DEFINING OF THE MINIMAL DOMAIN OF PROTEIN-4.1 INVOLVED IN SPECTRIN-ACTIN BINDING

The spectrin-actin-binding domain of protein 4.1 is encoded by a 21-am ino acid alternative exon and a 59-amino acid constitutive exon, To ch aracterize the minimal domain active for interactions with spectrin an d actin, we functionally characterized recombinant 4.1 peptides contai ning the 21-amino acid cassette plus varying portions of the 59-amino acid cassette (designated 21.10 to 21.59). Peptide 21.43 was shown ful ly functional in binary interactions with spectrin (by cosedimentation and coimmunoprecipitation experiments) and in ternary complex formati on with spectrin and actin (by an in vitro gelation assay), Further tr uncation produced peptides incapable of binary interactions but fully competent for ternary complex formation (peptides 21.36 and 21.31), sh orter peptides with reduced ternary complex activity and altered kinet ics (21.26 and 0.59), and inactive peptides (21.20 and 21.10), Binding studies and circular dichroism experiments suggested that residues 37 -43 of the constitutive domain were directly involved in spectrin bind ing. These data indicate that 4.1-spectrin binary interaction requires the 21-amino acid alternative cassette plus the 43 N-terminal residue s of the constitutive domain, Moreover, the existence of two possible ternary complex assembly pathways is suggested: one initiated by 4.1-s pectrin interactions, and a second by 4.1-actin interactions, The latt er may require a putative actin binding motif within the 26 N-terminal residues of the constitutive domain.