Spectrin oligomerization is cooperatively coupled to membrane assembly: a linkage targeted by many hereditary hemolytic anemias?
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 beta-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 beta-spectrin and a short NH(2) -terminal sequence in alpha-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, [(125)I]-labeled ankyrin was found by ND-PAGE3 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 beta-spectrin self-association site by an NH(2) -terminal 80-kDa alpha-spectrin peptide enhanced the affinity of spectrin dimer for ankyrin, indicating a direct relationship between ankyrin binding and the occupancy of the beta-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 alpha-spectrin repeats 4 to 6 that disrupt the ability of this region to trans-regulate ankyrin binding by the adjacent beta-spectrin repeats 14-15; and (iii) exon-skipping mutations that shorten alpha-spectrin and force repeats 4 to 6 to fall out-of-register with the ankyrin-binding motif in beta-spectrin. Collectively, these results demonstrate a molecular mechanism whereby a membrane receptor can directly promote cytoskeletal assembly.
Giorgi
M
M
Department of Pathology, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
Cianci
C D
CD
Gallagher
P G
PG
Morrow
J S
JS
D000743:Anemia, Hemolytic / Q000097:blood / Q000235:genetics*; D017487:Ankyrins / Q000737:chemistry* / Q000378:metabolism*; D003593:Cytoplasm / Q000502:physiology; D019281:Dimerization; D004612:Elliptocytosis, Hereditary / Q000097:blood / Q000235:genetics; D004910:Erythrocyte Membrane / Q000502:physiology* / Q000648:ultrastructure; D004912:Erythrocytes / Q000502:physiology; D006801:Humans; D046911:Macromolecular Substances; D008958:Models, Molecular; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D021122:Protein Subunits; D013049:Spectrin / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D013103:Spherocytosis, Hereditary / Q000097:blood / Q000235:genetics
D017487:Ankyrins; D046911:Macromolecular Substances; D021122:Protein Subunits; D013049:Spectrin