THE PDZ DOMAIN OF HUMAN ERYTHROCYTE P55 MEDIATES ITS BINDING TO THE CYTOPLASMIC CARBOXYL-TERMINUS OF GLYCOPHORIN-C - ANALYSIS OF THE BINDING INTERFACE BY IN-VITRO MUTAGENESIS

The PDZ domain, also known as the GLGF repeat/DHR domain, is an simila r to 90-amino acid motif discovered in a recently identified family of proteins termed MAGUKs (membrane-associated guanylate kinase homologu es), Sequence comparison analysis has since identified PDZ domains in over 50 proteins, Like SH2 and SH3 domains, the PDZ domains mediate sp ecific protein protein interactions, whose specificities appear to be dictated by the primary structure of the PDZ domain as well as its bin ding target, Using recombinant fusion proteins and a blot overlay assa y, we show that a single copy of the PDZ domain in human erythrocyte p 55 binds to the carboxyl terminus of the cytoplasmic domain of human e rythroid glycophorin C. Deletion mutagenesis of 21 amino acids at the amino terminus of the p55 PDZ domain completely abrogates its binding activity for glycophorin C. Using an alanine scan and surface plasmon resonance technique, we identify residues in the cytoplasmic domain of glycophorin C that are critical for its interaction with the PDZ doma in, The recognition specificity of the p55 PDZ domain appears to be un ique, since the three PDZ domains of hDlg (human lymphocyte homologue of the Drosophila discs large tumor suppressor) do not bind the cytopl asmic domain of glycophorin C. Taken together with our previous studie s, these results complete the identification of interacting domains in the ternary complex between p55, glycophorin C, and protein 4.1, Impl ications of these findings are discussed in terms of binding specifici ty and the regulation of cytoskeleton-membrane interactions.