Interaction between the N-terminal domain of gastric H,K-ATPase and the spectrin binding domain of ankyrin III

We screened a cDNA bank of rabbit gastric fundic mucosa by two-hybrid assay s looking for binding partners of the N-terminal domain of the rabbit gastr ic H,K-ATPase. We extracted five clones sharing more than 90% sequence iden tity. The longest clone codes for a protein sharing a high identity (96 and 96.8%, respectively) with a fragment of the membrane domain, from Arg-835 to Ser-873, plus the major part of the "spectrin binding domain" going from Glu-874 to Leu-1455 of human and mouse ankyrin III. We conclude that the m embrane and spectrin binding domains of the rabbit ankyrin III are candidat es for the binding partner of the N terminal domain of the rabbit gastric H ,K-ATPase. To validate the ankyrin-ATPase interaction and to test its speci ficity, we produced both domains in yeast and bacteria, coimmunoprecipitate d them with an anti-ATPase antibody, and copurified them by affinity chroma tography. The sequence of rabbit ankyrin III was not known, and this is the first report demonstrating that the ankyrin III and the H,K-ATPase interac t with no intermediate. The interaction involves the N-terminal domain of t he ATPase on one hand and the spectrin binding domain of the ankyrin on the other.