SPECIFIC AIMS Our overall objective is to characterize the functional and regulatory properties (common and distinct) of the AE anion exchanger gene family proteins, and to ascribe those properties to defined amino acid residues or domains of the proteins. The proposed experiments are designed to develop an understanding of the mechanisms by which the AE anion exchanger proteins function as regulated ion transporters and interact directly with other proteins. To accomplish these objectives, we propose: 1. To extend the functional analyses of AE1, AE2, and AE3 isoforms in two heterologous expression systems: the Xenopus oocyte and the transiently transfected CHOP cell. 2. To assess the functional and biochemical properties of proteins encoded by defined mutant AE1 cDNAs from patients with hereditary spherocytosis and other erythroid disorders. To mutate AE2 and AE3 in those conserved residues and assess the functional consequences. 3. To define the molecular bases of AE protein regulation by defining the amino acids which mediate various regulatory influences: 4. To ascertain the role of oligomerization state, glycosylation, and fatty acylation on the function and regulation of AE1, AE2, and AE3. 5. To create and select for second-site revertants in loss-of-function mutants, in order to provide information on secondary and tertiary structure of AE proteins. 6. To identify, clone, and characterize proteins which bind to the cytoplasmic domains of AE2 and AE3, and to delineate their binding sites within the AE protein sequences.
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