Localization of peripheral membrane protein binding sites on the cytoplasmic domain of erythrocyte band 3
Abstract
An important aspect of the erythrocyte membrane cytoskeleton structure and function is its ability to interact intimately with the membrane. Such an interaction is assured by the association of ankyrin, protein 4.1, and protein 4.2 with the cytoplasmic domain of the integral membrane protein, band 3. To better understand the structure and regulation of the interaction of cdb3 with these membrane cytoskeletal proteins, the binding sites of ankyrin and protein 4.1 were mapped using site-specific probes of cdb3 in peripheral protein binding inhibition assays. It was observed that modification of the two cdb3 sulfhydryls (residues 201 and 317) with number of different thiol reagents as well as native disulfide bond formation all blocked ankyrin binding. Experiments designed to distinguish which cysteine (201 or 317) was most important in ankyrin binding revealed that all four cysteines in the cdb3 dimer lie in a cluster near an ankyrin binding site. Seven antibodies were raised against cdb3 and used in competition studies to identify regions of close association of cdb3 with ankyrin and protein 4.1. For ankyrin, antibody inhibition was observed at sites near the cysteine 201-317 cluster and the proposed proline-rich hinge as well as at the acidic NH$\sb2$-terminus. Additional evidence for interaction with the NH$\sb2$-terminal region of cdb3 was shown by the ability of ankyrin to inhibit tyrosine phosphorylation of cdb3 at its N-terminus by tyrosine kinase p40. Antibodies to the membrane junction and to a highly conserved region of cdb3 (residues 142-154) exhibited no inhibition. For protein 4.1, antibody inhibition occurred at the membrane junction and also at the N-terminus. Antibodies to the hinge and conserved regions of cdb3 did not inhibit. Thus, ankyrin may bind to a site near the hinge and cysteine cluster, and protein 4.1 may bind to a site near the membrane junction with the N-terminal region participating in both interactions. Finally, the essential nature of the band 3$\cdot$ankyrin interaction in erythrocyte membrane stability was shown by specifically inhibiting this association in resealed ghosts (by pH 9.0 treatment or introduction of an inhibitory antibody) and measuring a decrease in membrane stability by ektacytometry.
Degree
Ph.D.
Advisors
Low, Purdue University.
Subject Area
Biochemistry|Immunology
Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server.