Carbonic anhydrase II binds to and enhances activity of the Na+/H+ exchanger.
We examined the ability of carbonic anhydrase II to bind to and affect the transport efficiency of the NHE1 isoform of the mammalian Na(+)/H(+) exchanger. The C-terminal region of NHE1 was expressed in Escherichia coli fused with an N-terminal glutathionine S-transferase or with a C-terminal polyhistidine tag. Using a microtiter ... plate binding assay we showed that the C-terminal region of NHE1 binds carbonic anhydrase II (CAII) and binding was stimulated by low pH and blocked by antibodies against the C-terminal of NHE1. The binding to NHE1 was confirmed by demonstrating protein-protein interaction using affinity blotting with CAII and immobilized NHE1 fusion proteins. CAII co-immunoprecipitated with NHE1 from CHO cells suggesting the proteins form a complex in vivo. In cells expressing CAII and NHE1, the H(+) transport rate was almost 2-fold greater than in cells expressing NHE1 alone. The CAII inhibitor acetazolamide significantly decreased the H(+) transport rate of NHE1 and transfection with a dominant negative CAII inhibited NHE1 activity. Phosphorylation of the C-terminal of NHE1 greatly increased the binding of CAII. Our study suggests that NHE1 transport efficiency is influenced by CAII, likely through a direct interaction at the C-terminal region. Regulation of NHE1 activity by phosphorylation could involve modulation of CAII binding.
Mesh Terms:
Animals, Binding Sites, Blotting, Western, CHO Cells, Carbonic Anhydrase II, Cricetinae, Dose-Response Relationship, Drug, Electrophoresis, Polyacrylamide Gel, Histidine, Hydrogen-Ion Concentration, Phosphorylation, Precipitin Tests, Protein Binding, Protein Structure, Tertiary, Recombinant Fusion Proteins, Sodium-Hydrogen Antiporter, Time Factors, Transfection
Animals, Binding Sites, Blotting, Western, CHO Cells, Carbonic Anhydrase II, Cricetinae, Dose-Response Relationship, Drug, Electrophoresis, Polyacrylamide Gel, Histidine, Hydrogen-Ion Concentration, Phosphorylation, Precipitin Tests, Protein Binding, Protein Structure, Tertiary, Recombinant Fusion Proteins, Sodium-Hydrogen Antiporter, Time Factors, Transfection
J. Biol. Chem.
Date: Sep. 27, 2002
PubMed ID: 12138085
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