Experimental and Molecular Pathology 70, 215–230 (2001) doi:10.1006/exmp.2001.2377, available online at http://www.idealibrary.com on Spectrin Oligomerization is Cooperatively Coupled to Membrane Assembly: A Linkage Targeted by Many Hereditary Hemolytic Anemias? Mauro Giorgi,* ,1 Carol D. Cianci,* Patrick G. Gallagher,² and Jon S. Morrow* ,2 *Department of Pathology and ² Department of Pediatrics, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06510 Received March 2, 2001 In the erythrocyte, ankyrin is the major adapter protein linking mutation of the linker sequences that join helices C and A in repeat units that intervene between the two functional sites, mutations that tetramers of band 3 to the spectrin–actin cytoskeleton. This linkage involves a direct interaction between ankyrin and the 14th–15th repeat presumably block repeat-to-repeat transfer of conformational informa- tion; (ii) mutations in -spectrin repeats 4 to 6 that disrupt the ability unit of -spectrin. The spectrin cytoskeleton itself is stabilized by the self-association of spectrin heterodimers into tetramers and larger of this region to trans-regulate ankyrin binding by the adjacent - spectrin repeats 14–15; and (iii) exon-skipping mutations that shorten oligomers, a process mediated by the 17th repeat unit of -spectrin and a short NH 2 -terminal sequence in -spectrin. The self-association -spectrin and force repeats 4 to 6 to fall out-of-register with the ankyrin-binding motif in -spectrin. Collectively, these results demon- of spectrin and its ankyrin-mediated membrane binding have generally been considered independent events. We now demonstrate that spectrin strate a molecular mechanism whereby a membrane receptor can di- rectly promote cytoskeletal assembly.  2001 Academic Press 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-PAGE 3 to enhance the affinity of spectrin self-association by 10-fold. The reciprocal process was also true, in that spectrin tetra- mers and oligomers bound ankyrin with enhanced affinity relative to INTRODUCTION dimer spectrin. Saturation of the -spectrin self-association site by an NH 2 -terminal 80-kDa -spectrin peptide enhanced the affinity of spectrin dimer for ankyrin, indicating a direct relationship between ankyrin binding and the occupancy of the -spectrin self-association During the process of erythroid maturation the cortical site. cdb3 accentuated these cooperative interactions. Several inherited cytoskeleton is formed by interactions between ankyrin, spectrin mutations that cause hemolytic disease but that do not directly band 3 (the membrane anion transporter 1, AE1), and spec- destabilize the self-association or ankyrin-binding sites can be ex- plained by these results. Three classes of mutations appear to disrupt trin (for reviews, see Lux and Palek, 1995; Morrow et al., cooperative coupling between self-association and ankyrin binding: (i) 1997). At some point in this process, spectrin heterodimers must also self-associate to form tetramers and larger oligo- 1 Current address: Dipartimento di Biologia di Base e Applicata, mers and to bind via ankyrin to band 3 at the membrane Universita’ di L’Aquila, L’Aquila, Italy. (Morrow and Marchesi, 1981; Woods and Lazarides, 1985; 2 To whom correspondence and requests for reprints should be ad- Lehnert and Lodish, 1988; Hanspal et al., 1992a; Hanspal dressed. Fax: (203) 785-7037. E-mail: jon.morrow@yale.edu. et al., 1992b; Nehls et al., 1993). Although recent evidence 3 Abbreviations used: BSA, bovine serum albumin; ND-PAGE, non- indicates that the assembly of the spectrin skeleton at the denaturing PAGE; PMSF, phenylmethylsulfonyl fluoride; EM, elec- tron micrograph. membrane is guided by ankyrin-independent interactions 0014-4800/01 $35.00 215 Copyright  2001 by Academic Press All rights of reproduction in any form reserved.