PROTEIN-4.1 DEFICIENCY ASSOCIATED WITH AN ALTERED BINDING TO THE SPECTRIN-ACTIN COMPLEX OF THE RED-CELL MEMBRANE SKELETON

Protein 4.1 has been defined as a major component of the subcortical s keleton of erythrocytes. It binds the spectrin-actin scaffold through a 10-kD internal domain. This binding requires an essential 21-amino a cid sequence motif, Motif I, which is retained by alternative splicing at the late stage of erythroid differentiation. We here analyze the m olecular basis of heterozygous 4.1(-) hereditary elliptocytosis, assoc iated with protein 4.1 partial deficiency, in nine related French fami lies. cDNA sequencing revealed a single codon deletion (AAA) resulting in a lysine residue deletion within the 10-kD binding domain, 3' of M otif I. The mutated allele was designated allele 4.1 Aravis. In order to assess the functional effect of the codon deletion, recombinant 10- kD constructs were made and various binding assays were performed usin g spectrin, purified spectrin-actin complex, or red cell membranes. Th ese experiments demonstrated that the deletion of the Lys residue clea rly prevents the binding capacity. Similar results were obtained with a construct containing the Lys residue but lacking Motif I. These data strongly suggest that the binding site to the spectrin-actin complex must contain the Lys 447 (or 448), and therefore resides not only on M otif I but extends 3' of this essential motif.