, as described [29]. Briefly, cells were transfected with the NHE1-containing plasmid using Lipofectamine™ 2000 reagent, as described above.…”