I. INTRODUCTION

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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 epithelial tissues, targeting of ion channels and cell adhesion molecules to specialized regions in myelinated axons, and sorting of Ca²⁺ homeostasis proteins to the Ca²⁺ 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 skeleton, and the review begins with a brief summary of this system.

The membrane skeleton of mammalian erythrocytes was first visualized in electron micrographs of detergent-extracted erythrocytes (440). The erythrocyte membrane skeleton is organized as a polygonal network formed by five to seven extended spectrin molecules linked to short actin filaments ~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 (305), 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 (442). 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. 1B).

View larger version (65K): [in this window] [in a new window]   Fig. 1. Organization of the erythrocyte membrane skeleton. A: electron microscopy of the membrane skeleton. Spectrin tetramers are cross-linked at nodes (junction points) by short filaments of F-actin and 4.1/adducin. Approximately six spectrins interact with each node. At the central region of the spectrin tetramers are bound ankyrin/anion exchanger complexes. [From Liu et al. (247).] B: membrane-cytoskeleton connections in erythrocytes. Ankyrin is linked through its ank-repeat region to dimers of the anion exchanger (gray). Each ankyrin is capable of cross-linking two anion exchanger dimers. The 15th helical repeat of -spectrin (yellow) interacts with ankyrin. At the NH2-terminal region of -spectrin is a binding site for 4.1 (red). 4.1 forms a ternary complex with the transmembrane protein glycophorin C and the membrane-associated guanylate kinase p55 (green). C: the spectrin-actin junction. Short filaments of F-actin containing 14-16 monomers tether spectrin at the nodes shown in A. The negative () end of the filaments is blocked by tropomodulin, and nonmuscle tropomyosin lies along the filament. -Spectrin binds actin via its NH2-terminal CH domains (CH1 and CH2) and the first two triple helical repeats (Rpt 1 and Rpt 2). Approximately six spectrins would fit a short actin filament if the spectrins were arranged along the side of the filament as indicated in this figure. This is consistent with the six spectrins per node indicated in A. Spectrin-actin interaction is promoted by protein 4.1 and adducin. Adducin has a high affinity for the positive (+) end of the filaments and possibly recruits other proteins to the positive end.

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 (127). 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 (125), protein 4.1 (68, 118), protein 4.2 (62), p55 (56), 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 - and -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 (395) 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, 94, 112). A substantial literature has documented naturally occurring mutations/deficiencies of skeletal proteins resulting in hereditary hemolytic anemias 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 -adducin null mouse, which exhibits spherocytosis (143).

	II. GENES, PROTEINS, AND PROTEIN INTERACTIONS

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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 (18) 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 ~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 - and -subunits, which are both related to -actinin (43, 105, 319, 387, 408). The - and -subunits are associated laterally to form antiparallel heterodimers, and heterodimers are assembled head-head to form heterotetramers (Fig. 2, Table 1).

View larger version (37K): [in this window] [in a new window]   Fig. 2. The domain structure of spectrin polypeptides and proteins. The - and -subunits are composed of discrete folding units or domains. Triple helical repeating units make up the length of each spectrin subunit. At the NH2 termini of -spectrins are tandem calponin homology domains, which bind actin. High-affinity interaction with actin requires the first two triple helices too (indicated as atypical repeats). At the COOH termini of the subunits are differentially spliced "tails": these can either contain pleckstrin homology domains or short regions of sequence rich on phosphorylation sites. The high molecular mass -H spectrins have additional triple helical repeats plus an internal SH3 domain. -Spectrins contain COOH-terminal EF hands in a "calmodulin-like" structure. Interactions between - and -subunits lead to the formation of tetramers. Lateral associations between  and  occur near the NH2 terminus of  and the COOH terminus of , such that the subunits lie anti-parallel to each other, and the EF hands are close to the CH domains. At the NH2 terminus of , the first helical repeat actually only comprises one helix; the most COOH terminal in  comprises two helices. These interact "head to tail" to form an intact triple helix bringing two laterally associated -dimers together to form an ()2-tetramer. Tetramers can form between  and -H in the same way.

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 (16) and Acanthamoeba (336). 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 (176) and Chlamydomonas (252). 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 modern form in simple metazoans.

The spectrin repertoire of the completed C. elegans and D. melanogaster genomes includes one -subunit (105), one -subunit (41, 159, 292), and one -H subunit (106, 276, 387). Currently characterized spectrins in humans include two -subunits (₁, ₂) (353, 419), four -subunits (₁, ₂, ₃, ₄) (24, 187, 261, 286, 310, 372, 427, 428), and a -H subunit (also referred to as ₅) (371) (Table 1). -Spectrins also include isoforms that have not been characterized at a molecular level. _NM is a -type subunit identified at neuromuscular junctions based on immunoreactivity (31). _Golgi-Spectrin has been discovered by Beck et al. (9) to be an immunoreactive form of -spectrin associated with Golgi structures. _Golgi shares epitopes with ₁-erythrocyte spectrin, but establishing the relationship of _Golgi spectrin with other -spectrins will require molecular characterization. Recently, ₃-spectrin has been proposed to function as the Golgi spectrin (372). However, the pattern of expression and cellular localization of ₃-spectrin do not support a general role in Golgi function (310).

Alternative splicing provides additional diversity among - and -spectrins.  I,  II, and  IV spectrins are all differentially spliced.  I,  II, and  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  III polypeptides described to date have a long COOH-terminal region that includes a pleckstrin homology (PH) domain (310, 372). One nomenclature refers to -spectrin spliceoforms by the order of their discovery (429): short  I is  I  I, and long  I is  I  II. However, this system has become confusing as new family members have been discovered: long  IV is  IV  1 and short  IV is  IV  4, while long  II is  II  1 and short  II is  II  2. We will refer instead to spliceoforms by molecular weight or in some way to help describe their domain composition.

The long COOH-terminal regions of -spectrins have a PH domain (see below) linked by an apparently unstructured region of ~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  I and  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  I COOH-terminal region (i.e., in erythrocyte -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 -spectrins. Thus differential splicing modulates both the interactive and regulatory properties of the -spectrins.

II is subject to further differential splicing. A splice variant termed ELF1 represents a truncated -spectrin, consisting of little more than a calponin homology domain (the CH1 domain) and the COOH-terminal region of the short  II (287).

Combinatorial association of -spectrins with various - and -H subunits yields / and /-H heterotetramers with distinct functions and patterns of expression (Fig. 2, Table 1). Human ₁/₁-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 ₂/₂-spectrin (343). ₂/₂, ₂/₃, and ₂/₄ represent the major forms of spectrin in nonerythroid vertebrate tissues. Terminal web spectrin comprised of ₂ and a presumed -H spectrin are localized in apical domains of epithelial tissues such as small intestine, while ₂/₂-spectrins are associated with basolateral domains (147).

-Spectrins contain 22 domains with the following features: domains 1-9 and 11-21 are comprised of triple helical repeats also found in  and -H spectrins (see below); domain 10 is an SH₃ (src homology domain 3) motif; the COOH-terminal domain 22 is related to calmodulin (393) (Figs. 2 and 3). Domain 11 of vertebrate ₂-subunits contains a 35-residue extension with the cleavage site for Ca²⁺-activated protease and a calmodulin-binding site (162, 235). D. melanogaster spectrin contains a predicted calmodulin-binding site at a different position than vertebrates (105).

View larger version (34K): [in this window] [in a new window]   Fig. 3. Spectrin domains. A: triple helical repeats. Ribbon representation of two triple helical repeats of chicken -spectrin (from the PDB file 1CUN; Ref. 155). B: calponin homology domains. The figure shows a ribbon representation of the tandem calponin homology domains of utrophin (taken from the PDB file 1QAG; Ref. 291). The equivalent sequence in human -G spectrin is 68% identical and is likely to adopt a similar overall conformation, based on the known structures of individual spectrin CH domains (6, 46). Three actin binding sequences (ABS) have been mapped in utrophin and are shown in red (ABS1), purple (ABS2), and cyan (ABS3). ABS1 and ABS2 are in the first calponin homology domain (CH1); ABS3 is in the second (CH2). ABS1 and ABS2 are highly conserved between utrophin and -G spectrin (80 and 68% identical, respectively) and probably present the primary site of interaction with filamentous actin. ABS3 is less conserved in sequence between spectrin and utrophin (25% identical), even though the helical conformation of this sequence is preserved in spectrin. C: pleckstrin homology domain. The pleckstrin homology domain of -spectrins represents a major binding site for protein and lipid ligands. This figure shows the interaction of the PH domain of mouse -G spectrin with inositol 1,4,5-trisphosphate (IP3) (shown as the small molecule in ball and stick, bottom right). The PH domain makes contact with the 4- and 5-phosphate groups through salt bridges and hydrogen bonds; the 1-phosphate group (on the right of the IP3 molecule) is mostly solvent exposed, and the inositol ring has virtually no interactions with the protein. Residues shown likely to interact with the ligand are (from left) Tyr-69, Lys-8, Arg-21, and Ser-22. (from the PDB file 1BTN; Ref. 190). D: SH3 domain. SH3 domains are thought to interact with their targets via hydrophobic interactions with proline-rich sequences. The spectrin SH3 has a "stripe" of hydrophobic residues along one surface that are thought to mediate interactions with targets such as e3B1 (456). The figure shows the residues of this stripe (from left to right: Phe-52, Trp-41, Pro-54, Tyr-57, Tyr-13) (from the PDB file 1SHG; Ref. 298).

-Spectrins contain 19 domains beginning with a highly conserved NH₂-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 ₁-spectrin (210). Repeat 17 is a partial helical repeat that pairs with the COOH terminus of -spectrins to form a noncovalent triple-helical structure (see below).

-H spectrins of D. melanogaster (106, 387), C. elegans (276), and humans (371) have NH₂-terminal actin-binding domains and COOH-terminal domains closely related to -spectrins but contain 30 triple-helical repeats instead of 17. -H spectrins of Drosophila and C. elegans also have an SH₃ domain inserted in the fifth triple helical domain. -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 -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 -actinin are comprised of antiparallel -helices connected by an extended -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 -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 NH₂-terminal actin-binding domains of the -spectrins are comprised of a pair of calponin homology (CH) domains (47). These are similar in sequence to a region of the smooth muscle actin-binding protein calponin; such tandem pairs occur in other proteins that have lateral associations with actin filaments, including dystrophin, utrophin, -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 NH₂-terminal (45) and COOH-terminal (6) CH domains of -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 -spectrin CH domains are also likely to interact with actin. The actin binding activity of -spectrin is restricted to the -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 NH₂-terminal CH domain (known as CH₁), 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. -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 ~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 (340). 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 -spectrins (447) reveal similar folds to PH domains of other proteins (340) (Fig. 3C). PH domains include a seven-stranded -sheet arranged as a -barrel with a COOH-terminal -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 (447). 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 (340).

D) CALMODULIN-RELATED DOMAIN. -Spectrins contain EF-hand motifs located at the NH₂ terminus of that are juxtaposed to the actin-binding domain on the adjacent -subunit. The EF-hand domain of -spectrin shares structural homology with calmodulin and also exhibits a Ca²⁺-dependent conformational change (392, 393). An important distinction between spectrin and calmodulin is that spectrin only contains the two NH₂-terminal EF hands and lacks the two COOH-terminal EF hands present in calmodulin. Intact human erythrocyte spectrin or recombinant  I or  II EF hands bind Ca²⁺ 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 Ca²⁺ at many sites of micromolar affinity, which presumably are not related to EF hands (414). Fowler and Taylor (123) reported that low micromolar levels of Ca²⁺ influenced human erythrocyte spectrin-actin interactions, although spectrin-actin and spectrin-4.1-actin interactions were described as Ca²⁺ insensitive by Ohanian et al. (307). Clearly much remains to be understood about the significance of Ca²⁺ binding by spectrins.

E) SH₃ domain. SH₃ 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 (321). SH₃ domains are inserted in -spectrins (419) and in invertebrate -H spectrins (106, 276) (Fig. 2). The structure of the -spectrin SH₃ domain has been resolved at an atomic level by X-ray crystallography (298) and NMR (30, 352) (Fig. 3D). The three-dimensional structure of spectrin SH₃ domains is a compact -barrel and exhibits the same overall fold as other SH₃ domains.

Spectrin heterotetramers are assembled through the following interactions between - and -subunits: 1) a lateral and antiparallel association between -subunits and -subunits; 2) head-head association between laterally associated heterodimers by linkage between partial triple-helical repeats at the COOH-terminal end of the -subunits and the NH₂-terminal end of -subunits (see Fig. 2). Structural requirements for lateral association between - and -spectrins have been analyzed for erythrocyte (14, 368, 403) and D. melanogaster spectrin (409, 411). The minimal domains required for - complexes are the first two triple-helical domains of -spectrin and the last two triple helical domains of -spectrin (368). The first two triple helical domains of -spectrin and last two of -spectrin are closely related to the four triple helical domains of -actinin, which also associate laterally in an antiparallel orientation (100). The atomic structure of tandem -actinin domains reveals a dimer formed stabilized by electrostatic interactions provided by complementary surfaces (100). Association between four triple helical domains of ₁- and ₁-spectrins is of high affinity with a dissociation constant (K_D) of 10 nM (403). Further stabilization of - complexes is provided by interaction between the calmodulin-related domain of -spectrin and the calponin homology domains of -spectrin (409, 411).

The lateral association of - and -spectrin subunits is highly conserved and occurs between D. melanogaster and vertebrate spectrins (43). The high affinity of - and -subunits implies that spectrin will not exist as independent - or -subunits but is an obligatory heterodimer or tetramer. Reports of -spectrin unaccompanied by an -subunit in striated muscle and at neuromuscular junctions (31, 338) suggest the possiblity of a heretofore unrecognized -subunit or an immunoreactive but otherwise highly diverged -spectrin.

Head-to-head contacts between - and -spectrin subunits are believed to occur through contacts resembling pairing between helices in triple helical bundles (89, 211, 221, 366, 395, 433). The NH₂ terminus of -spectrin provides one helical segment, and the COOH-terminal repeat of -spectrin provides two antiparallel helices, with - pairing resulting in a noncovalent triple helical segment. Flanking residues on -spectrin also contribute to - association (55).

Defects in spectrin tetramer formation have been established in erythrocytes of patients with hereditary elliptocytosis (315, 395). Mutations in - and -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 -helix, as well as mutations in residues predicted to provide contacts between helices. Human mutations in the partial helical domain of -spectrin have been introduced into D. melanogaster -spectrin and result in a temperature-sensitive phenotype (89) (see below).

Proteins that contain NH₂-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 -actinins and dystrophins. In addition, trabeculin/macrophin/MACF of vertebrates (236) and Kakapo of D. melanogaster (154) 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 (83). Trio/Unc 73 is required for axon pathfinding in C. elegans and D. melanogaster (259, 375).

View larger version (34K): [in this window] [in a new window]   Fig. 4. Representatives of the spectrin superfamily. Members of the spectrin superfamily have in common multiple triple helical repeats, a pair of calponin homology domains at the NH2-terminal of one subunit, and a calmodulin-like domain in the COOH-terminal region of one subunit. Specific functions that distinguish each member of the family are conferred by variations in the number of triple helical repeats, and the presence of varying additional ligand-binding domains that include SH3 and PH domains in spectrin, plakin, and GAR22 domains in trabeculin/kakapo and a cysteine-rich domain in dystrophin. In addition, individual helices can contain specific binding activities, for example, the ankyrin binding activity of the 15th repeat in -spectrins.

Triple-helical repeats of -actinin are most similar to the first repeats of -spectrin (repeats 1 and 2) and the last repeats of -spectrin (domains 20 and 21) (43). -Actinin thus contains within a single polypeptide the COOH-terminal domain of -spectrin (calmodulin-related domain and last two triple helical repeats) and the NH₂-terminal domain of -spectrin (CH domains and first two triple helical repeats) (Fig. 4). These considerations have led to suggestions that - and -spectrins evolved from a homodimeric -actinin-like precursor polypeptide (41, 319, 387, 408). One possible evolutionary scenario is that -actinin became elongated through insertion of seven repeats by an unspecified mechanism, followed by duplication events resulting in a giant -actinin-like protein. Insertion of a transcriptional promoter within a repeat has been proposed to provide the basis for the modern split repeat at the - tetramerization site and the origin of separate - and -spectrin genes (387, 408).

A) SPECTRIN-ACTIN INTERACTION. The site of contact of F-actin with the actin-binding domains of -actinin (273), fimbrin (161), and utrophin (291) 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 (161), suggesting the possibility that -spectrin could also perturb F-actin structure.

Comparison of actin-binding activities of the -spectrin actin-binding domain alone and with triple-helical repeats suggests that the first triple helical repeat also participates in spectrin-actin complexes (238) (Fig. 1C). These results support a model where -spectrin actually lies along the actin filament, thus engaging in contacts involving two actin monomers (238). 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) (351).

There is evidence that different -spectrins exhibit varying modes of interaction with actin. It is interesting to note that a very small  II-spectrin splice variant ELF1 (a "mini-spectrin") contains a single CH domain that is similar to the CH₁ domain of other -spectrins. Since ₁-spectrin CH₁, isolated as a tryptic fragment, binds actin avidly in vitro (207), it is likely that ELF1 is a true actin-binding protein, even though some other proteins with single CH domains do not necessarily bind actin (6). Intriguingly, the CH₁ tryptic fragment of  I-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 µM, while the intact erythrocyte dimer binds to actin with a K_D of 250 µM (309). This indicates a higher affinity interaction than native erythrocyte  I/ I-spectrin dimer. One possibility might be that  I-spectrin has a suppressive effect on the interaction of 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 -spectrins may have additional roles in actin binding. CH₁, the NH₂-terminal CH domain, probably provides the primary interaction with actin (see Ref. 291). CH₂, the COOH-terminal of the pair, binds actin only weakly (46). In utrophin, CH₂ provides a specificity for actin subtypes: utrophin binds -actin (cytoskeletal) with higher affinity than -actin (sarcomeric). The CH domains of -spectrin therefore have the potential to target spectrin to particular isoforms of actin, as is the case with utrophin (426). In this context, it is interesting to note that erythrocyte membranes contain only -actin, rather than the - mixture typical of most cytoskeletal systems (333).

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). -Spectrin associates with membranes through two distinct classes of sites. One is regulated by calmodulin and is localized in the NH₂-terminal region. The other -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 -spectrin PH domain (415). One class of spectrin-PH domain interactions is likely to involve PI lipids, since the PH domain of ₁-spectrin associates with sites in brain membranes stripped of peripheral proteins that are blocked by inositol 1,4,5-trisphosphate (IP₃) and presumably represent phosphatidylinositol sites (416).

C) SPECTRIN-ION CHANNEL INTERACTIONS. Candidates for spectrin-binding proteins in brain synaptosomes include the NR₂ and NR₁ subunits of the NMDA receptor (422). Spectrin binds to the NR_2B subunit at sites distinct from those of -actinin-2 and members of the PSD95/SAP90 family. The spectrin-NR_2B interactions are inhibited by Ca²⁺ and fyn-mediated NR_2B phosphorylation, but not by Ca²⁺/calmodulin or by calmodulin kinase II-mediated phosphorylation of NR2B. The spectrin-NR₁ interactions are unaffected by Ca²⁺ but inhibited by Ca²⁺/calmodulin and by phosphorylation of NR₁ by protein kinases A and C. The NR₁ subunit thus is a candidate to interact with the Ca²⁺/calmodulin-regulated site on -spectrin (80, 374).

Spectrin associates via the -spectrin SH₃ domain with the -subunit of the amiloride-sensitive Na⁺ channel, EnNaC (349, 457), and with the Na⁺/H⁺ exchanger, NHE₂ (59). The amiloride-sensitive Na⁺ channel/spectrin complexes also contain the protein Apx, which is an apically localized protein identified in Xenopus (457). Association with -spectrin may be responsible for apical targeting of NHE₂, since deletion of the spectrin-binding loop results in basolateral localization of the channel (59). /H (TW) is the most likely form of spectrin to associate with the apically targeted NHE₂ and amiloride-sensitive Na⁺ channels since /-spectrin generally is localized to the basolateral domains of epithelial cells (438).

The cGMP-gated cation channel of rod photoreceptor plasma membranes copurifies with a 240-kDa polypeptide subsequently identified as spectrin based on immunoreactivity (290). Direct linkage between spectrin and the cation channel is likely since antibodies against each protein coimmunoprecipitates the other from relatively pure preparations of the channel. The 240-kDa polypeptide was localized to the inner surface of the rod outer segment plasma membrane and excluded from the disk membranes, as would be expected for an association of this protein with the cGMP-gated cation channel in vivo.

D) SPECTRIN AS A MEMBRANE ADAPTOR FOR CYTOPLASMIC PROTEINS. Cytoplasmic ligands for spectrin include a protein termed HsSH₃bp1 that binds to tyrosine kinases and binds to spectrin through association with the -spectrin SH₃ domain (432, 456). HsSH₃bp1 is associated with macropinosomes and may couple spectrin to these intracellular organelles (432). Spectrin PH domains are likely to associate with proteins in addition to their interactions with PI lipids (above). An interesting example is RACK1, a protein kinase C-anchoring protein, which associates selectively with the -spectrin PH domain and activated protein kinase C to form a ternary complex (348).

A search for annexin VI-binding proteins revealed spectrin as one of ~14 membrane-associated proteins that associate with annexin VI in blot overlays (420). Annexin VI has subsequently been implicated in directing proteolytic degradation of spectrin during receptor-mediated endocytosis (204). The interpretation of these experiments was that annexin VI promoted proteolysis of spectrin, which resulted in relaxation of restraints of budding of a subset of coated pits. Annexin VI, according to this model, utilizes spectrin as an adaptor to direct and possibly activate a protease.

E) SPECTRIN AS A REGULATOR PROTEIN. Spectrin, at nanomolar concentrations, inhibits phospholipase D as well as phospholipases C and A₂ in in vitro assays (254, 255). The basis for inhibition was most likely not a direct interaction of spectrin with these enzymes, but rather competition for their phospholipid substrates (255). Spectrin in lymphocytes associates with CD45 (192, 248), which is a member of a family of membrane-spanning glycoproteins with protein-tyrosine phosphatase activity. Spectrin binds to CD45 with a nanomolar affinity and upregulates the tyrosine phosphatase activity of CD45 (248).

Ankyrin in human erythrocytes provides a high-affinity link between the cytoplasmic domain of the anion exchanger and the spectrin/actin network (Fig. 1). The ankyrin family has a general role as an adapter between a variety of integral membrane proteins and the spectrin skeleton. The C. elegans genome contains a single ankyrin gene, (312), whereas the D. melanogaster genome has two ankyrin genes, Dank1 and Dank2 (35, 110). The ankyrin gene family of mammals currently includes three members: ankyrin-R (R for restricted; also termed ankyrin1) (229, 260), first characterized in erythrocytes, (227); ankyrin-B (B for broadly expressed, also termed ankyrin 2) first characterized in brain (313); and ankyrin-G (G for general or giant; also termed ankyrin 3) independently discovered in searches for components of the node of Ranvier (219) and for epithelial ankyrins (93, 327, 385). Ankyrins are expressed in most tissues, and in many cases all three ankyrins are found in the same cell type.

Diseases of humans attributed to ankyrins include hereditary spherocytosis, which can result from decreased expression and/or mutated forms of ankyrin-R (112). In mice, a similar disorder (the nb/nb mutation) results in a nearly complete deficiency of 210-kDa isoforms of ankyrin-R in erythrocytes, neurons, and striated muscle (452) with a phenotype of severe anemia (33) and degeneration of a subset of Purkinje cell neurons accompanied by cerebellar dysfunction (326). Ankyrin mutations in C. elegans result in the unc44 phenotype, which includes abnormal axon guidance and uncoordinated movements (312).

Ankyrins are modular proteins comprised of three domains conserved among family members as well as specialized domains found in alternatively spliced isoforms (see Fig. 5). Conserved domains are an NH₂-terminal membrane-binding domain, a 62-kDa spectrin-binding domain, and a 12-kDa death domain. The membrane-binding domains are comprised of 24 copies of a 33-residue repeat known as the ANK repeat that is involved in protein recognition in many types of proteins (34, 359) (Fig. 6). The 24 ANK repeats form 4 subdomains, each comprised of the basic folding unit of 6 repeats (280). The role of six-repeat subdomains as protein binding sites is discussed below. Death domains were first reported in proteins such as Fas and the tumor necrosis factor receptor that participate in apoptosis pathways (394). These domains can associate with related death domains in other proteins. The protein interactions of the ankyrin death domain could involve self-association and/or interactions with other proteins and are not yet resolved. The death domain is followed by a regulatory domain subject to alternative splicing that in the case of ankyrin-R modulates both binding of the anion exchanger and of spectrin (81, 157). A regulatory function for this domain has not been demonstrated for ankyrin-B or ankyrin-G. However, the COOH-terminal domains are the most divergent among ankyrin family members and are likely to have distinct functions that are yet to be defined.

View larger version (27K): [in this window] [in a new window]   Fig. 5. Summary of human ankyrin genes and their alternatively spliced variants. Canonical ankyrins are of 190-220 kDa and have membrane-binding, spectrin-binding, and death domains. Giant ankyrins of 270-480 kDa have insertions of up to 2,400 amino acid residues between the spectrin-binding and death domains and are abundant in axons. Small ankyrins of 26-120 kDa are missing various domains found in canonical ankyrins and are associated with intracellular organelles.

View larger version (31K): [in this window] [in a new window]   Fig. 6. Schematic view of ankyrin domains and their protein interactions (top) and atomic structure of Ank repeats (bottom). The NH2-terminal membrane-binding domain of ankyrins is composed of 24 Ank repeats folded into 6-repeat subdomains. These domains associate with a variety of membrane proteins as well as tubulin and clathrin. The bottom panel shows a stack of six ankyrin repeats in the protein I-B (PDB file 1NFI; Ref. 193). The two images (a and b) show the same structures rotated by 90°. The sites of protein interactions are likely to be the loops of Ank repeats, based on structures of complexes of I-B and several other Ank repeat proteins with their protein partners (see Ref. 359).

Ankyrin genes are expressed and processed in a complex tissue-dependent and developmentally regulated fashion, giving rise to a large number of isoforms that utilize different combinations of functional domains. For example, alternative splicing generates ankyrin-G isoforms missing all or portions of their membrane-binding domain (93, 178, 219, 327). Alternate exon usage is also used to generate an unusual isoform of ankyrin-R in striated muscle. This isoform lacks both the membrane-binding and spectrin-binding domains and contains only the COOH-terminal 26 kDa plus a hydrophobic stretch (452). The functions of these small ankyrins are likely to involve intracellular as opposed to plasma membrane roles, since these truncated ankyrins are associated with Golgi, lysosomal, and sarcoplasmic reticulum membranes (11, 88, 92, 452).

Ankyrin-B and ankyrin-G spliceoforms also include polypeptides with insertions of up to 2,500 amino acid residues and molecular mass of up to 480 kDa, which are abundantly expressed in axons (49, 219, 224, 225) (see below). The inserted sequences in both genes are placed between the spectrin-binding and death domains (see Fig. 5). Ankyrin-G polypeptides of 270 and 480 kDa contain inserted sequences beginning with a serine/threonine-rich stretch of ~400 residues, that is followed by sequence with similarity to that of the inserted sequence of 440-kDa ankyrin-B. The 270-kDa ankyrin-G lacks a 190-kDa stretch of this sequence resulting from the use of an alternate splice donor site.

Much of the 220-kDa inserted sequence of 440-kDa ankyrin-B has the configuration of an extended random coil based on physical properties of expressed polypeptides (49). Because the alternatively spliced insert of ankyrin-G has a highly polar hydrophilicity profile similar to that of ankyrin-B, it is likely that the 480-kDa ankyrin-G also contains an extended stretch of random coil inserted between its spectrin-binding and death domains. The properties of the inserted sequences suggest a structural model for 440-kDa ankyrin-B and 480-kDa ankyrin-G where the membrane-associated head domain is separated from the death domain by an extended filamentous tail domain encoded by the inserted sequence (see Fig. 5). The length of the tail domain could be up to 0.5 µm if fully extended, which is a distance that, in principle, could be resolved in the light microscope.

Ankyrin isoforms possessing the tail domain are highly localized to axons, whereas isoforms lacking this domain are localized to the neuron cell body (49, 224). Possible functions of the tail domains include axonal targeting motifs, deregulation of ankyrin membrane-binding domains by physical separation from the regulatory domain, and long-range connections between molecules interacting with the death domain/COOH-terminal domains and membrane-binding domains. Given the potential distance spanned by the inserted sequence, these putative ankyrin-binding molecules could be in distinct membrane domains or even different cellular compartments.

A defining feature of the ankyrin family is their ability to interact through the membrane-binding domain with structurally diverse proteins with apparently unrelated primary sequences. Currently identified ion channels/pumps that associate with ankyrin and colocalize in cells include anion exchanger isoforms (AE1, AE2, AE3) (22, 23, 196, 293), the Na⁺-K⁺-ATPase (216, 297, 304), the voltage-dependent Na⁺ channel (263, 269), and the Na⁺/Ca²⁺ exchanger (240). IP₃ receptor and ryanodine receptor Ca²⁺-release channels also associate with ankyrins (36, 37, 199). Evidence for an in vivo interaction between ankyrin-B and Ca²⁺ release channels is based on altered targeting of these proteins in striated muscle of ankyrin-B-deficient mice (397) (see below). Finally, ankyrin also associates with cell adhesion molecules (CAMs) including CD44 (203, 249) and the L1 CAM family (L1/neurofascin/NrCAM/CHL1/NgCAM) (76, 79, 107, 182, 450). Evidence for physiologically important interactions between ankyrins and L1 CAMs include downregulation of L1 in axons of ankyrin-B (/) mice (358), and reduction of Dank2 in cell bodies of neurons of neuroglian mutant flies (35).

The ankyrin membrane-binding domain also interacts with cytoplasmic proteins including tubulin (19, 75, 82) and clathrin (283). Ankyrin associates with microtubules with a relatively low affinity in the micromolar range, and the physiological significance of this interaction remains to be determined. However, ankyrin association with clathrin occurs with a K_D in the nanomolar range and is specific for the fourth subdomain of ankyrin and the terminal knob domain of clathrin. Evidence that ankyrin-clathrin interactions are functionally important is that microinjection of the fourth subdomain inhibits receptor-mediated endocytosis of low-density lipoprotein (283).

The basis for diversity of binding partners for the ankyrin membrane-binding domains is due to special properties of the ankyrin repeats, a motif which mediates protein recognition for a large number of proteins including the transcription factors GABP-, Nf/IB, and SwI 6, p53 binding protein and p16 cyclin inhibitor (reviewed in Ref. 359). Atomic structures for these proteins reveal that ankyrin repeats are folded into a series of antiparallel -helices connected by loops that are arranged perpendicular to the helices (Fig. 6). Residues located at the tips of the loops are the most variable between repeats and contain recognition sites for diverse proteins. The four repeat subdomains of ankyrins each have distinct binding properties, and these subdomains also cooperate with each other to generate a further level of diversity (281, 282). An additional feature of ankyrin is that binding interactions can occur simultaneously with different membrane proteins, resulting in multiprotein complexes (281, 282).

Sites of ankyrin interactions have been evaluated for the erythrocyte anion exchanger (AE1) (78, 96, 424), the Na⁺-K⁺-ATPase (197, 453), and the L1 CAM family (181, 450). A general conclusion from these studies is ankyrin is capable of structurally distinct interactions with diverse proteins. This conclusion is reinforced by the fact that ankyrin has independent binding sites for AE1 and L1 CAMs (281, 282). Another conclusion from analysis of ankyrin-binding sites of the Na⁺-K⁺-ATPase (450) and L1 CAMs (450) is that ankyrin-binding sites of these proteins share a conformation as a random coil even though they lack obvious sequence similarity. An ankyrin-membrane protein complex has not yet been resolved at an atomic level, but these considerations suggest a model where random coils of at least certain ankyrin-binding proteins associate with loops of ankyrin repeats (see above) (450). Given that ankyrin repeats fold into subdomains, it is likely that loops of ankyrin repeats in different subdomains also could collaborate to create a "binding pocket."

Cytoplasmic domains of the erythrocyte anion exchanger (5) and neurofascin (450) are both dimers in solution. Considered together with the finding that ankyrin has two binding sites for neurofascin as well as for two sites for the anion exchanger, the existence of dimers implies that these proteins and ankyrin are capable of forming oligomeric complexes. One predicted configuration of the anion exchanger/ankyrin complex at low ratios of ankyrin to the anion axchanger is as a pseudo-anion exchanger tetramer with two dimers independently associated with distinct sites on the ankyrin membrane-binding domain (281). Evidence that such an arrangement occurs in erythrocyte membranes is provided by observations that the anion exchanger behaves as a mixture of dimers and tetramers in native membranes and only as a dimer in ankyrin-deficient membranes (439). One could also imagine that at higher ankyrin-to-membrane protein ratios it would be possible to form linear arrays of dimeric membrane proteins cross-linked by the multivalent ankyrin membrane-binding domain. These complexes could be further immobilized by coupling to the spectrin-based membrane skeleton through the spectrin-binding domain of ankyrin. Ability to form such large immobilized complexes between ankyrin and neurofascin could be important for the assembly of specialized membrane domains such as axon initial segments and nodes of Ranvier where these proteins are localized (see below).

A) L1 CAMS AS GENERAL CORECEPTORS FOR ANKYRINS. The major class of ankyrin-binding proteins in mammalian brain is a group of CAMs in the Ig/FnIII superfamily known as L1 CAMs. These molecules together comprise over 1% of the membrane protein in adult brain tissue and are abundantly expressed on axons in the developing nervous system (reviewed in Ref. 181). The L1 CAM family of cell adhesion molecules in the vertebrate nervous system is comprised of L1, NgCAM, NrCAM, CHL1, and neurofascin, in D. melanogaster by neuroglian (181), and in C. elegans by LAD1 (L. Chen and V. Bennett, unpublished data). L1 CAMs possess variable extracellular domains comprised of six Ig and three to five fibronectin type III domains, along with a relatively conserved cytoplasmic domain (181). Extracellular domains of L1 CAMs participate in homophilic interactions as well as a variety of interactions with soluble proteins and other CAMs (39, 121, 350). Extensive diversity in extracellular interactions of L1 CAM family members is provided by divergence between extracellular domains encoded by different genes as well as multiple alternatively spliced variants of each gene (as many as 50 estimated in the case of neurofascin) (165). Consistent with the diverse extracellular domains, a range of functions has been attributed to the L1 CAM family, including axon fasciculation, axonal guidance, neurite extension, a role in long-term potentiation, synaptogenesis, and myelination (39).

Analysis of LAD-1, the single L1 CAM gene in C. elegans, has provided a global view of the pattern of expression in a metazoan and suggests a general expression at sites of cell-cell contact early in embryonic development and in essentially all cell types (Chen and Bennett, unpublished data). LAD-1 binds to GFP-tagged worm ankyrin (unc44) in cultured cells and colocalizes with ankyrin in most cell types. These findings suggest that LAD-1 is the major receptor for ankyrin (unc-44) in the worms. Given that ankyrin is multivalent with respect to neurofascin and the anion exchanger (282), it is possible that ankyrin forms heterocomplexes with LAD-1 and various ion channels (see Fig. 7 for an example of vertebrate L1 CAM/Na⁺ channel complexes).

View larger version (20K): [in this window] [in a new window]   Fig. 7. Schematic model for organization of 480/270-kDa ankyrin-G and ankyrin-associated proteins (spectrin, L1 CAMs, voltage-gated Na+ channel) at axon initial segments and nodes of Ranvier. 480/270-kDa ankyrin-G (219), 186-kDa neurofascin and NrCAM (77), and / IV spectrin (24) are colocalized with voltage-gated Na+ channels at nodes of Ranvier and axon initial segments. Ankyrins are multivalent in in vitro assays (281, 282) and are proposed in this model to interact with both the channel and adhesion molecules. Neurofascin and possibly NrCAM are palmitoylated at a cysteine residue in the membrane-spanning domain (342), which could contribute to a phospholipid microdomain. The extended tail domain of 480-kDa ankyrin-G cou