Summary The actin crosslinking proteins exhibit marked diversity in size and shape and crosslink actin filaments in different ways. Amino acid sequence analysis of many of these proteins has provided clues to the origin of their diversity. Spectrin, a-actinin, ABP-120, ABP-280, fimbrin, and dystrophin share a homologous sequence segment that is implicated as the common actin binding domain. The remainder of each protein consists of repetitive and non-repetitive sequence segments that have been shuffled and multiplied in evolution to produce a variety of proteins that are related in function and in composition, but that differ significantly in structure. introduction The actin-based cytoskeleton is thought to play an important role in the structure and motility of nonmuscle cells in addition to its well known role in muscle contraction. The precise arrangement of actin filaments (F-actin) in a given cell type is tailored to the functional requirements of that cell. Single filaments of actin can serve as the track for myosin-based motility, but actin is often crosslinked into isotropic gels (e.g. cortical actin) and into bundles (e.g. the cores of microvilli and stercocilia). The state of actin in a given cell or subcellular domain is in large part a function of its associated actin binding proteins. large numbers of which have been found in extracts of muscle and nonmuscle cells. These proteins are often bivalent molecules that can crosslink actin into gcls or lateral bundles of filaments in vztro, much like the gels and bundles that are observed in cells (reviewed in refs 1,2). Why are there so many different actin crosslinking proteins? Dcspite their similar behavior in v i m , the function of actin crosslinkers in cells is likely to be far more diverse. In addition to their crosslinking roles. many of these proteins are also responsible for the interactions between actin filaments and other cellular structures such as membranes, Z discs, microtubules. and cell junctions. Diversity also provides for differential regulation, so that calcium and protein cofactors such as calmodulin and protein 4.1 affect the actin interaction of some crosslinkers but not others(3-4). Thus it is not surprising that actin crosslinkers come in
many shapes and sizes, ranging from one to four subunits per native molecule and from 23 kD (Acanrhamoeba Gelactin I) to 430 kD (Drosophila spectrin) in molecular mass('.'). Recent attention has turned to the origins of diversity among the actin crosslinkers. Comparisons of the amino acid sequences of a-actinin, spectrin, the 'actin binding proteins' (ABP-280 and ABP-120) and fimbrin have revealed a few types of sequence segments or cassettes that have been shuffled in evolution to produce related proteins with rcrnarkably different structural properties. Sequence comparisons have also led to the tentative identification of dystrophin, the defective gene product in Duchenne and Becker muscular dystrophies, as an actin crosslinking prot e i ~ ~ (In~ )this , review, I will summarize the recent sequence data and their implication for the structure and evolution of the actin crosslinking proteins. Particular emphasis is placed on the evolution of members of the spectrin superfamily, for which the greate5t number of sequences are available. a-Act ini n a-Actinin was first described as an actin crosslinker in ckeletal muscle, but additional family membcrs have been found in smooth muscle and non-muscle cells (reviewed in ref. 3). Muscle and non-muscle isoforms arc similar in mass (about 100kD), but differ in their sensitivity to calcium. Calcium binding diminishes the ability of non-muscle a-actinins to crosslink actin, a regulatory function that is probably important in cells that assemble and disassemble their cytoskeleton, whereas muscle a-actinins are calcium-insensitive("). By electron microscopy, a-actinin appears as a 30-40 nm rod-shaped dimeric molecule and antibody labelling experiments have shown that the strands of thc dimcr are antiparallel('). a-Actinin increases the viscosity of F-actin solutions by forming loose lateral bundles of actin filaments("). The amino acid sequences of muscle and non-muscle actini in ins("-'^) (shown schematically in Fig. 1) can each be subdivided into six sequence segments that have been conserved in evolution. Actin binding activity is attributed to the amino terminal region of the mo1ecule(l6), although the activity of proteolytic fragrncnts is not as striking as the activity of intact aactinin. Interestingly, the first -250 residues of the polypeptide constitute a domain that is conserved among many different actin crosslinking proteins (filled rectangle). A very differcnt domain is found at the carboxy terminus of the polypeptide (open rectangle). Tn the native dimer, this carboxy terminal domain (which includes two helix-loop-helix calcium binding sequences or EF hands) is apposed to the amino terminal domain of its neighboring subunit. Calciumbinding is thought to trigger a structural change in the carboxy terminal domain that affects the activity of the actin binding site in the apposed amino terminal
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d o m ~ i i n ( l ~ ’Muscle ~ ~ ) . a-actinin apparently lacks calcium binding activity(17),and therefore calcium sensitivity, because of deleterious amino acid substitutions in the EF hands(’2). The middle region of a-actinin is made up of four quasi-repetitive segments, each -120 amino acids in length (shaded ellipses), that have some resemblance to the repetitive segments found in aand fl spectrins(12714,1s) (open ellipses, see below). But the quasi-repetitive segments of a-actinin bear only limited similarity to one anothed3), suggesting that substantial divergence occured after their multiplication from an ancestral sequence segment. Spectrin Spectrins are another well characterized family of actin crosslinking proteins, first recognized as a major component of the human erythrocyte membrane. Genetic, structural and biochemical evidcnce all suggest that spectrin forms a submembrane network in the erythrocyte that is crosslinked by short actin filaments, and that this complex is essential for normal cell shape and membrane stability (reviewed in ref. 19). Several isoforms of spectrin (also known as fodrin and TW260/240) have been identified in various nonerythroid cell types, and all have a similar structure (reviewed in ref. 20). They consist of antiparallel heterodimers that are joined head to head to form an elongated tetramer (-200-250 nm) that crosslinks actin filaments into isotropic gels. a-Spectrhs are about 280 kD in molecular mass, and /3 spectrins range from 246kD (human erythroid 43 spectrin) to 430kD (Drosophila & spectrin)(‘. I). The /3 subunit is responsible for much of the diversity among spectrin isoforms in such properties as molecular length,
Fig. 1. Schematic sequence comparison of the actin crosslinking proteins. Rectangles distinguish the non-repetitive sequence segments from repetitive sequences. Solid rectangles indicate the conserved actiii binding domain. segments marked ‘EF’ include thc EF hand calcium binding sequence, and segments marked ‘S’ include the SH3 sequence motif. Arrows mark the position of ‘hinge’ regions in the sequence of dystrophin and ABP-280. Sequence data is not available Lor the carboxy terminal domain of pH spcctrin.
membrane binding, and regulation of actin binding activities(4). Complete sequence data is available for the w and /3 subunits of erythrocyte spectrin(21.22)(shown schematically as a tetramer in Fig. 1). Complete sequences of three non-erythroid LY spectrins are also known, and these have the same general structure as erythrocyte a spectrin(””-25)).But, while a spectrins are conserved in length and segmented structure, they show marked diversity in their amino acid se uences. As suggested from properties of the protein.?’6) and confirmed by comparisons of the deduced protein sequence^(^^^'^)), erythroid spectrin appears to be the product of a recent branchpoint in spcctrin evolution. Thus, human nonerythroid a’ specti-in is more closely related to Drosophila a spectrin(24)than to human erythroid N spectrin(”*”). Partial sequence comparisons of nonerythroid j3 s ectrins to erythroid /? spectrin suggest a similar trendg3’”, although a complete analysis must await sequence data for vertebrate non-erythroid /s spectrin. In contrast to aspectrins, therc arc likely to be differences in the segmented organization of 6 spectrins to account for their heterogeneity in size (as in fiH spectrin, described below). The sequences of aand {3 spectrin are similar in many ways to a-actinin. One striking feature is that the ends of the native spectrin molecule resemble the ends of aactinin. In fact, homologs of all of the a-actinin segments can be found within a and spectrin: the three amino terminal segments of /? spectrin resemble the three amino terminal segments of a-actinin(21329) and the three carboxy terminal segments of a spectrin resemble the three carboxy terminal segments of aa~tinin(’~,~”’~). Spectrin differs from a-actinin in the
repetitive sequence domain. Spectrin not only includes more repetitive segments than a-actinin, thus accounting for its greater size, but it also includes a distinct lineage of repetitive segments that are shorter (-106 residues, open ellipses) and more closely related to one another than are the quasi-repetitive segments that occur in both a-actinin and spectrin (shaded ellipses). The length difference between the two segment types is partly due to an &residue insertion in the quasirepetitive segments that is recognized when the repetitive segments of a and /? spectrins are aligned and compared with the quasi-repetitive segments(21-2). Presumably, the quasi-repetitive segments evolved from an ancestor that included this insert, and the repetitive scgments evolved from an ancestor that lacked the insert. Biochemical studies of erythrocyte /3 spectrin suggest that the amino-terminal non-repetitive segment (filled rectangle is responsible for spectrin’s actin-binding activity(30. Similar studies place the actin binding site within a homologous domain of the protein ABP- 120(31) (described below). Conservation of this sequence segment in a number of actin binding proteins is also consistent with its proposed actin-binding activity. Based on these lines of evidence, the conserved amino terminal segment is referred to here as the ‘actinbinding domain.’ The roles of the other non-repetitive spectrin sequences remain to be determined. First, segment 22, at the carboxy terminus of a spectrin, includes two EF hand sequences(23),(as does segment 6 of ru-actinin) (I4) and recombinant fragments of Drumphila a spectrin bind calcium in blot overlay experiments(”). Interestingly, human erythrocyte spectrin and sea urchin spectrin both ap ear to be calcium-sensitive in their actin-interactionsCg.’”) and future studies should address the possible role of the EF hand sequences. Although vertebrate non-erythroid LU spectrin is also predicted to bind calcium(z3),calcium does not appear Second, a 36to affect its actin intcraction in residue domain between segments 11 and 12 of vertebrate non-erythroid ~pectrins(’~)includes a calmodulin bindin site and a calcium-dependent protease cleavage site(3 36). These sites act synergistically to affect the structure and actin-binding activity of spectrin and may be important to its regulation in non-erythroid cells(34).However, Drosophila LU spectrin and human erythrocyte a spectrin both lack this 36-residue sequence(22.B). Third, segment 10, from the middle region of a spectrins, resembles the SH3 domain found in oncogenes such as v-src and v-crk, and in phospholipase C(37). Other actin-binding proteins, including yeast ABPlp and myosin I(3*), include a similar sequence and these proteins may bind a common ligand in the cortical region of the cell. Finally, there appears to be heterogeneity in the protein products of a single a spectrin gene. Short sequences in segments 10 and 21 of human non-erythroid N spectrin are thought to arise by alternative splicing, and these
1
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sequences may affect specific functional sites in the spectrin molecule(” 17). Recently, a novel isoform of spectrin that has an unuwally large /3 subunit (pH)and is 25 % longer than ~ tetramer was identified in Drosopliila(6). the I X spectrin Partial cDNA sequence data from pHrevealed that it is closely related to the 13 amino terminal segments o f /3 spectrin, although lJHincludes a non-repetitive segment with an SH3 sequence motif (Fig. 1) that is not found in other p spectrins. The large size (430 kD) and sequence organization of /jH are similar to the vertebrate protein dystrophin (described below). but additional sequence data from the carboxy end of pH. a region that is conserved among dystrophins. will be necessary to further resolve the structural rclation between spectrin and dystrophin. Dystrophin Using an elegant genetic strategy, human genomic DNA encoding the gene product associated with Duchenne and Becker muscular dystrophies was isolated and used to recover cDNA encoding the normal gene product(39). The gene product, named dystrophin, is extraordinarily large (427 kD)(7’. Sequence comparisons between dystrophin, spectrin and a-actinin revealed that all three of these proteins share signihcant sequence homology, both in the amino terminal actin binding domain and in the repetitive sequence region(4o41). Therefore, the dystrophin molecule i? presumed to be an elongated actin binding protein(’), although the native dystrophin molecule has not yet been purified and characterized. The carboxy terminal domain of dystrophin (Fig. 1) includes a short region that is similar to Dictyostelium aa ~ t i n i n ((shaded ~) rectangle) and a unique region (open rectangle) that is conserved in chicken and human dyqtro hins and in an autosomal homolog of dystro~ h i n .43). ( ~ Dystrophin also includes four potential ‘hinge’ sites (arrows) that might accommodate folds within the native molecule(“). Dystrophin lacks the quasi-repetitive lineage of segments found in spectrins and a-actinin. Instead, the dystrophin repctitive segments (open ellipses) coniorm to a 109-residue repeating unit(“).
F
-
The Spectrin Superfamily of Proteins The repetitive and non-repetitive sequence similarities among spectrins, a-actinins and dystrophin support their classification a5 a spectrin superfamily of proteins(29345).One of the diagnostic features of this superfamily is the 106 to 120-recidue repctitivc sequence unit that was first identified in peptide sequences of erythrocyte spectrin(36).Structural predictions based on the amino acid sequences of spectrin, aactinin, and dystrophin suggest that each repetitive sequence unit folds to produce a repetitive structural unit, largely a helical, that independently contributes to the length of the native protein.(7,”,46).
Actin Binding Protein-280 Actin binding protein (ABP-280) is an elongated actin crosslinking protein that causes actin filaments to form isotropic gels and promotes perpendicular branching of filaments (reviewed in ref. 2). It was originally purified from rabbit macrophage and a related protein (filamin) was identified in smooth muscle(’7). ABP-280 is found in a variety of cell types and is especially prominent in platelets where it promotes interactions between the actin cytoskeleton and the membrane glycoprotein GPIb/IX(“). Like spectrin and a-actinin, ABP-280 is an elongated molecule with actin binding sites displayed at its termini (contour length= 160nln). A significant difference is that ABP-280 exists as a tail-to-tail dimer rather than an antiparallel dimed“). The sequence of ABP-280 revealed an interestin trend in the evolution of the actin crosslinkers(56 (Fig. 1). ABP-280 has an amino terminal actin binding domain followed by a domain of 23 repetitive segments that are each -100 amino acids long. Thus, like members of the spectrin superfamily, ABP-280 appears to have evolved through multiplication of an ancestral segment to form a repetitive structural domain that determines the relative placement of the aminoterminal actin binding sites in the native molecule. But the ABP-280 repeating unit does not appear to be related to the segments of the spectrin superfamily in sequence or its predicted structure. While the s ectrin segments are predicted to be largely a-heIical(’), the repetitive segments of ABP-280 are predicted to have /3sheet structure(5”).
in the cores of intestinal microvilliijJ). Fimbrin differs from the above crosslinkers in a few important ways. First, it is much smaller; at 68 kD i t is little more than twice the expected size of the conserved actin binding domain (-30kD). Second, it is a monomeric protein and thus must possess two aclin binding sites per polypeptide(”). Third. fimbrin forms tight lateral bundles of filaments(”), whereas the other proteins form loose bundles or isotropic gels. The sequence of fimbrin, while it shares the same actin binding domain found in thc above proteins, reveals a striking difference in primary structure(”) (Fig. 1). The molecule consists of two tandem actin binding domains, preceded by a short segment with two conserved EF hand sequences. Like non-muscle aactinin, fimbrin binds calcium, and calcium alters the The F-actin bundling properties of fimbrin iiz ~,’ifm(’.’). fimbrin sequence also revealed that it is homologous to the more widely expressed proteins L-plastin and T-pla~tin(~~).
Actin Binding Protein-120 ABP-120 is found in the cortical cytoplasm of Dictyostelium, a region of the cell that is rich in crosslinked actin filaments(51). The purified protein is similar in size and shape to a-actinin and is thought to be an antiparallel homodimer(”z). Functionally, ABP120 behaves much like ABP-280 in that it forms an isotropic actin gel with perpendicular branches(”). The amino acid sequence of ABP-120 resemblcs ABP-280 (Fig. 1). It consists of the amino terminal actin binding domain followed by six -100-residue repetitive segments that are predicted to have /3-sheet structure(53). Therefore, the relation of ABP-120 to ABP-280 is analogous to the relation of a-actinin to spectrin. In both cases, variation in repetitive segment num ber produces homologous molecules that differ in length and presumably link actin filaments over different distances in the cytoplasm. Given the predicted similarities in secondary structure between ABP-280 and ABP-120, it is surprising that these molccules form dimcrs in different way^(^^.^'). It will be interesting to resolve the molecular bases for their apparently distinct modes of self association.
A Superfamily of Actin Crosslinking Proteins The spectrin superfamily of proteins is clearly a subset of a larger group of actin crosslinking proteins that I will refer to here as the ’actin crosslinker superfamily.’ Thc unique primary structure of fimbrin emphasizcs the plastic, modular nature of the actin binding domain that relates these proteins in evolution. The minimum requirement for actin crosslinking appears to be the presence of two of these actin binding sites. The relative positioning of the two sites, whether they occur on a single polypeptide chain, on two chains, or on four chains, appears to determine the specific crocslinking behavior of the individual molecule. Sequence alignments of the actin binding domains from different members of the superfamily reveal a number of conserved residues that are likelv to have a direct role in the actin i n t e r a c t i ~ n ( ~ ’j 3~, j~f ).. ~Some ’ of the conserved residues may be important to the correct ~). folding of the actin binding d ~ r n a i n ( ~ > ’However, there are also significant sequence differences between the actin binding domains that may account for some of their differences in regulation and activity. For example, protein 4.1 and Ca+*/calmodulin influence the actin crosrlinking activity of some spectrin isoformrC4),but are not known to affect the activity of other proteins such as a-actinin and fimbrin. The actin binding activit of some crosslinkers (such as a-actinin and filamin)(5J is inhibited by tropornyoin. a protein that binds to thc sides of actin filaments(’), whereas the activity of the spectrin isoform TW260/240 is not inhibited by tropomyosin(”). The sequence of TW260/240 is not yet available, but in the future it is likely to provide useful insights into the structure and activity of the actin binding domain.
Fimbrin Fimbrin is an actin bundling protein that was first found
Evolution of the Actin Crosslinking Proteins Multigene families probably arise through stochastic
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Fig. 2. Dot-plot comparisons of members of the spectrin siipcrfamily. Sequences of Drusoyhiku a spectrin(*'), human erythrocyte p spectrin("), and Drosophila a-actinin"') are shown schematically along the plot axes. The stringencies of comparison were 20 identical matches per window of 100 residues in A, 20 and 34 identical matches per window of 100 in B, and 26 identical matches per window o f 100 in C. The programs 'Compare' and 'Dot-plot' were used to generate the comparisons@*).Arrows indicate the duplicated domain of a spectrin.
gene duplication evend6") and the rcsulting products are thought to diverge through processes such as exon shuffling. unequal crossing over (multiplication), and genetic Thesc three processes can account for much of the observed diversity ainong the actin crosslinkers. (1) Sequence shuffling produced structurally distinct proteins that share a single conserved domain - the actin binding site. (2) Sequence multiplication provided variation in the number of segments within each protein, either before or after shuffling. and this produced proteins that differ in shape and size. ( 3 ) Sequence drift provided further variation in the products of shuffling and multiplication. For example, sequence drift can account for the differential calcium sensitivity of muscle and non-muscle a-actinins. Dot-plot sequence compari5ons reveal the boundaries of shuffling events and multiplication events, and through varied stringency, they also provide a measure of sequence drift. In a computer-generated dot-plot, the two sequences to be compared are pkdced on the axes of a comparison grid and a sliding window along each axis is used to compare every region of one sequence with every region of a second sequence. The resulting diagonals indicate the coordinates of similarity between the two sequences. By varying the stringency of comparison, diffcrent degrees of similarity can be rcsolvcd. (Stringency refers to the minimal number of identical matches per sequence window that are required to generate a signal.) When the sequence of Drosophila cwaclinin('3)is compared to the sequence of erythrocyte fi spectrin(21),the diagonal line that spans the first 250 residues of both proteins describes thc boundary of the conserved actin binding domain (Fig. 2A). Most of the other diagonals relate segment 3 of a-actinin to the repetitive segments of ?/J spectrin. When Dvosophila a spectrin is compared with itself,
both the boundaries and the periodicity of repeating sequences appear in the resulting dot-plot (Fig. 2B). At low stringency (20 % identity; Fig. 2B, upper left), nearly all of the 106-residue repetitive se ments in a spectrin score as similar to onc another("? However, thc repctitivc sequence is degenerate, and high stringency dot-plots (34 % identity; Fig. 2B, lower right) reveal only a subset of the low stringency pattern(") (Fig. 2B, arrow). One implication of these patterns is that a minimal framework of residues is conserved within the repeating unit, but residues outside of this framework are free to drift according to other selective pressures. As a result, most of the repetitive segments are only -20-30 % identical to one another("). The most prominent high stringency signal corresponds to the sequence of segments 2-8 and 11-17. These boundaries are not easily discerned in the dot-plot of any one protein but are most obvious in segment-by-segment comparisons, as described by Wasenius et al.(23).Segment 2 is more like segment 11 than any other segment, segment 3 is more like segment 12 than any other segment, and so on. These two groups of segments probably arose by partial duplication of a common ancestor, and sequence drift has not yet erased the sequence similarities that reflect their common origin. We recently proposed a scheme by which spcctrin might have evolved from an a-actinin-like ancestor through successive duplication events("). The rationale for the scheme stems from two observations. First, the quasi-repetitive segments of a-actinin show greater differences among themselves than any of the other repetitive segments, which suggests that they arose through the most ancient duplication events (although a difference in selective pressures might also account for their divergence), Second, the repetitive segments of
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from chicken a spectrin, erythrocyte a and /3 spectrin and dystrophin (using the sequence alignment of Koenig and K ~ n k e l ) (were ~ ~ ) also identified on the basis of a conserved residue in at least half of the repetitive segments (Fig. 4). Many of the consensus positions that are conserved in all five consensus sequences (Fig. 4: vertical bars) are also part of the a spectrin strict consensus. These residues are probably important to the structural framework of the repeating unit. i";spectrin has fewer consensus positions than LY spectrins, and all except one of the p positions (#S3) are also found in the a consensus. Dystrophin has fewer consensus positions than a spcctrin, but seven of its consensus positions are unique to dystrophin. Dystrophin is further distinguished from the spectrins by the length of its repeating unit (-109 residues vs. 106 residues). Three positions (marked by parentheses) were omitted from the dystrophin consensus to maximize its alignment with the spectrin conscnsus. These two properties of the dystrophin consensus suggest that the dystrophin repetitive segments are a distinct lineage that evolved independently of the repetitive segments of spectrin. However, the segments of dystrophin and spectrin are related in a third important property: they both lack the 8-residue sequence that, in part, distinguishes the quasi-repetitive lineage found in spectrin and acactinin from the repetitive lineage of segments in spectrin. Thus, dystrophin may also have evolved from an a-actininlike ancestor, perhaps the same ancestor that produced spectrin, but diverged before multiplication of the repetitive segments (Fig. 3). This scheme suggests a remarkable feat of convergence since the /3 spectrin lineage produced a protein (/kspectrin) that is ncarly identical in size to dystrophin('). The predicted structural similarities between ABP120 and ABP-280(50353)suggest that they too arose by differential multiplication steps. But sequence compaiisons do not reveal the lineal relations that are observed among members of the spectrin superfamily. The sequencc of additional inembers of this group, such as Glamin, will provide a broader basis for future sequence comparisons.
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Fig. 3. ELolution of the spectrin superfamily. See text for details.
spectrins and dystrophin show greater similarity to the third segment of a-actinin than to the other a-actinin segments (Fig. 2A). These observations are consistent with an early evolutionary event in which segment 3 of a-actinin was duplicated, modified and subsequently multiplied to generate the spectrins and dystrophin (Fig. 3). While the scheme is hypothetical in detail, it illustrates some of the interesting questions in the evolution of the spectrin superfamily. The a spectrin dotplot result (Fig. 2B) suggests that one of the steps in its evolution was a large-scale duplication(23) (Fig. 3, horizontal bars). Did the repetitive domains of ac and i"; spectrin arise through the same duplication steps, or through independent steps starting from a common ancestor? Comparison of erythrocyte p spectrin(*') to Drosoyhila a spectrin(24)in dot-plots suggests that the answer is both. The large duplicated sequence that is present in two copies in a spectiin appears to be present in only one copy near the middle of the repetitive domain in j3 spectrin (Fig. 2C, arrows). Thus, a and /3 spectrin may have had a common ancestor that was intermediate in size between spectrin and a-actinin (Fig. 3). The repetitive segments of dystrophin are not sufficiently similar to one another or to spectrin to be able to trace their evolution using dot-plots, but information on their origin can be obtained through consensus comparisons. Alignment of the repetitive segments of Dvosophilu a spectrin reveals 14 positions in the repeating unit that have a conserved amino acid residue that is rarely substituted, and there are 54 positions within the repeating unit that are conserved in at least half of the repetitive We refer to the latter positions as the consensus and the former positions as the 'strict consensus.' Consensus sequences Position:
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Conclusions Many of the actin crosslinking proteins are related through a common actin-binding domain. Additional 50
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sequence motifs are conscrvcd among these proteins, but in the course of evolution they have been shuffled to produce proteins that vary remarkably in structure. With the possible exception of dystrophin, the actin binding site is always present in two copies per native molecule. Biochemical studies have also failed to detect monovalent members of the superfamily that are mechanistically analo ous to some of the microtubuleassociated proteins(63. Future studies should address whether or not such proteins exist. Still other actin crosslinkers, such as the gelactins(')). have yet to be sequenced and these proteins may provide further insight into crosslinker structure and evolution. I have used the term 'actin crosslinker superfamily' to refer to the protcins that hhare the conserved actin binding domain, but it is probably premature to adopt a specific nomenclature until the boundaries of the superfamily are better defined. There are other protein se uences For associated with actin-binding example, MAP2 and caldesmon are actin crosslinkers that appcar to be unrelated to the superfamily in seq~ence(~~,~~). The identification of sequence similarities among the actin crosslinking proteins has significantly advanced our knowledge of their structure and evolution. But to understand the cellular roles of these proteins, it will be necessary to understand the functional differences betwcen them. The non-repetitive segments are probably important to the specialized roles of these proteins. and attention should be focused on their contribution to function. The widely distributed repetitive segments and the actin binding domain also may have acquired specialized functions through sequence drift. The modular structure of these proteins should allow specific repetitive and non-repetitive segments to be isolated and tested for functional homology with one another. Genetic studies of some of these proteins have been initiatcd in DictyosteZium and DrosophiZa(13.522), and these experimental systems are likely to be valuable in future studies of functional conservation among the actin crosslinkers.
7
Acknowledgements I thank Dan Branton and Dan Kiehart for critical reading of the manuscript. This work was supported by
NIH GM39686. References 1 POLLARD, 1.D . AND C O O P ~1. K A. , (1986). Actin and actin-binding proteins: A critical evaluatioii of mechanisms and functions. Annu. Rei,. Biochenr. 55. 987-1038. 2 STOSSEL. T. P., CHAPONNIER, C., EZZEL.R. M.. HARTWIG. J. H.. J.ANMEY, P. A , , K\VI-\TKoW~SKI. D . J.. LlUD. S. E., SMITH, D . B., SOUTHWICK, F. S., Ylii, H. L. A N D ZANFR, K. S. (1985). Nonmusclc actin-bindingprotcins. Annu. Rev. Cell B d . 1. 353-402. 3 RLANCHARD. A , . OHANIAN: V. A N D CRITCHLEP, D. (1989). Thc str-ncturc and function of a-actinin. J. Muscle Rea. und Ccdl Mofdiry 10, 280-289. 4 COLLMAN. T. K.. FISHKIND, D. J . . MOOSEKER. M. S. AND MORROW,J. S. (1989). Contributions of thc /+subunit to spectrin structure and function. Cell Woril. CjXoskeleroii 12, 248-263.
5 MARCIA.11. AUD KOKN.E. D. (l'W7). Purification from Acnrzrharnoeba cwrellunii ol proteins that induce gelation and syncresis of F-actin. .I. Bzol. Chcm. 252. 399-402. 6 DUBREUL, R. R., BYERS, T. .I., STFWART. C. T. A N D KICHART,I). P. (19913). A specrrin isoforin from D m s n p i d u is similar in size to vertehraie dystrophin. J. Cell B i d . 111, 1849-1858. 7 KOENIC, M., MOYACO. A. P. AND K U N k h L , 1.M. (1988). The complete sequence of dystrophin predicts a rod-shaped cytoskeletal protein. Cell 53, 219-228. 6 BTJRRIDGE, K. AND FERAMISCO, J . R. (1981). Non-muscle a-actinins are calcium-sensitive actin-binding proteins. Nnfure 294. 365-367. 9 WALRAFF, E., SCHLEJDLR, M., M~DERSITZKI, M., KILGER, D . , ISLNBLRCI, G. A N D GERISCII. G . (1986). Selection of Dicfyosreliton mutants defective in cytoskeletal proteins: Use of an antibody that binds to the ends of cr-actinin rods. EMBU J . 5. 61-67. 10 POULUBYAYA, Z. A., TSKHOVRLBOVA, L. A , , ZAALISIIVILI, M. R.1. AND STWANLVKO, G. A. (1975). Electron microscopic study of a-actinin. J. Moi. Bioi. 92. 357-359. 11 ARIMURA. C., SCZUKI: T . , Y4KAGISAWA, M., IhlAMURA, M,, FIAMAD.4, Y . AND MAS-AKI, 'I. (1Y88). Primary structure of chicken skeletal muscle and fibroblast a-actinins deduced from cDNA sequences. Eur. J . Biochem. 177, 649-655. 12 R ~ R O N M., D., DAWSON, M. U.. JONES, P. , PATEL,B. AND CRITCHLEY, L). K. (1987). The sequencs of chick wactinin reveals homologies to spectrin and calmodulin. J. Bioi. Chern. 262, 17 623-17 629. 13 FVRRERC, E., KFI I.Y, M.. BAIL , E.. FYRRERG, C. AND RFEDY,M. C. (1990). blolecular genetics of sarcomeric Drosophifn a-aclinin: mutant allcle~disrupL Z disk integrity and muscle insertions, but do not affect nonmuscle tissues. J , C'eN Bioi. 110, 1999-2011. 14 NOEGEL.A,. WITKE.W. AND SCHLEICHBR. M. (1987). Calcium-scnsitivc non-inusclc a-actinin contains EF-hand structurcs and highly conserved rcgions. Fehs Left 221. 391-396. 15 YOUSSOUPIAN. H.. McAi M. mu KWIAIWWSKL. D. J . (1990). Cloning and chromosomal localimtion 01. the human cytoskeletal a-actinin gene reveals linkage to the /3 spectrin gene. Am. J . H u m . Gen. 47. 62-72. 16 MIMVRA, N. 4ND ASAUO, A. (1987). Further characterizalion of a conserved actin-binding 27 kDa fragment of actinogelin and a-actinins and mapping of their binding sites on the actin molecule by chemical crosa-linking. J. B i d Chem. 262. 4117-4723 17 D L H R ~ L IR. I LR., . BRANDIN, E.. SUN REISBEKG. J . H., GOLDSThIN. 1.S. B. AND BRANTOK, D. [ 1991). Structure. calmodulin-binding. and calcium-binding properties of recombinant (Y spcctriii polypcptidcs. J . Bioi. Chen?., in press. 18 W'ASEPIIUS. V.-M.. N.GWANEN, 0..L ~ H T O V.-P. , AND SARASTE,M. (1987). a-actinin and spectrin have common structural domains. FLBS Leu. 221. 73-76. 19 B ~ N N ~ IV-.I(1985). . 'The membrane skeleton of buman erythrocytes and its R e. v . Biochrm. 54, 273-3114. implication for more complex cells. A ~ I ? u 20 COLEMAN, 1. R., FIYHKIVIJ: T). J., MOOSEKER: M. s. AKD MORROW, J. S. (1989). Functional diversily among yxxtrin isoforms. Cell ~bfo~il. Cytorkelelocz 12. 225-247. J. C., C H A N G , J.-G.. TSF, w.T., M A R C H E S I , V. T. AND 21 WIUKEIMAN. FORGET. B , G. (19YO). Pull length sequence of the cDNA for human erythroid fi spectrin. J . Bioi. (%ern. 265, 11827 -11832. 22 SAHR.K. E.. LAURILA, P.. K O T ~ L L., A , SCARPA, A. L.. COLPAL, E.. LHO. T. I... I,INNENR4CH, A. J., WINKELMAY, J . C.. SPEICHER, D. W'..MARCHESI. V. T., CLRTIS,P. .I. AYD FORGET,B. G. (1990). The complete cDNA and polypeptide qequence of human erythroid n spectrin. J. Biol. Cheni. 265. 4434--443. 23 WASENUS,V.-M., SARASIE, M., SALVEX, P., EKAMAA, M., HOLM.L. AND LEHTO, V.-P. (1989). Primary structure ofthe brain aspectrin. J . Ce/iBio/.108, 79-93, 24 DUBKLLIIL. R. K.. UYEKS, T. J . , SILLMAN. A . 1.. BAR-ZVI.D . , G o I D s T F I N , L. S. B. AND BRANrOii. L). (1989). 'The complete sequence of Drosuphifa a) spectrin: Conservation of structural domains between a spectrim and aactinin. 1. Cell B i d . 109. 2197-2206. 25 h l o o ~R. , T. AND MCMAHON.A. P. (1990). Gciieration of diversity in noncrythroid spectrim. J. B i d Cheni. 265. 3427-4433. 26 GLONNLY. J . R.. J K ;\NU GLONAEI.P. (1984). Comparison of spectrin isolatcd from crpthroid and non-erythroid sources. Eur. 1. Biochenz. 144, 529-539. 27 hkIL?AHVN,A. P.. GI~BELHAUS, D. H.. CH.%MPION. J. E., B ~ L L EJS. . A , , L:ACEY. C~RRI'IT. S . B., HENCHhLAN. S . K . AND MOON,R. T. (1987). CDNA cloning, sequencing and chromosonic mapping of a non-erythroid spectrin. human cr-fodrin. Differenrimion 34, 68-78. 28 Lmo. 7 . L., FORTUGNO-EKIKSUN. D., BARTON, D.. YANG-FENG, 'I. L., FKANLKO. L., HARKIS, A. S.,MURKOW, J. S . , MARCHESI, V. 'I. AND BENZ,E. J., JR(1988). Comparison of nnnerythroid espectrin genes revcals strict homology among diverse upecies. Mol. Cell. B i d . 8, 1-9. 29 BYERS, T. J . , HUSAIN-CHISHTI, A,, DIIBREUIL, R. R., BRANTON, D . AND G~I.DSTFIN. L. S. B. (1989). Dro.~ophilu spectrin: Sequence similarity to the
(r?)
amino-terminal tlomain of wattinin and dystrophin. J. Cell 5iol. 109, 1633-1641. W . F. .AND GOODMAN, s. R. (1990). The 30 K ~ R I U C H A., hf., ZIMMOR, identifica ti017 and sequence of the actin-binding domain of human red blood cell 0 spectrin. J. Biol. Chem. 265, 21833-11840. 31 BKESNICK, A.R.,WARREN, V. AND C~ NDE E IX. J. (1990). Identification of a short sequence essential for actin binding by Dicryosfeliuni ABP120. J. Bioi. Cliern. 265, 9236-9240. 32 FOWLER. V. A X D 'I'AYLOK. L). L. (1980). Spectrin plus band 4.1 cross-link actin. J. Cell B i d . 85, 361-376. 33 FISHKIND, D . J.. B O N U ~E K., 31. AND BFGG,I). A. (1987). Isolation and characterization of sea urchin egg spectrin: calcium modulation of thc spcctrinactin interaction. Cell Motility und thr C.yluskeletoii 7, 304-314. 34 HARKIS,A. S. .AND MORKOW. J. S. (1990). Calinoduliu and calciumdcpcndcnt protease I coordinately regdale the interaction of fodrin with actin. Proc. Xarl Acad. Sci. U S A 87, 3007-3013. 35 H ~ RRI S, A. S.,CROALL, D . E. ~ N M D ~ K K O J. WS. , (1988). The calmodulinbinding
many shapes and sizes, ranging from one to four subunits per native molecule and from 23 kD (Acanrhamoeba Gelactin I) to 430 kD (Drosophila spectrin) in molecular mass('.'). Recent attention has turned to the origins of diversity among the actin crosslinkers. Comparisons of the amino acid sequences of a-actinin, spectrin, the 'actin binding proteins' (ABP-280 and ABP-120) and fimbrin have revealed a few types of sequence segments or cassettes that have been shuffled in evolution to produce related proteins with rcrnarkably different structural properties. Sequence comparisons have also led to the tentative identification of dystrophin, the defective gene product in Duchenne and Becker muscular dystrophies, as an actin crosslinking prot e i ~ ~ (In~ )this , review, I will summarize the recent sequence data and their implication for the structure and evolution of the actin crosslinking proteins. Particular emphasis is placed on the evolution of members of the spectrin superfamily, for which the greate5t number of sequences are available. a-Act ini n a-Actinin was first described as an actin crosslinker in ckeletal muscle, but additional family membcrs have been found in smooth muscle and non-muscle cells (reviewed in ref. 3). Muscle and non-muscle isoforms arc similar in mass (about 100kD), but differ in their sensitivity to calcium. Calcium binding diminishes the ability of non-muscle a-actinins to crosslink actin, a regulatory function that is probably important in cells that assemble and disassemble their cytoskeleton, whereas muscle a-actinins are calcium-insensitive("). By electron microscopy, a-actinin appears as a 30-40 nm rod-shaped dimeric molecule and antibody labelling experiments have shown that the strands of thc dimcr are antiparallel('). a-Actinin increases the viscosity of F-actin solutions by forming loose lateral bundles of actin filaments("). The amino acid sequences of muscle and non-muscle actini in ins("-'^) (shown schematically in Fig. 1) can each be subdivided into six sequence segments that have been conserved in evolution. Actin binding activity is attributed to the amino terminal region of the mo1ecule(l6), although the activity of proteolytic fragrncnts is not as striking as the activity of intact aactinin. Interestingly, the first -250 residues of the polypeptide constitute a domain that is conserved among many different actin crosslinking proteins (filled rectangle). A very differcnt domain is found at the carboxy terminus of the polypeptide (open rectangle). Tn the native dimer, this carboxy terminal domain (which includes two helix-loop-helix calcium binding sequences or EF hands) is apposed to the amino terminal domain of its neighboring subunit. Calciumbinding is thought to trigger a structural change in the carboxy terminal domain that affects the activity of the actin binding site in the apposed amino terminal
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d o m ~ i i n ( l ~ ’Muscle ~ ~ ) . a-actinin apparently lacks calcium binding activity(17),and therefore calcium sensitivity, because of deleterious amino acid substitutions in the EF hands(’2). The middle region of a-actinin is made up of four quasi-repetitive segments, each -120 amino acids in length (shaded ellipses), that have some resemblance to the repetitive segments found in aand fl spectrins(12714,1s) (open ellipses, see below). But the quasi-repetitive segments of a-actinin bear only limited similarity to one anothed3), suggesting that substantial divergence occured after their multiplication from an ancestral sequence segment. Spectrin Spectrins are another well characterized family of actin crosslinking proteins, first recognized as a major component of the human erythrocyte membrane. Genetic, structural and biochemical evidcnce all suggest that spectrin forms a submembrane network in the erythrocyte that is crosslinked by short actin filaments, and that this complex is essential for normal cell shape and membrane stability (reviewed in ref. 19). Several isoforms of spectrin (also known as fodrin and TW260/240) have been identified in various nonerythroid cell types, and all have a similar structure (reviewed in ref. 20). They consist of antiparallel heterodimers that are joined head to head to form an elongated tetramer (-200-250 nm) that crosslinks actin filaments into isotropic gels. a-Spectrhs are about 280 kD in molecular mass, and /3 spectrins range from 246kD (human erythroid 43 spectrin) to 430kD (Drosophila & spectrin)(‘. I). The /3 subunit is responsible for much of the diversity among spectrin isoforms in such properties as molecular length,
Fig. 1. Schematic sequence comparison of the actin crosslinking proteins. Rectangles distinguish the non-repetitive sequence segments from repetitive sequences. Solid rectangles indicate the conserved actiii binding domain. segments marked ‘EF’ include thc EF hand calcium binding sequence, and segments marked ‘S’ include the SH3 sequence motif. Arrows mark the position of ‘hinge’ regions in the sequence of dystrophin and ABP-280. Sequence data is not available Lor the carboxy terminal domain of pH spcctrin.
membrane binding, and regulation of actin binding activities(4). Complete sequence data is available for the w and /3 subunits of erythrocyte spectrin(21.22)(shown schematically as a tetramer in Fig. 1). Complete sequences of three non-erythroid LY spectrins are also known, and these have the same general structure as erythrocyte a spectrin(””-25)).But, while a spectrins are conserved in length and segmented structure, they show marked diversity in their amino acid se uences. As suggested from properties of the protein.?’6) and confirmed by comparisons of the deduced protein sequence^(^^^'^)), erythroid spectrin appears to be the product of a recent branchpoint in spcctrin evolution. Thus, human nonerythroid a’ specti-in is more closely related to Drosophila a spectrin(24)than to human erythroid N spectrin(”*”). Partial sequence comparisons of nonerythroid j3 s ectrins to erythroid /? spectrin suggest a similar trendg3’”, although a complete analysis must await sequence data for vertebrate non-erythroid /s spectrin. In contrast to aspectrins, therc arc likely to be differences in the segmented organization of 6 spectrins to account for their heterogeneity in size (as in fiH spectrin, described below). The sequences of aand {3 spectrin are similar in many ways to a-actinin. One striking feature is that the ends of the native spectrin molecule resemble the ends of aactinin. In fact, homologs of all of the a-actinin segments can be found within a and spectrin: the three amino terminal segments of /? spectrin resemble the three amino terminal segments of a-actinin(21329) and the three carboxy terminal segments of a spectrin resemble the three carboxy terminal segments of aa~tinin(’~,~”’~). Spectrin differs from a-actinin in the
repetitive sequence domain. Spectrin not only includes more repetitive segments than a-actinin, thus accounting for its greater size, but it also includes a distinct lineage of repetitive segments that are shorter (-106 residues, open ellipses) and more closely related to one another than are the quasi-repetitive segments that occur in both a-actinin and spectrin (shaded ellipses). The length difference between the two segment types is partly due to an &residue insertion in the quasirepetitive segments that is recognized when the repetitive segments of a and /? spectrins are aligned and compared with the quasi-repetitive segments(21-2). Presumably, the quasi-repetitive segments evolved from an ancestor that included this insert, and the repetitive scgments evolved from an ancestor that lacked the insert. Biochemical studies of erythrocyte /3 spectrin suggest that the amino-terminal non-repetitive segment (filled rectangle is responsible for spectrin’s actin-binding activity(30. Similar studies place the actin binding site within a homologous domain of the protein ABP- 120(31) (described below). Conservation of this sequence segment in a number of actin binding proteins is also consistent with its proposed actin-binding activity. Based on these lines of evidence, the conserved amino terminal segment is referred to here as the ‘actinbinding domain.’ The roles of the other non-repetitive spectrin sequences remain to be determined. First, segment 22, at the carboxy terminus of a spectrin, includes two EF hand sequences(23),(as does segment 6 of ru-actinin) (I4) and recombinant fragments of Drumphila a spectrin bind calcium in blot overlay experiments(”). Interestingly, human erythrocyte spectrin and sea urchin spectrin both ap ear to be calcium-sensitive in their actin-interactionsCg.’”) and future studies should address the possible role of the EF hand sequences. Although vertebrate non-erythroid LU spectrin is also predicted to bind calcium(z3),calcium does not appear Second, a 36to affect its actin intcraction in residue domain between segments 11 and 12 of vertebrate non-erythroid ~pectrins(’~)includes a calmodulin bindin site and a calcium-dependent protease cleavage site(3 36). These sites act synergistically to affect the structure and actin-binding activity of spectrin and may be important to its regulation in non-erythroid cells(34).However, Drosophila LU spectrin and human erythrocyte a spectrin both lack this 36-residue sequence(22.B). Third, segment 10, from the middle region of a spectrins, resembles the SH3 domain found in oncogenes such as v-src and v-crk, and in phospholipase C(37). Other actin-binding proteins, including yeast ABPlp and myosin I(3*), include a similar sequence and these proteins may bind a common ligand in the cortical region of the cell. Finally, there appears to be heterogeneity in the protein products of a single a spectrin gene. Short sequences in segments 10 and 21 of human non-erythroid N spectrin are thought to arise by alternative splicing, and these
1
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sequences may affect specific functional sites in the spectrin molecule(” 17). Recently, a novel isoform of spectrin that has an unuwally large /3 subunit (pH)and is 25 % longer than ~ tetramer was identified in Drosopliila(6). the I X spectrin Partial cDNA sequence data from pHrevealed that it is closely related to the 13 amino terminal segments o f /3 spectrin, although lJHincludes a non-repetitive segment with an SH3 sequence motif (Fig. 1) that is not found in other p spectrins. The large size (430 kD) and sequence organization of /jH are similar to the vertebrate protein dystrophin (described below). but additional sequence data from the carboxy end of pH. a region that is conserved among dystrophins. will be necessary to further resolve the structural rclation between spectrin and dystrophin. Dystrophin Using an elegant genetic strategy, human genomic DNA encoding the gene product associated with Duchenne and Becker muscular dystrophies was isolated and used to recover cDNA encoding the normal gene product(39). The gene product, named dystrophin, is extraordinarily large (427 kD)(7’. Sequence comparisons between dystrophin, spectrin and a-actinin revealed that all three of these proteins share signihcant sequence homology, both in the amino terminal actin binding domain and in the repetitive sequence region(4o41). Therefore, the dystrophin molecule i? presumed to be an elongated actin binding protein(’), although the native dystrophin molecule has not yet been purified and characterized. The carboxy terminal domain of dystrophin (Fig. 1) includes a short region that is similar to Dictyostelium aa ~ t i n i n ((shaded ~) rectangle) and a unique region (open rectangle) that is conserved in chicken and human dyqtro hins and in an autosomal homolog of dystro~ h i n .43). ( ~ Dystrophin also includes four potential ‘hinge’ sites (arrows) that might accommodate folds within the native molecule(“). Dystrophin lacks the quasi-repetitive lineage of segments found in spectrins and a-actinin. Instead, the dystrophin repctitive segments (open ellipses) coniorm to a 109-residue repeating unit(“).
F
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The Spectrin Superfamily of Proteins The repetitive and non-repetitive sequence similarities among spectrins, a-actinins and dystrophin support their classification a5 a spectrin superfamily of proteins(29345).One of the diagnostic features of this superfamily is the 106 to 120-recidue repctitivc sequence unit that was first identified in peptide sequences of erythrocyte spectrin(36).Structural predictions based on the amino acid sequences of spectrin, aactinin, and dystrophin suggest that each repetitive sequence unit folds to produce a repetitive structural unit, largely a helical, that independently contributes to the length of the native protein.(7,”,46).
Actin Binding Protein-280 Actin binding protein (ABP-280) is an elongated actin crosslinking protein that causes actin filaments to form isotropic gels and promotes perpendicular branching of filaments (reviewed in ref. 2). It was originally purified from rabbit macrophage and a related protein (filamin) was identified in smooth muscle(’7). ABP-280 is found in a variety of cell types and is especially prominent in platelets where it promotes interactions between the actin cytoskeleton and the membrane glycoprotein GPIb/IX(“). Like spectrin and a-actinin, ABP-280 is an elongated molecule with actin binding sites displayed at its termini (contour length= 160nln). A significant difference is that ABP-280 exists as a tail-to-tail dimer rather than an antiparallel dimed“). The sequence of ABP-280 revealed an interestin trend in the evolution of the actin crosslinkers(56 (Fig. 1). ABP-280 has an amino terminal actin binding domain followed by a domain of 23 repetitive segments that are each -100 amino acids long. Thus, like members of the spectrin superfamily, ABP-280 appears to have evolved through multiplication of an ancestral segment to form a repetitive structural domain that determines the relative placement of the aminoterminal actin binding sites in the native molecule. But the ABP-280 repeating unit does not appear to be related to the segments of the spectrin superfamily in sequence or its predicted structure. While the s ectrin segments are predicted to be largely a-heIical(’), the repetitive segments of ABP-280 are predicted to have /3sheet structure(5”).
in the cores of intestinal microvilliijJ). Fimbrin differs from the above crosslinkers in a few important ways. First, it is much smaller; at 68 kD i t is little more than twice the expected size of the conserved actin binding domain (-30kD). Second, it is a monomeric protein and thus must possess two aclin binding sites per polypeptide(”). Third. fimbrin forms tight lateral bundles of filaments(”), whereas the other proteins form loose bundles or isotropic gels. The sequence of fimbrin, while it shares the same actin binding domain found in thc above proteins, reveals a striking difference in primary structure(”) (Fig. 1). The molecule consists of two tandem actin binding domains, preceded by a short segment with two conserved EF hand sequences. Like non-muscle aactinin, fimbrin binds calcium, and calcium alters the The F-actin bundling properties of fimbrin iiz ~,’ifm(’.’). fimbrin sequence also revealed that it is homologous to the more widely expressed proteins L-plastin and T-pla~tin(~~).
Actin Binding Protein-120 ABP-120 is found in the cortical cytoplasm of Dictyostelium, a region of the cell that is rich in crosslinked actin filaments(51). The purified protein is similar in size and shape to a-actinin and is thought to be an antiparallel homodimer(”z). Functionally, ABP120 behaves much like ABP-280 in that it forms an isotropic actin gel with perpendicular branches(”). The amino acid sequence of ABP-120 resemblcs ABP-280 (Fig. 1). It consists of the amino terminal actin binding domain followed by six -100-residue repetitive segments that are predicted to have /3-sheet structure(53). Therefore, the relation of ABP-120 to ABP-280 is analogous to the relation of a-actinin to spectrin. In both cases, variation in repetitive segment num ber produces homologous molecules that differ in length and presumably link actin filaments over different distances in the cytoplasm. Given the predicted similarities in secondary structure between ABP-280 and ABP-120, it is surprising that these molccules form dimcrs in different way^(^^.^'). It will be interesting to resolve the molecular bases for their apparently distinct modes of self association.
A Superfamily of Actin Crosslinking Proteins The spectrin superfamily of proteins is clearly a subset of a larger group of actin crosslinking proteins that I will refer to here as the ’actin crosslinker superfamily.’ Thc unique primary structure of fimbrin emphasizcs the plastic, modular nature of the actin binding domain that relates these proteins in evolution. The minimum requirement for actin crosslinking appears to be the presence of two of these actin binding sites. The relative positioning of the two sites, whether they occur on a single polypeptide chain, on two chains, or on four chains, appears to determine the specific crocslinking behavior of the individual molecule. Sequence alignments of the actin binding domains from different members of the superfamily reveal a number of conserved residues that are likelv to have a direct role in the actin i n t e r a c t i ~ n ( ~ ’j 3~, j~f ).. ~Some ’ of the conserved residues may be important to the correct ~). folding of the actin binding d ~ r n a i n ( ~ > ’However, there are also significant sequence differences between the actin binding domains that may account for some of their differences in regulation and activity. For example, protein 4.1 and Ca+*/calmodulin influence the actin crosrlinking activity of some spectrin isoformrC4),but are not known to affect the activity of other proteins such as a-actinin and fimbrin. The actin binding activit of some crosslinkers (such as a-actinin and filamin)(5J is inhibited by tropornyoin. a protein that binds to thc sides of actin filaments(’), whereas the activity of the spectrin isoform TW260/240 is not inhibited by tropomyosin(”). The sequence of TW260/240 is not yet available, but in the future it is likely to provide useful insights into the structure and activity of the actin binding domain.
Fimbrin Fimbrin is an actin bundling protein that was first found
Evolution of the Actin Crosslinking Proteins Multigene families probably arise through stochastic
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Fig. 2. Dot-plot comparisons of members of the spectrin siipcrfamily. Sequences of Drusoyhiku a spectrin(*'), human erythrocyte p spectrin("), and Drosophila a-actinin"') are shown schematically along the plot axes. The stringencies of comparison were 20 identical matches per window of 100 residues in A, 20 and 34 identical matches per window of 100 in B, and 26 identical matches per window o f 100 in C. The programs 'Compare' and 'Dot-plot' were used to generate the comparisons@*).Arrows indicate the duplicated domain of a spectrin.
gene duplication evend6") and the rcsulting products are thought to diverge through processes such as exon shuffling. unequal crossing over (multiplication), and genetic Thesc three processes can account for much of the observed diversity ainong the actin crosslinkers. (1) Sequence shuffling produced structurally distinct proteins that share a single conserved domain - the actin binding site. (2) Sequence multiplication provided variation in the number of segments within each protein, either before or after shuffling. and this produced proteins that differ in shape and size. ( 3 ) Sequence drift provided further variation in the products of shuffling and multiplication. For example, sequence drift can account for the differential calcium sensitivity of muscle and non-muscle a-actinins. Dot-plot sequence compari5ons reveal the boundaries of shuffling events and multiplication events, and through varied stringency, they also provide a measure of sequence drift. In a computer-generated dot-plot, the two sequences to be compared are pkdced on the axes of a comparison grid and a sliding window along each axis is used to compare every region of one sequence with every region of a second sequence. The resulting diagonals indicate the coordinates of similarity between the two sequences. By varying the stringency of comparison, diffcrent degrees of similarity can be rcsolvcd. (Stringency refers to the minimal number of identical matches per sequence window that are required to generate a signal.) When the sequence of Drosophila cwaclinin('3)is compared to the sequence of erythrocyte fi spectrin(21),the diagonal line that spans the first 250 residues of both proteins describes thc boundary of the conserved actin binding domain (Fig. 2A). Most of the other diagonals relate segment 3 of a-actinin to the repetitive segments of ?/J spectrin. When Dvosophila a spectrin is compared with itself,
both the boundaries and the periodicity of repeating sequences appear in the resulting dot-plot (Fig. 2B). At low stringency (20 % identity; Fig. 2B, upper left), nearly all of the 106-residue repetitive se ments in a spectrin score as similar to onc another("? However, thc repctitivc sequence is degenerate, and high stringency dot-plots (34 % identity; Fig. 2B, lower right) reveal only a subset of the low stringency pattern(") (Fig. 2B, arrow). One implication of these patterns is that a minimal framework of residues is conserved within the repeating unit, but residues outside of this framework are free to drift according to other selective pressures. As a result, most of the repetitive segments are only -20-30 % identical to one another("). The most prominent high stringency signal corresponds to the sequence of segments 2-8 and 11-17. These boundaries are not easily discerned in the dot-plot of any one protein but are most obvious in segment-by-segment comparisons, as described by Wasenius et al.(23).Segment 2 is more like segment 11 than any other segment, segment 3 is more like segment 12 than any other segment, and so on. These two groups of segments probably arose by partial duplication of a common ancestor, and sequence drift has not yet erased the sequence similarities that reflect their common origin. We recently proposed a scheme by which spcctrin might have evolved from an a-actinin-like ancestor through successive duplication events("). The rationale for the scheme stems from two observations. First, the quasi-repetitive segments of a-actinin show greater differences among themselves than any of the other repetitive segments, which suggests that they arose through the most ancient duplication events (although a difference in selective pressures might also account for their divergence), Second, the repetitive segments of
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t
Dystrophin
-
Jn
h
Y
from chicken a spectrin, erythrocyte a and /3 spectrin and dystrophin (using the sequence alignment of Koenig and K ~ n k e l ) (were ~ ~ ) also identified on the basis of a conserved residue in at least half of the repetitive segments (Fig. 4). Many of the consensus positions that are conserved in all five consensus sequences (Fig. 4: vertical bars) are also part of the a spectrin strict consensus. These residues are probably important to the structural framework of the repeating unit. i";spectrin has fewer consensus positions than LY spectrins, and all except one of the p positions (#S3) are also found in the a consensus. Dystrophin has fewer consensus positions than a spcctrin, but seven of its consensus positions are unique to dystrophin. Dystrophin is further distinguished from the spectrins by the length of its repeating unit (-109 residues vs. 106 residues). Three positions (marked by parentheses) were omitted from the dystrophin consensus to maximize its alignment with the spectrin conscnsus. These two properties of the dystrophin consensus suggest that the dystrophin repetitive segments are a distinct lineage that evolved independently of the repetitive segments of spectrin. However, the segments of dystrophin and spectrin are related in a third important property: they both lack the 8-residue sequence that, in part, distinguishes the quasi-repetitive lineage found in spectrin and acactinin from the repetitive lineage of segments in spectrin. Thus, dystrophin may also have evolved from an a-actininlike ancestor, perhaps the same ancestor that produced spectrin, but diverged before multiplication of the repetitive segments (Fig. 3). This scheme suggests a remarkable feat of convergence since the /3 spectrin lineage produced a protein (/kspectrin) that is ncarly identical in size to dystrophin('). The predicted structural similarities between ABP120 and ABP-280(50353)suggest that they too arose by differential multiplication steps. But sequence compaiisons do not reveal the lineal relations that are observed among members of the spectrin superfamily. The sequencc of additional inembers of this group, such as Glamin, will provide a broader basis for future sequence comparisons.
-
1
1
1
v p-spectnn
a-spectrin
Fig. 3. ELolution of the spectrin superfamily. See text for details.
spectrins and dystrophin show greater similarity to the third segment of a-actinin than to the other a-actinin segments (Fig. 2A). These observations are consistent with an early evolutionary event in which segment 3 of a-actinin was duplicated, modified and subsequently multiplied to generate the spectrins and dystrophin (Fig. 3). While the scheme is hypothetical in detail, it illustrates some of the interesting questions in the evolution of the spectrin superfamily. The a spectrin dotplot result (Fig. 2B) suggests that one of the steps in its evolution was a large-scale duplication(23) (Fig. 3, horizontal bars). Did the repetitive domains of ac and i"; spectrin arise through the same duplication steps, or through independent steps starting from a common ancestor? Comparison of erythrocyte p spectrin(*') to Drosoyhila a spectrin(24)in dot-plots suggests that the answer is both. The large duplicated sequence that is present in two copies in a spectiin appears to be present in only one copy near the middle of the repetitive domain in j3 spectrin (Fig. 2C, arrows). Thus, a and /3 spectrin may have had a common ancestor that was intermediate in size between spectrin and a-actinin (Fig. 3). The repetitive segments of dystrophin are not sufficiently similar to one another or to spectrin to be able to trace their evolution using dot-plots, but information on their origin can be obtained through consensus comparisons. Alignment of the repetitive segments of Dvosophilu a spectrin reveals 14 positions in the repeating unit that have a conserved amino acid residue that is rarely substituted, and there are 54 positions within the repeating unit that are conserved in at least half of the repetitive We refer to the latter positions as the consensus and the former positions as the 'strict consensus.' Consensus sequences Position:
10
30
20
4a
Conclusions Many of the actin crosslinking proteins are related through a common actin-binding domain. Additional 50
aa
70
60
..+...L...U..L......++.+L......PPF.+.......UI..+....T...YG+.L..VP.LI++H..F...L.A...+L..L...A..LI....Y.... I . .+. .. C . . .U- . L . .....+. ..L. .... .Q.F.. ...... .UI .- + - ...T. . G - L . . ...LL++H.
Drosophila a I Erythrocyte 3\
. ...+. ..L..
.L- - L . .
Chicken brain n I ..+... L...U..L...A..++.+L..S..LPPF.+.......YI..+..I..T..YG+.L..V..LL++H..F...L.A...+L..L...A..LI...HYA... Erythrocyte rr I ..+...L...U..L......+...L........F.........UI......L.T...G..L..V....++H......L.........L......L.......... Dystrophin L L..LN .+ U..L......+...L...()...F...L..L..UL......L..O.p.........L...+.L...L.........L......L...().....
...
I
I
I 1
I
I
I
90
1aa
. ..H.. ...
.A- L . .
54 43 59 29 27
II
Fig. 4. Repeat consensus sequences of the spectrin superfalnily. Consensus sequences were determined as previously described'24).Positively and ncgatively charged residues arc indicated by '+' and '--', rcspectivcly. Parentheses mark positions in thc dystrophin Consensus where a single residue must be omitted to maximize its alignment with the spectrim. Vertical lines mark residues that are conserved in all five consensus qequences and the number of consensus positions in each sequence is indicated to the right of each row.
sequence motifs are conscrvcd among these proteins, but in the course of evolution they have been shuffled to produce proteins that vary remarkably in structure. With the possible exception of dystrophin, the actin binding site is always present in two copies per native molecule. Biochemical studies have also failed to detect monovalent members of the superfamily that are mechanistically analo ous to some of the microtubuleassociated proteins(63. Future studies should address whether or not such proteins exist. Still other actin crosslinkers, such as the gelactins(')). have yet to be sequenced and these proteins may provide further insight into crosslinker structure and evolution. I have used the term 'actin crosslinker superfamily' to refer to the protcins that hhare the conserved actin binding domain, but it is probably premature to adopt a specific nomenclature until the boundaries of the superfamily are better defined. There are other protein se uences For associated with actin-binding example, MAP2 and caldesmon are actin crosslinkers that appcar to be unrelated to the superfamily in seq~ence(~~,~~). The identification of sequence similarities among the actin crosslinking proteins has significantly advanced our knowledge of their structure and evolution. But to understand the cellular roles of these proteins, it will be necessary to understand the functional differences betwcen them. The non-repetitive segments are probably important to the specialized roles of these proteins. and attention should be focused on their contribution to function. The widely distributed repetitive segments and the actin binding domain also may have acquired specialized functions through sequence drift. The modular structure of these proteins should allow specific repetitive and non-repetitive segments to be isolated and tested for functional homology with one another. Genetic studies of some of these proteins have been initiatcd in DictyosteZium and DrosophiZa(13.522), and these experimental systems are likely to be valuable in future studies of functional conservation among the actin crosslinkers.
7
Acknowledgements I thank Dan Branton and Dan Kiehart for critical reading of the manuscript. This work was supported by
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