Spectrin Oligomerization is Cooperatively Coupled to Membrane Assembly: A Linkage Targeted by Many Hereditary Hemolytic Anemias?

Abstract

In the erythrocyte, ankyrin is the major adapter protein linking tetramers of band 3 to the spectrin-actin cytoskeleton. This linkage involves a direct interaction between ankyrin and the 14th–15th repeat unit of β-spectrin. The spectrin cytoskeleton itself is stabilized by the self-association of spectrin heterodimers into tetramers and larger oligomers, a process mediated by the 17th repeat unit of β-spectrin and a short NH2 -terminal sequence in α-spectrin. The self-association of spectrin and its ankyrin-mediated membrane binding have generally been considered independent events. We now demonstrate that spectrin self-association, the binding of spectrin to ankyrin, and the binding of ankyrin to the 43-kDa cytoplasmic domain of band 3 (cdb3) are coupled in a positively cooperative way. In solution, [125I]-labeled ankyrin was found by ND-PAGE33Abbreviations used: BSA, bovine serum albumin; ND-PAGE, nondenaturing PAGE; PMSF, phenylmethylsulfonyl fluoride; EM, electron micrograph. to enhance the affinity of spectrin self-association by 10-fold. The reciprocal process was also true, in that spectrin tetramers and oligomers bound ankyrin with enhanced affinity relative to dimer spectrin. Saturation of the β-spectrin self-association site by an NH2 -terminal 80-kDa α-spectrin peptide enhanced the affinity of spectrin dimer for ankyrin, indicating a direct relationship between ankyrin binding and the occupancy of the β-spectrin self-association site. cdb3 accentuated these cooperative interactions. Several inherited spectrin mutations that cause hemolytic disease but that do not directly destabilize the self-association or ankyrin-binding sites can be explained by these results. Three classes of mutations appear to disrupt cooperative coupling between self-association and ankyrin binding: (i) mutation of the linker sequences that join helices C and A in repeat units that intervene between the two functional sites, mutations that presumably block repeat-to-repeat transfer of conformational information; (ii) mutations in α-spectrin repeats 4 to 6 that disrupt the ability of this region to trans-regulate ankyrin binding by the adjacent β-spectrin repeats 14–15; and (iii) exon-skipping mutations that shorten α-spectrin and force repeats 4 to 6 to fall out-of-register with the ankyrin-binding motif in β-spectrin. Collectively, these results demonstrate a molecular mechanism whereby a membrane receptor can directly promote cytoskeletal assembly.