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 er ythrocyte plasma membrane and, via association with the adapter molecu le, ankyrin (Ank1), forms the major link between the membrane and the underlying spectrin cytoskeleton. The major kidney isoform of AE1 (kAE 1), a protein that is otherwise identical to erythroid AE1 but lacks t he 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-spectri n cytoskeleton. In this study, we have used cell-free binding assays t o investigate the interaction of anion exchangers with an Ank1 fragmen t, R13-H, that contains the AE1 binding site. Microsomes from cells ex pressing full-length erythroid AE1 bound I-125-labeled R13-H, revealin g the presence of both high (K-d = 12.5 nM) and low (K-d = 166 nM) aff inity sites. This binding was specific, as evidenced by the failure to observe high affinity binding of I-125-R13-H to microsomes from cells transfected with vector alone or with AE1m, a mutant lacking the simi lar to 400 amino acid NH2-terminal cytoplasmic ankyrin binding domain, Using this assay, we could detect no high affinity association betwee n kAE1 and I-125-R13-H. We conclude that the NH2-terminal 79 amino aci ds play an essential role in high affinity and specific binding of AE1 to Ank1.