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Emergence of metazoans from their unicellular ancestors required solutions to a new set of problems imposed by function of cells in the context of tissues and in motile organisms of immense size compared with individual cells. These requirements of communal life include ability of cells to develop micron-scale spatial organization of cell surfaces and of intracellular compartments to optimize cell-cell interactions and intercellular signaling. In addition, cells incorporated into an actively moving organism must deal with enormous mechanical stresses at the plasma membrane-cytoskeleton interface compared with free-living cells. Understanding the molecular basis for such metazoan adaptations represents an interdisciplinary challenge involving biochemistry, cell biology, physiology, and molecular medicine. This review focuses on the molecular physiology of spectrin and the spectrin-associated proteins ankyrin, adducin, and protein 4.1. These proteins were first discovered as components of the membrane skeleton of human erythrocytes (see below) and are required for survival of erythrocytes in the circulation. The erythrocyte membrane proteins are members of closely related families that are associated with membranes in simple metazoans, including Caenorhabditis elegans and Drosophila melanogaster, and are expressed in most vertebrate tissues. Spectrin, ankyrin, adducin, and protein 4.1 are modular proteins that are not present in their assembled state in the completed Saccharomyces cerevisea or Arabidopsis thaliana genomes and so far have not appeared in Zea mays genomic sequences. These proteins therefore are likely to have evolved early in evolution of metazoans, following divergence of plants and fungi, and represent candidates for roles in specialized activities of multicellular animals.
Recent discoveries based on studies involving C. elegans and D. melanogaster as well as gene knock-outs in mice will be reviewed that demonstrate functions of spectrin- and ankyrin-based protein assemblies in diverse roles that are all related to multicellular life. Functions that will be described include morphogenesis of epithellal tissues, targeting of ion channels and cell adhesion molecules to specialized regions in myelinated axons, and sorting of [Ca.sup.2+] homeostasis proteins to the [Ca.sup.2+] compartment of the endoplasmic reticulum (ER) of striated muscle. The clinical implications of these observations are only beginning to be appreciated and are discussed. Elucidation of physiological roles of spectrin and ankyrin-associated proteins is based on a strong foundation at a molecular level. The review includes current information regarding gene family members, atomic structures, oligomeric state, and protein interactions. The human erythrocyte remains the best understood in terms of its membrane s keleton, and the review begins with a brief summary of this system.
A. Overview of the Erythrocyte Membrane Skeleton
The membrane skeleton of mammalian erythrocytes was first visualized in electron micrographs of detergent-extracted erythrocytes . The erythrocyte membrane skeleton is organized as a polygonal network formed by five to seven extended spectrin molecules linked to short actin filaments [sim]40 nm in length [42, 247, 360] (Fig. 1A). The spectrin-actin network of erythrocytes is coupled to the membrane bilayer primarily by association of spectrin with ankyrin, which in turn is bound to the cytoplasmic domain of the anion exchanger [17, 20, 23, 256, 400, 441]. The anion exchanger is associated into dimers , which associate with separate sites on the membrane-binding domain of ankyrin to form pseudo-tetramers [53, 281, 332, 437]. Anion exchanger dimers also are associated on their cytoplasmic surface with band 4.2 . Additional membrane connections are provided at the spectrin-actin junction by a complex between protein 4.1, p55, a member of the MAGUK family, and glycophorin C [170, 266, 267] (Fig. 1 B).
Several proteins responsible for capping actin and defining the length of actin filaments as well as stabilizing spectrin-actin complexes have been localized to spectrin-actin junctions by electron microscopy [91, 402]. Protein 4.1 stabilizes spectrin-actin complexes [400, 401]. Adducin associates with the fast-growing end of actin filaments in a complex that caps the filament and promotes assembly of spectrin [135, 222, 223, 239] (Fig. 1C). A nonmuscle isoform of tropomyosin is associated with the sides of actin filaments . Tropomyosin is of the same length as actin filaments visualized in electron micrographs and is a candidate to function as a morphometric ruler defining the length of actin filaments in erythrocyte membranes. Tropomodulin caps the slow-growing end of actin filaments in a ternary complex involving tropomyosin [124-126, 421].
Components of the erythrocyte membrane skeleton have been the subject of recent reviews or papers that include discussions of tropomodulin , protein 4.1 [68, 118], protein 4.2 , p55 , spectrin [164, 410], the anion exchanger, and glycophorin C [4, 381]. The contributions of these proteins to mechanical properties of erythrocyte membranes have also been summarized [97, 289].
A major function of the spectrin skeleton in erythrocytes is to provide mechanical support for the membrane bilayer and allow survival of these cells in the circulation. The essential nature of the spectrin-skeleton in red cell biology was first demonstrated in mutant mice with deficiencies in [alpha] and [beta]-spectrin and ankyrin [33, 152]. Numerous mutations have subsequently been catalogued in humans with hereditary hemolytic anemias. Defects in lateral associations of the spectrin-actin network result in abnormally shaped cells in elliptocytosis and poikilocytosis and include loss of spectrin dimer-tetramer interactions  and deficiency of protein 4.1 [67, 120, 383]. Defects in membrane associations result in loss of unsupported phospholipid bilayer and spherocytosis. Molecular defects include spectrin deficiency from a variety of causes [1, 2, 52, 04, 112]. A substantial literature has documented naturally occurring mutations/deficiencies of skeletal proteins resulting in hereditary hemolytic anem ias in humans and mice (reviewed in Refs. 87, 166, 396). An emerging area of interest is mutations resulting from targeted gene knock-outs in mice resulting in hemolytic anemias that may foreshadow human disorders. An example is the [beta]-adducin null mouse, which exhibits spherocytosis .
II. GENES, PROTEINS, AND PROTEIN INTERACTIONS
Solution of the organization of the spectrin-based membrane skeleton of the human erythrocyte membrane has provided the biochemical equivalent of a high-resolution genetic pathway of interacting membrane structural proteins. The discovery that other tissues express isoforms of ankyrin  and spectrin [20, 40, 146, 151, 237, 343] suggested that the erythrocyte membrane skeleton had a broad relevance for other cell types. However, although the basic structural principles established in erythrocytes are likely to apply in other tissues, the organization, protein interactions, and functions of spectrin-based structures are considerably more diverse in other cells. Nevertheless, understanding the physiological roles of these proteins begins with their structure and biochemistry. This section focuses on genes, alternatively spliced variants, protein structure, and protein interactions of generally expressed forms of spectrin, and the proteins that interact with spectrin: ankyrin, protein 4.1, and adducin.
Spectrins are extended, flexible molecules [sim]200-260 nm in length and 3-6 nm across with actin-binding domains at each end [20, 146, 362, 400]. Spectrins are comprised of [alpha]- and [beta]-subunits, which are both related to a-actinin [43, 105, 319, 387, 408]. The [alpha]- and [beta]-subunits are associated laterally to form antiparallel heterodimers, and heterodimers are assembled head-head to form heterotetramers [Fig. 2, Table 1].
Metazoan spectrins exhibit 50-60% sequence similarity along the length of the predicted polypeptide chains, when compared between Drosophila, C. elegans, and vertebrates, with some regions with 70-80% identity. Candidates for prototypic spectrins, possibly comprised of a single subunit, have also been characterized biochemically and by electron microscopy in Dictyostelium  and Acanthamoeba . However, sequence information is not yet available for these presumed spectrin ancestors. Spectrin subunits are absent from completed S. cerevisiae and Arabidopsis thaliana genomes, although individual domains of spectrin are represented. Similarly, no spectrin sequence has yet appeared in genome of Zea mays. Polypeptides cross-reacting with spectrin have been reported in higher plants [118, 284, 364] as well as green algae  and Chlamydomonas . However, these immunoreactive forms of spectrin have not been characterized in terms of primary sequence or visualized by electron microscopy. Definition of the full scope of the spectrin family awaits completion of genome sequencing. However, the available data demonstrate that spectrins are ancient proteins present in their modem form in simple metazoans.
The spectrin repertoire of the completed C. elegans and D. melanogaster genomes includes one a-subunit , one 13-subunit [41, 159, 292], and one [beta]-H subunit [106, 276, 387]. Currently characterized spectrins in humans include two [alpha]-subunits ([[alpha].sub.1], [[alpha].sub.2) [353, 419], four [beta]-subunits ([[beta].sub.1], [[beta].sub.2], [[beta].sub.3], [[beta].sub.4]) [24, 187, 261, 286, 310, 372, 427, 428], and a [beta]-H subunit [also referred to as [[beta].sub.5])  [Table 1]. [beta]-Spectrins also include isoforms that have not been characterized at a molecular level. [[beta].sub.NM] is a [beta]-type subunit identified at neuromuscular junctions based on immunoreactivity . [[beta].sub.Golgi]-Spectrin has been discovered by Beck et al.  to be an immunoreactive form of [beta]-spectrin associated with Golgi structures. [[beta].sub.Golgi] shares epitopes with [[beta].sub.1]-erythrocyte spectrin, but establishing the relationship of [[beta].sub.Golgi] spectrin with other [beta]-spe ctrins will require molecular characterization. Recently, [[beta].sub.3]-spectrin has been proposed to function as the Golgi spectrin . However, the pattern of expression and cellular localization of [[beta].sub.3]-spectrin do not support a general role in Golgi function .
Alternative splicing provides additional diversity among [alpha]- and [beta]-spectrins. [beta] I, [beta] II, and [beta] IV spectrins are all differentially spliced. [beta] I, [beta] II, and [beta] IV spectrins have COOH-terminal regions that are subject to differential mRNA splicing to generate "short" or "long" COOH-terminal regions [24, 168, 427]. The [beta] III polypeptides described to date have a long COOH-terminal region that includes a pleckstrin homology (PH) domain [310, 372]. One nomenclature refers to [beta]-spectrin spliceoforms by the order of their discovery : short [beta] I is [beta] I [sigma] I, and long [beta] I is [beta] I [sigma] II. However, this system has become confusing as new family members have been discovered: long [beta] IV is [beta] IV [sigma] 1 and short [beta] IV is [beta] IV [sigma] 4, while long [beta] II is [beta] II [sigma] 1 and short [beta] II is [beta] IV [sigma] 2. We will refer instead to spliceoforms by molecular weight or in some way to help describe their domai n composition.
The long COOH-terminal regions of [beta]-spectrins have a PH domain (see below) linked by an apparently unstructured region of [sim]100 amino acid residues to the last (partial) triple helical repeat. The polypeptide chain terminates 50-60 residues after the PH domain. Short COOH-terminal isoforms do not contain a PH domain. About halfway through the linker region after the last (partial) triple helical repeat, the sequences diverge and terminate after 22-28 residues. In both [beta] I and [beta] II spectrins, the short COOH-terminal region contains multiple Ser or Thr residues that are potential substrate sites for casein kinase II. In the case of the short [beta] I COOH-terminal region [i.e., in erythrocyte [beta]-spectrin], at least six of these residues are substrates for casein kinase II [163, 323]. As described more fully in section IIC, the PH domain probably represents a major ligand-binding site in [beta]-spectrins. Thus differential splicing modulates both the interactive and regulatory properties o f the [beta]-spectrins.
[beta] II is subject to further differential splicing. A splice variant termed ELF1 represents a truncated [beta]-spectrin, consisting of little more than a calponin homology domain [the CH1 domain] and the COOH-terminal region of the short [beta] H .
Combinatorial association of [alpha]-spectrins with various [beta]- and [beta]-H subunits yields [alpha]/[beta] and [alpha]/[beta]-H heterotetramers with distinct functions and patterns of expression (Fig. 2, Table 1). Human [[alpha].sub.1]/[[beta].sub.1]-spectrins were first characterized in mammalian erythrocytes, and also are expressed in striated muscle and a subset of neurons in the central nervous system (227, 300, 345). Avian erythrocytes, in contrast, have [[alpha].sub.2]/[[beta].sub.2]-spectrin (343). [[alpha].sub.2]/[[beta].sub.2], [[alpha].sub.2]/[[beta].sub.3], and [[alpha].sub.2]/[[beta].sub.4] represent the major forms of spectrin in nonerythroid vertebrate tissues. Terminal web spectrin comprised of [[alpha].sub.2] and a presumed [beta]-H spectrin are localized in apical domains of epithelial tissues such as small intestine, while [[alpha].sub.2]/[[beta].sub.2]-spectrins are associated with basolateral domains (147).
[alpha]-Spectrins contain 22 domains with the following features: domains 1-9 and 11-21 are comprised of triple helical repeats also found in [beta] and [beta]-H spectrins (see below); domain 10 is an [SH.sub.3] (src homology domain 3) motif the COOH-terminal domain 22 is related to calmodulin (393) (Figs. 2 and 3). Domain 11 of vertebrate [[alpha].sub.2]-subunits contains a 35-residue extension with the cleavage site for [Ca.sup.2+]-activated protease and a calmodulinbinding site (162, 235). D. melanogaster spectrin contains a predicted calmodulin-binding site at a different position than vertebrates (105).
[beta]-Spectrins contain 19 domains beginning with a highly conserved [NH.sub.2]-terminal actin-binding domain comprised of two adjacent calponin homology domains (6, 46), followed by 17 consecutive triple-helical repeat domains and ending with a COOH-terminal domain which includes a PH domain (Figs. 2 and 3). Ankyrin-binding sites are at a site in the midregion of the tetramer (75, 400) and have been assigned to repeat number 15 of [[beta].sub.1]-spectrin (210). Repeat 17 is a partial helical repeat that pairs with the COOH terminus of [alpha]-spectrins to form a noncovalent triple-helical structure (see below).
[beta]-H spectrins of D. melanogaster (106, 387), C. elegans (276), and humans (371) have [NH.sub.2]-terminal actinbinding domains and COOH-terminal domains closely related to [beta]-spectrins but contain 30 triple-helical repeats instead of 17. [beta]-H spectrins of Drosophila and C. elegans also have an [SH.sub.3] domain inserted in the fifth triple helical domain. [beta]-H spectrins lack an ankyrin-binding site (276, 371, 387).
A) TRIPLE-HELIcAL DOMAINS. Atomic resolution of the structure of triple-helical repeat domains obtained by X-ray crystallography (100, 155, 434) as well as NMR (319) provides a striking confirmation of the structure originally predicted by Speicher and Marchesi (367). Triple helical repeats are comprised of two parallel and one antiparallel [alpha]-helices, which are stabilized by interactions between hydrophobic residues spaced in a heptad repeat pattern found in other examples of paired helical structures. Tandem triple helical repeats of spectrin and [alpha]-actinin are comprised of antiparallel [alpha]-helices connected by an extended [alpha]-helix (100, 155) (Fig. 3A). Comparison of hydrodynamic properties of single and multiple domains suggests that serial repeats are flexible and configured such that the average end-end length is reduced compared with values predicted for rigid rods (403). One possible source of flexibility is bending of the [alpha]-helix interconnecting domains. A novel mechanism for shortening end-end distances by rearrangement of helices has been proposed by Grum et al. (155) based on alternative structures observed by crystallography. An extended series of triple helical repeats may also exhibit a superhelical twist (155, 274).
Spectrin repeats have recently been demonstrated by atomic force microscopy to reversibly unfold and refold when subjected to forces in the range of 35 pN (347). Spectrin and other proteins (see below) with triple helical domains therefore have the potential to function as molecular springs that can store energy and dampen deformations resulting from mechanical stress. The potential role of this elastic behavior in spectrin function is discussed below.
B) ACTIN-BINDING DOMAIN. The [NH.sub.2]-terminal actin-binding domains of the [beta]-spectrins are comprised of a pair of calponin homology (CH) domains (47). These are similar in sequence to a region of the smooth muscle actinbinding protein calponin; such tandem pairs occur in other proteins that have lateral associations with actin filaments, including dystrophin, utrophin, [alpha]-actinin, and fimbrin (Fig. 3B). Related calponin-based actin-binding domains have been recently found in the plakin family of proteins involved in connection of actin filaments with intermediate filaments and microtubules (435, 436), and in cortexillins, which bundle actin filaments (116).
X-ray crystallography has resolved the atomic structures of the [NH.sub.2]-terminal (45) and COOH-terminal (6) CH domains of [beta]-spectrin, and of tandem CH domains of fimbrin (149) and utrophin (291) (Fig. 3B). The tandem pairs of CH domains of fimbrin and utrophin interact with actin, and their binding to actin filaments has been resolved at atomic resolution (149, 161, 208, 291). The [beta]-spectrin CH domains are also likely to interact with actin. The actin binding activity of [beta]-spectrin is restricted to the [beta]-chains (44, 238). A minimal actin-binding fragment of erythrocyte spectrin has been produced by limited trypsin digestion and derives from residues 47-186 (207). These residues represent the [NH.sub.2]-terminal CH domain (known as [CH.sub.1]), which in utrophin and fimbrin is the highest affinity actin binding site (161, 291).
Several considerations suggest that the site of contact with F-actin involves the junction between CH domains, with the first domain providing most of the interactions (6, 161). The second CH domain may contribute by enhancing the affinity for F-actin and/or have a regulatory role (6) (see below). Ironically, the single calponin domain of calponin itself lacks actin-binding activity (145), suggesting the likely possibility that CH domains have additional unresolved functions.
C) PH DOMAIN. [beta]-Spectrins contain a PH domain located in the COOH-terminal segement, which is deleted in certain alternatively spliced isoforms (see above). These domains extend out from spectrin rods in the midregion of spectrin tetramers and are placed within 10 nm of each other (see Fig. 2). PH domains are [sim]100-residue folding units first resolved in pleckstrin, which is a major protein kinase C substrate in platelets, and subsequently been found in many proteins . A unifying feature of proteins with PH domains is a role in signaling and proximity to plasma membranes. Ligands for PH domains may include polyphosphatidylinositol lipids as well as proteins.
The three-dimensional structures of the PH domains of mouse [190, 262] and D. melanogaster [beta]-spectrins  reveal similar folds to PH domains of other proteins  (Fig. 3C). PH domains include a seven-stranded [beta]-sheet arranged as a [beta]-barrel with a COOH-terminal [alpha]-helix. Solution of PH domain structure from other proteins indicates that while the overall folding of PH domains are conserved, variations in loop lengths and composition provide substantial variability in potential interaction surfaces . Consistent with structural predictions, binding activities of PH domains of spectrin and other proteins are distinct both with respect to interactions with various phosphatidylinositol lipids and to proteins .
D) CALMODULIN-RELATED DOMAIN. [alpha]-Spectrins contain EF-hand motifs located at the [NH.sub.2] terminus of that are juxtaposed to the actin-binding domain on the adjacent [beta]-subunit. The EF-hand domain of [alpha]-spectrin shares structural homology with calmodulin and also exhibits a [Ca.sup.2+]-dependent conformational change [392, 393]. An important distinction between spectrin and calmodulin is that spectrin only contains the two [NH.sub.2]-terminal EF hands and lacks the two COOH-terminal EF hands present in calmodulin. Intact human erythrocyte spectrin or recombinant [alpha] I or [alpha] II EF hands bind [CA.sup.2+] selectively with a stoichiometry corresponding to the number of EF hands but with an unphysiological affinity in the range of hundreds of micromolar [8, 257, 258]. However, intact horse spectrin molecules have been reported to bind [CA.sup.2+] at many sites of micromolar affinity, which presumably are not related to EF hands . Fowler and Taylor  reported that low micromolar l evels of [CA.sup.2+] influenced human erytbrocyte spectrin-actin interactions, although spectrin-actin and spectrin-4.1-actin interactions were described as [CA.sup.2+] insensitive by Ohanian et al. . Clearly much remains to be understood about the significance of [CA.sup.2+] binding by spectrins.
E) [SH.sub.3] DOMAIN. [SH.sub.3] domains, initially observed in the Src protein tyrosine kinase, are present in many proteins involved in cell signaling and mediate interactions with proline-rich stretches in a variety of target proteins . [SH.sub.3] domains are inserted in [alpha]-spectrins  and in invertebrate [beta]-H spectrins [106, 276] (Fig. 2). The structure of the a-spectrin [SH.sub.3] domain has been resolved at an atomic level by X-ray crystallography  and NMR [30, 352] (Fig. 3D). The three-dimensional structure of spectrin [SH.sub.3] domains is a compact [beta]-barrel and exhibits the same overall fold as other [SH.sub.3] domains.
2. Subunit interactions
Spectrin heterotetramers are assembled through the following interactions between [alpha]- and [beta]-subunits: 1) a lateral and antiparallel association between [beta]-subunits and [alpha]-subunits; 2) head-head association between laterally associated heterodimers by linkage between partial triple-helical repeats at the COOH-terminal end of the [beta]-subunits and the [NH.sub.2]-terminal end of [alpha]-subunits (see Fig. 2). Structural requirements for lateral association between [alpha]- and [beta]-spectrins have been analyzed for erythrocyte [14, 368, 403] and D. melanogaster spectrin [409, 411]. The minimal domains required for [alpha]-[beta] complexes are the first two triple-helical domains of [beta]-spectrin and the last two triple helical domains of [alpha]-spectrin . The first two triple helical domains of [beta]-spectrin and last two of [alpha]-spectrin are closely related to the four triple helical domains of [alpha]-actinin, which also associate laterally in an antiparallel orientation [100 ]. The atomic structure of tandem [alpha]-actinin domains reveals a dimer formed stabilized by electrostatic interactions provided by complementary surfaces . Association between four triple helical domains of [[alpha].sub.1]- and [[beta].sub.1]-spectrins is of high affinity with a dissociation constant [(K.sub.D)] of 10 nM . Further stabilization of [alpha]-[beta] complexes is provided by interaction between the calmodulin-related domain of [alpha]-spectrin and the calponin homology domains of [beta]-spectrin [409, 411].
The lateral association of [alpha]- and [beta]-spectrin subunits is highly conserved and occurs between D. metanogaster and vertebrate spectrins . The high affinity of [alpha]- and [beta]-subunits implies that spectrin will not exist as independent [alpha]- or [beta]-subunits but is an obligatory heterodimer or tetramer. Reports of [beta]-spectrin unaccompanied by an [alpha]-subunit in striated muscle and at neuromuscular junctions [31, 338] suggest the possiblity of a heretofore unrecognized [alpha]-subunit or an immunoreactive but otherwise highly diverged [beta]-spectrin.
Head-to-head contacts between [alpha]- and [beta]-spectrin subunits are believed to occur through contacts resembling pairing between helices in triple helical bundles [89, 211, 221, 366, 395, 433]. The [NH.sub.2] terminus of [alpha]-spectrin provides one helical segment, and the COOH-terminal repeat of [beta]-spectrin provides two antiparallel helices, with [alpha]-[beta] pairing resulting in a noncovalent triple helical segment. Flanking residues on [alpha]-spectrin also contribute to [alpha]-[beta] association .
Defects in spectrin tetramer formation have been established in erythrocytes of patients with hereditary elliptocytosis [315, 395]. Mutations in [alpha]- and [beta]-spectrin defined in these patients would be predicted to disrupt helical pairing predicted from biochemical studies [89, 396, 434]. These mutations include substitution of prolines, which would be expected to disrupt an [alpha]-helix, as well as mutations in residues predicted to provide contacts between helices. Human mutations in the partial helical domain of [alpha]-spectrin have been introduced into D. melanogaster [alpha]-spectrin and result in a temperature-sensitive phenotype  (see below).
3. Spectrin superfamily
Proteins that contain [NH.sub.2]-terminal CH domains, COOH-terminal calmodulin-related domains, and intervening triple helical domains comprise the spectrin superfamily. Currently recognized proteins with these combined features include [alpha]-actinins and dystrophins. In addition, trabeculin/macrophin/MACF of vertebrates  and Kakapo of D. melanogaster  are newly recognized members of the plakin family that have CH domains, 23-29 triple helical domains, and a calmodulin-related domain, as well as domains related to plectin and the plakin family (Fig. 4). The COOH termini of these proteins contain a microtubule-binding domain [206, 236]. These proteins thus can interact with actin filaments, cadherins, or integrins through the plakin domain and microtubules. Spectrin-like repeats also are present in eight to nine tandem copies in unc 73/kalirin/Trio, which also contain a dbl/pleckstrin homology domain . Trio/Unc 73 is required for axon pathfinding in C. elegans and D. melanogaster [259, 375].
Triple-helical repeats of [alpha]-actinin are most similar to the first repeats of [beta]-spectrin (repeats 1 and 2) and the last repeats of a-spectrin (domains 20 and 21) . [alpha]-Actinin thus contains within a single polypeptide the COOH-terminal domain of [alpha]-spectrin (calmodulin-related domain and last two triple helical repeats) and the [NH.sub.2]terminal domain of [beta]-spectrin (OH domains and first two triple helical repeats) (Fig. 4). These considerations have led to suggestions that [alpha]- and [beta]-spectrins evolved from a homodimeric [alpha]-actinin-like precursor polypeptide [41, 319, 387, 408]. One possible evolutionary scenario is that a-actinin became elongated through insertion of seven repeats by an unspecified mechanism, followed by duplication events resulting in a giant [alpha]-actinin-like protein. Insertion of a transcriptional promoter within a repeat has been proposed to provide the basis for the modem split repeat at the [alpha]- [beta] tetramerization site and the orig in of separate [alpha]- and [beta]-spectrin genes [387, 408].
4. Spectrin-protein interactions
A) SPECTRIN-ACTIN INTERACTION. The site of contact of F-actin with the actin-binding domains of [alpha]-actinin (173], fimbrin , and utrophin  has been mapped to actin subdomains 1 and 2 as well as subdomain 1 of the adjacent actin monomer by image analysis of electron micrographs. These conclusions are supported by genetic mapping of actin contact sites with fimbrin in yeast [175, 177]. The fimbrin actin-binding domain induces a conformational change in F-actin , suggesting the possibility that [beta]-spectrin could also perturb F-actin structure.
Comparison of action-binding activities of the [beta]-spectrin actin-binding domain alone and with triple-helical repeats suggests that the first triple helical repeat also participates in spectrin-actin complexes  (Fig. 1C). These results support a model where [beta]-spectrin actually lies along the actin filament, thus engaging in contacts involving two actin monomers . Interestingly, similar studies of dystrophin-actin interactions have concluded that lateral associations occur between actin and dystrophin triple helical domains (in this case repeat number 11) .
There is evidence that different [beta]-spectrins exhibit varying modes of interaction with actin. It is interesting to note that a very small [beta] II-spectrin splice variant ELF1 (a "mini-spectrin") contains a single CH domain that is similar to the [CH.sub.1] domain of other [beta]-spectrins. Since [[beta].sub.1]-spectrin [CH.sub.1], isolated as a tryptic fragment, binds actin avidly in vitro , it is likely that ELF1 is a true actinbinding protein, even though some other proteins with single CH domains do not necessarily bind actin . Intriguingly, the [CH.sub.1] tryptic fragment of [beta] 1-spectrin appears to have a higher affinity interaction with actin than might have been expected: it inhibits interaction of whole erythrocyte spectrin with actin with a half-maximal effect at 5 [micro]M, while the intact erythrocyte dimer binds to actin with a [K.sub.D] of 250 [micro]M . This indicates a higher affinity interaction than native erythrocyte [alpha] I/[beta] I-spectrin dimer. One possibility might be that [alpha] I-spectrin has a suppressive effect on the interaction of [[beta].sub.1] CH domains with actin. As we note below, the weak interaction of erythrocyte spectrin with actin is enhanced by proteins 4.1 and adducin.
The tandem CH domains in [beta]-spectrins may have additional roles in actin binding. [CH.sub.1], the [NH.sub.2]-terminal CH domain, probably provides the primary interaction with actin (see Ref. 291). [CH.sub.2], the COOH-terminal of the pair, binds actin only weakly . In utrophin, [CH.sub.2] provides a specificity for actin subtypes: utrophin binds [beta]-actin (cytoskeletal) with higher affinity than [alpha]-actin (sarcomeric). The CH domains of [beta]-spectrin therefore have the potential to target spectrin to particular isoforms of actin, as is the case with utrophin . In this context, it is interesting to note that erythrocyte membranes contain only [beta]-actin, rather than the [beta]-[gamma] mixture typical of most cytoskeletal systems .
B) SPECTRIN-MEMBRANE INTERACTIONS. Spectrins are coupled to membranes by multiple pathways including direct association with membrane-spanning proteins, interaction with phospholipids, and through interactions with ankyrins (see below). Binding of spectrin to ankyrin-independent protein sites has been measured in brain membranes [80, 251, 373, 374]. [beta]-Spectrin associates with membranes through two distinct classes of sites. One is regulated by calmodulin and is localized in the [NH.sub.2]-terminal region. The other [beta]-spectrin site is located in the COOH-terminal domain, which includes the PH domain.
Association of the spectrin PH domain with membranes has been demonstrated in living cells using green fluorescent protein-tagged [beta]-spectrin PH domain . One class of spectrin-PH domain interactions is likely to involve PI lipids, since the PH domain of [[beta].sub.1]-spectrin associates with sites in brain membranes stripped of peripheral proteins that are blocked by inositol 1,4,5-trisphosphate ([IP.sub.3]) and presumably represent phosphatidylinositol sites .
C) SPECTRIN-ION CHANNEL INTERACTIONS. Candidates for spectrin-binding proteins in brain synaptosomes include the [NR.sub.2] and [NR.sub.1] subunits of the NMDA receptor . Spectrin binds to the [NR.sub.2B] subunit at sites distinct from those of [alpha]-actinin-2 and members of the PSD95/SAP90 family. The spectrin-[NR.sub.2B] interactions are inhibited by [Ca.sup.2+] and fyn-mediated [NR.sub.2B] phosphorylation, but not by [Ca.sup.2+]/calmodulin or by calmodulin kinase II-mediated phosphorylation of NR2B. The spectrin-[NR.sub.1] interactions are unaffected by [Ca.sup.2+] but inhibited by [Ca.sup.2+]/calmodulin and by phosphorylation of [NR.sub.1] by protein kinases A and C. The [NR.sub.1] subunit thus is a candidate to interact with the [Ca.sup.2+]/calmodulin-regulated site on [beta]-spectrin [80, 374].
Spectrin associates via the [alpha]-spectrin [SH.sub.3] domain with the [alpha]-subunit of the amiloride-sensitive [Na.sup.+] channel, EnNaC [349, 457], and with the [Na.sup.+]/[H.sup.+] exchanger, [NHE.sub.2] . …