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 assays
looking for binding partners of the N-terminal domain of the rabbit gastric
H,K-ATPase. We extracted five clones sharing more than 90% sequence identity. 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 membrane and
spectrin binding domains of the rabbit ankyrin III are candidates 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 specificity, we produced both
domains in yeast and bacteria, coimmunoprecipitated them with an anti-ATPase
antibody, and copurified them by affinity chromatography. 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 interact with no intermediate. The interaction
involves the N-terminal domain of the ATPase on one hand and the spectrin binding
domain of the ankyrin on the other.