title: Identification of a band-3 binding site near the N-terminus of erythrocyte membrane protein 4.2.
abstract: Protein 4.2 (P4.2) is a major component of the erythrocyte plasma membrane accounting for approx. 5% of total membrane protein. The major membrane binding site for P4.2 is contained within the cytoplasmic domain of band 3 (cdb3), although the precise location of the cdb3 binding site is not known. To identify the cdb3 binding site, we used synthetic P4.2 peptides (15-mers) that spanned the entire 721-amino-acid large isoform of P4.2, and determined the binding of these peptides to cdb3 in an in vitro binding assay. One peptide, P8 (L61FVRRGQPFTIILYF), bound strongly to cdb3 and four others bound less strongly (P22, L271LNKRRGSVPILRQW; P27, G346EGQRGRIWIFQTST; P41, L556WRKKLHLTLSANLE; P48, I661HRERSYRFRSVWPE). These peptides have in common a cluster of two or three basic amino acid residues (arginine or lysine), in a region without nearby acidic residues. Cdb3 bound saturably to P8 with a Kd of 0.16 microM and a capacity of 0.56 mol of cdb3 monomer/mol of P8. Use of overlapping synthetic peptides further defined the cdb3 site as being contained within V63RRGQPFTIILYF. Replacement of R64R with R64G, G64R or G64G almost completely abolished cdb3 binding, suggesting that R64R is essential for cdb3 binding. P8 competitively inhibited binding of purified human erythrocyte P4.2 to cdb3. In blot overlay assays, cdb3 bound to a 23 kDa N-terminal P4.2 tryptic peptide containing V63RRGQPFTIILYF but not to other P4.2 tryptic peptides lacking this site. The V63RRGQPFTIILYF site is highly conserved in mouse and human erythrocyte P4.2 as well as between P4.2 and transglutaminase proteins, which are evolutionarily related to P4.2.
authors.affiliation: Albert Einstein College of Medicine-Montefiore Medical Center, Division of Hematology, Bronx, NY 10467, USA.
authors.forename: A C
authors.initials: AC
authors.lastname: Rybicki
authors.forename: S
authors.initials: S
authors.lastname: Musto
authors.forename: R S
authors.initials: RS
authors.lastname: Schwartz
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001457:Anion Exchange Protein 1, Erythrocyte / Q000378:metabolism*; D001665:Binding Sites; D001798:Blood Proteins / Q000737:chemistry / Q000378:metabolism*; D003598:Cytoskeletal Proteins; D004910:Erythrocyte Membrane / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014357:Trypsin
chemical_list: D001457:Anion Exchange Protein 1, Erythrocyte; D001798:Blood Proteins; D003598:Cytoskeletal Proteins; D008565:Membrane Proteins; D010455:Peptides; C044791:erythrocyte membrane band 4.2 protein; D014357:Trypsin