Nodes of Ranvier and axon initial segments of myelinated nerves, sites of cell–cell contact in early embryos and epithelial cells, and neuromuscular junctions of skeletal muscle all perform physiological functions that depend on clustering of functionally related but structurally diverse ion transporters and cell adhesion molecules within microdomains of the plasma membrane. These specialized cell surface domains appeared at different times in metazoan evolution, involve a variety of cell types, and are populated by distinct membrane-spanning proteins. Nevertheless, recent work has shown that these domains all share on their cytoplasmic surfaces a membrane skeleton comprised of members of the ankyrin and spectrin families. This review will summarize basic features of ankyrins and spectrins, and will discuss emerging evidence that these proteins are key players in a conserved mechanism responsible for assembly and maintenance of physiologically important domains on the surfaces of diverse cells.Spectrins are flexible rods 0.2 microns in length with actin-binding sites at each end (
Shotton et al. 1979;
Bennett et al. 1982) (A). Spectrins are assembled from α and β subunits, each comprised primarily of multiple copies of a 106-amino acid repeat (
Speicher and Marchesi 1984). In addition to the canonical 106-residue repeat, β spectrins also have a carboxy-terminal pleckstrin homology domain (
Zhang et al. 1995;
Macias et al. 1994) and tandem amino-terminal calponin homology domains (
Bañuelos et al. 1998), whereas α spectrins contain an Src homology domain 3 (SH3) site (
Musacchio et al. 1992), a calmodulin-binding site (
Simonovic et al. 2006), and EF hands (
Travé et al. 1995) (A). Spectrin α and β subunits are assembled antiparallel and side-to-side into heterodimers, which in turn are associated head-to-head to form tetramers (
Clarke 1971;
Shotton et al. 1979;
Davis and Bennett 1983) (A). In human erythrocytes, in which spectrin was first characterized (
Marchesi and Steers 1968;
Clarke 1971), actin oligomers containing 10–14 monomers are each linked to five to six spectrin tetramers by accessory proteins to form a geodesic domelike structure that has been resolved by electron microscopy (
Byers and Branton 1985). The principal proteins at the spectrin–actin junction are protein 4.1, adducin, tropomyosin, tropomodulin, and dematin (
Bennett and Baines 2001) (
Open in a separate windowDomain structure and variants of spectrin and ankyrin proteins. (
A) Molecular domains of spectrins: Two α spectrins and five β spectrins are shown. Spectrins are comprised of modular units called spectrin repeats (yellow). Other domains such as the ankyrin binding domain (purple), Src-homology domain 3 (SH3, blue), EF-hand domain (red), and calmodulin-binding domain (green) promote interactions with binding targets important for spectrin function. The pleckstrin homology domain (black) promotes association with the plasma membrane and the actin binding domain (grey) tethers the spectrin-based membrane skeleton to the underlying actin cytoskeleton. (
B) The spectrin tetramer, the fundamental unit of the spectrin-based membrane skeleton. The spectrin repeat domains of α and β spectrin associate end-to-end to form heterodimers. Heterodimers associate laterally in an antiparallel fashion to form tetramers. The tetramers can then associate end-to-end to form extended macromolecules that link into a geodesic dome shape directly underneath the plasma membrane. (
C) Molecular domains present in canonical ankyrins. The membrane binding domain of ankyrin isoforms (orange) is comprised of 24 ANK repeats. The spectrin binding domain (green-blue) allows ankyrins to coordinate integral membrane proteins to the membrane skeleton. The death domain (pink) is the most highly conserved domain. The regulatory domain (brown) is the most variable region of ankyrins. The regulatory domain interacts intramolecularly with the membrane binding domain to modulate ankyrin’s affinity for other binding partners. All ankyrins and spectrins are subject to alternative splicing, which further increases their functional diversity.
Table 1.
Binding partners of spectrin and ankyrins
Spectrin Binding Partners | | | |
Alpha | Beta | | |
Transporters/ion channels EnNaC (sodium) NHE2 (ammonium) | Membrane anchors PI lipids Band 4.1 Ankyrin EAAT4 (glutamate) | | |
Membrane receptors NMDA receptor | Signaling RACK-1 | | |
Signaling HsSH3pb1 Calmodulin | Cytoskeleton/cellular transport F-actin Adducin Dynactin | | |
Ankyrin Binding Partners | | | |
Membrane BD | Spectrin BD | DD | REG D |
Ion channels: Anion exchanger Na+/K+ATPase Voltage-gated Na+ channels Na+/Ca2+ Exchanger KCNG2/3 Rh antigen IP3 receptor Ryanodine receptor Cell adhesion molecules: L1-CAMs CD44 E-cadherin Dystroglycan Cellular transport: Tubulin Clathrin | Spectrin | FasL | Hsp40 Obscurin PP2A |
Open in a separate windowSpectrin is coupled to the inner surface of the erythrocyte membrane primarily through association with ankyrin, which is in turn linked to the cytoplasmic domains of the anion exchanger (
Bennett 1978;
Bennett and Stenbuck 1979a,
b) and Rh/RhAG ammonium transporter (
Nicolas et al. 2003). The spectrin-based membrane skeleton and its connections through ankyrin to membrane-spanning proteins are essential for survival of erythrocytes in the circulation, and mutations in these proteins result in hereditary hemolytic anemia (
Bennett and Healy 2008). The ankyrin-binding sites of β spectrins 1–4 are located in the 15th spectrin repeat, which is folded identically to other repeats but has distinct surface-exposed residues (
Davis et al. 2008;
Ipsaro et al. 2009;
Stabach et al. 2009) (A, A). Mammalian β-5 spectrin and its ortholog β-H spectrin in
Drosophila and
Caenorhabditis elegans are the only β spectrins lacking ankyrin-binding activity (
Dubreuil et al. 1990;
Thomas et al. 1998;
McKeown et al. 1998;
Stabach and Morrow 2000).
Open in a separate windowAnkyrins and spectrins organize macromolecular complexes in diverse types of specialized membranes. (
A) Ankyrin-G forms a complex with β-IV spectrin, neurofascin (a cell adhesion protein), and ion channels (KCNQ2/3 and voltage-gated sodium channel) at axon initial segments in Purkinje neurons. (
B) In force buffering costameres of skeletal muscle, ankyrins -B and -G cooperate to target and stabilize key components of the dystroglycoprotein complex. At the membrane, ankyrin-G binds to dystrophin and β-dystroglycan. (
C) In cardiomyocyte transverse tubules, ankyrins -B and -G coordinate separate microdomains. Ankyrin-B binds Na+/K+ ATPase, Na+/Ca
2+ exchanger (NCX-1), and the inositol triphosphate receptor (IP3R). Ankyrin-G forms a complex with Nav1.5 and spectrin. (
D) Ankyrin-G in epithelial lateral membrane assembly. Ankyrin-G binds to E-cadherin, β-2 spectrin, and the Na+/K+ ATPase. Spectrins are connected via F-actin bridges bound to α/γ adducin and tropomodulin.Ankyrin interacts with β spectrins through a ZU5 domain (
Mohler et al. 2004a;
Kizhatil et al. 2007a;
Ipsaro et al. 2009) (B), and with most of its membrane partners through ANK repeats (
Bennett and Baines 2001) (C,D). In addition, ankyrins have a highly conserve “death domain” and a carboxy-terminal regulatory domain (see the following discussion). The 24 ANK repeats are stacked in a superhelical array to form a solenoid (
Michaely et al. 2002). Interestingly, the ANK repeat stack behaves like a reversible spring when stretched by atomic force microscopy, and may function in mechano-coupling in tissues such as the heart (
Lee et al. 2006). ANK repeats are components of many proteins and participate in highly diverse protein interactions (
Mosavi et al. 2004) (C). This versatile motif currently is being exploited using designed ANK repeat proteins (DARPins) engineered to interact with specific ligands that can function as substitutes for antibodies (
Stumpp and Amstutz 2007;
Steiner et al. 2008).Spectrin and ankyrin family members are expressed in most, if not all, animal (metazoan) cells, but are not present in bacteria, plants, or fungi. Spectrins are believed to have evolved from an ancestral α-actinin containing calponin homology domains and two spectrin repeats but not other domains (
Thomas et al. 1997;
Pascual et al. 1997). Ankyrin repeats are expressed in all phyla, presumably because of a combination of evolutionary relationships and in cases of bacteria and viruses by horizontal gene transfer. However, the spectrin-binding domain of ankyrin is present only in metazoans (B). It is possible that evolution of ankyrins and spectrins could have been one of the adaptations required for organization of cells into tissues in multicellular animals.The human spectrin family includes two α subunits and five β subunits, whereas
Drosophila and
C. elegans have a single α subunit and two β subunits (
Bennett and Baines 2001). Vertebrate ankyrins are encoded by three genes: ankyrin-R (ANK1) (the isoform first characterized in erythrocytes and also present in a restricted distribution in brain and muscle), ankyrin-B (ANK2), and ankyrin-G (ANK3). Vertebrate ankyrins evolved from a single gene in early chordates (
Cai and Zhang 2006).
C. elegans ankyrin is encoded by a single gene termed
unc-44 (
Otsuka et al. 1995), whereas the
Drosophila genome contains two ankyrin genes:
ankyrin (
Dubreuil and Yu 1994) and
ankyrin2 (
Bouley et al. 2000).Mammalian ankyrins -B and -G are co-expressed in most cells, although they have distinct functions (
Mohler et al. 2002;
Abdi et al. 2006). Ankyrins -B and -G are closely related in their ANK repeats, and spectrin-binding domains, but diverge in their carboxy-terminal regulatory domains. Regulatory domains are natively unstructured and extended (
Abdi et al. 2006). These flexible domains engage in intramolecular interactions with the membrane-binding and spectrin-binding domains (
Hall and Bennett 1987;
Davis et al. 1992;
Abdi et al. 2006) that modulate protein associations and provide functional diversity between otherwise conserved ankyrins.In addition to the standard versions of ankyrins and spectrin subunits depicted in , many variants of these proteins are expressed with the addition and/or deletion of functional domains because of alternative splicing of pre-mRNAs. For example, β spectrins can lack PH domains (
Hayes et al. 2000), and giant ankyrins have insertions of up to 2000 residues (
Kordeli et al. 1995;
Chan et al. 1993;
Pielage et al. 2008;
Koch et al. 2008), whereas other ankyrins lack either the entire membrane-binding domain (
Hoock et al. 1997), or both membrane- and spectrin-binding domains (
Zhou et al. 1997). The insertions in 440 kDa ankyrin-B and 480 kDa ankyrin-G (B) have an extended conformation that potentially could have specialized roles in connections between the plasma membrane and cytoskeleton of axons where these giant ankyrins reside (
Chan et al. 1993;
Kordeli et al. 1995) (B). Interestingly, the inserted sequences in
Drosophila giant ankyrins interact with microtubules at the presynaptic neuromuscular junction (
Pielage et al. 2008) (see the following section).
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