The major kidney AE1 isoform does not bind ankyrin (Ank1) in vitro. An essential role for the 79 NH2-terminal amino acid residues of band 3.
The AE1 (band 3) protein mediates the exchange of anions across the erythrocyte plasma membrane and, via association with the adapter molecule, ankyrin (Ank1), forms the major link between the membrane and the underlying spectrin cytoskeleton. The major kidney isoform of AE1 (kAE1), a protein that is otherwise identical to erythroid AE1 but lacks the NH2-terminal 79 amino acids, is localized to the basolateral plasma membrane of acid-secreting (alpha-type) intercalated cells of distal nephron. It has been proposed that this polarized distribution of kAE1 is due, at least in part, to its association with the ankyrin-spectrin cytoskeleton. In this study, we have used cell-free binding assays to investigate the interaction of anion exchangers with an Ank1 fragment, R13-H, that contains the AE1 binding site. Microsomes from cells expressing full-length erythroid AE1 bound 125I-labeled R13-H, revealing the presence of both high (Kd = 12.5 nM) and low (Kd = 166 nM) affinity sites. This binding was specific, as evidenced by the failure to observe high affinity binding of 125I-R13-H to microsomes from cells transfected with vector alone or with AE1m, a mutant lacking the approximately 400 amino acid NH2-terminal cytoplasmic ankyrin binding domain. Using this assay, we could detect no high affinity association between kAE1 and 125I-R13-H. We conclude that the NH2-terminal 79 amino acids play an essential role in high affinity and specific binding of AE1 to Ank1.
Ding
Y
Y
Department of Biological Sciences, Stanford University, California 94305-5020.
Casey
J R
JR
Kopito
R R
RR
D000596:Amino Acids / Q000378:metabolism*; D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry / Q000378:metabolism*; D000837:Anion Exchange Resins; D017487:Ankyrins / Q000378:metabolism*; D001483:Base Sequence; D002478:Cells, Cultured; D006801:Humans; D007668:Kidney / Q000378:metabolism*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D011485:Protein Binding
D000596:Amino Acids; D001457:Anion Exchange Protein 1, Erythrocyte; D000837:Anion Exchange Resins; D017487:Ankyrins; D009838:Oligodeoxyribonucleotides