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.
Festy
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INSERM U410, Faculté de Médecine X. Bichat, 16 Rue Henri Huchard, 75018 Paris, France.
Robert
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JC
Brasseur
R
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Thomas
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A
D000595:Amino Acid Sequence; D000818:Animals; D017487:Ankyrins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D002846:Chromatography, Affinity; D017506:H(+)-K(+)-Exchanging ATPase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D011817:Rabbits; D013049:Spectrin / Q000378:metabolism*; D013270:Stomach / Q000201:enzymology*
D017487:Ankyrins; D013049:Spectrin; D017506:H(+)-K(+)-Exchanging ATPase