Identification of the carbonic anhydrase II binding site in the Cl(-)/HCO(3)(-) anion exchanger AE1.
The human Cl(-)/HCO(3)(-) anion exchanger (AE1) possesses a binding site within its 33 residue carboxyl-terminal region (Ct) for carbonic anhydrase II (CAII). The amino acid sequence comprising this CAII binding site was determined by peptide competition and by testing the ability of truncation and point mutants of the Ct sequence to bind CAII with a sensitive microtiter plate binding assay. A synthetic peptide consisting of the entire 33 residues of the Ct (residues 879-911) could compete with a GST fusion protein of the Ct (GST-Ct) for binding to immobilized CAII, while a peptide consisting of the last 16 residues (896-911) could not. A series of truncation mutants of the GST-Ct showed that the terminal 21 residues of AE1 were not required for binding CAII. Removal of four additional residues (887-890) from the Ct resulted in loss of CAII binding. Acidic residues in this region (D887ADD) were critical for binding since mutating this sequence in the GST-Ct to DAAA, AAAA, or NANN caused loss of CAII binding. A GST-Ct construct mutated to D887ANE, the homologous sequence in AE2, could bind CAII. AE2 is a widely expressed anion exchanger and has a homologous Ct region with 60% sequence identity to AE1. A GST fusion protein of the 33 residue Ct of AE2 could bind to CAII similarly to the Ct of AE1. Tethering of CAII to an acidic motif within the Ct of anion exchangers may be a general mechanism for promoting bicarbonate transport across cell membranes.
Vince
J W
JW
Medical Research Council Group in Membrane Biology, Departments of Medicine and Biochemistry, Room 7344, Medical Sciences Building, University of Toronto, Toronto, Canada M5S 1A8.
Reithmeier
R A
RA
D000595:Amino Acid Sequence; D027321:Anion Transport Proteins; D017920:Antiporters / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D001667:Binding, Competitive; D001692:Biological Transport / Q000235:genetics; D002256:Carbonic Anhydrases / Q000737:chemistry*; D027963:Chloride-Bicarbonate Antiporters; D004800:Enzymes, Immobilized; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments / Q000378:metabolism; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000378:metabolism; D064606:SLC4A Proteins; D016415:Sequence Alignment
D027321:Anion Transport Proteins; D017920:Antiporters; D027963:Chloride-Bicarbonate Antiporters; D004800:Enzymes, Immobilized; D008565:Membrane Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D064606:SLC4A Proteins; D002256:Carbonic Anhydrases