Howard Hughes Medical Institute, Departments of Cell Biology and
Biochemistry, Duke University Medical Center, Durham, NC 27513, USA
* These authors contributed equally to this work
Author for correspondence (e-mail:
p.mohler{at}cellbio.duke.edu
)
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Introduction |
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Canonical ankyrins are 190-220 kDa proteins expressed in most tissues and
cell types and comprise a membrane-binding domain (MBD) of 24 ANK
repeats, a spectrin-binding domain, a death domain and a C-terminal domain.
Whereas death domains in other proteins may function in activation of
NF-B, caspase proteases and cell death, this domain has no known role
within ankyrins. Ankyrin expression is regulated by both tissue- and
developmental-stage-specific cues that also give rise to numerous ankyrin
polypeptides due to alternative splicing. 440 kDa ankyrin-B and 480 kDa
ankyrin-G polypeptides result from an insertion of a 220 kDa random coil
between the spectrin-binding domain and the death domain, which results in a
predicted extended length of up to 0.5-0.6 µm. These giant isoforms have
specialized functions in unmyelinated axons (ankyrin-B) and in the targeting
of voltage-dependent Na+ channels to axon initial segments and
nodes of Ranvier (480 kDa and 270 kDa ankyrin-G also contain a 40 kDa
serine/threonine-rich domain glycosylated with GlucNAc monosaccharide
residues). Finally, small ankyrin isoforms lacking large portions of canonical
ankyrins are localized to specialized membrane sites, for example, 119 kDa
ankyrin-G (Golgi), 100/120 kDa ankyrin-G (lysosomes) and 26 kDa ankyrin-R
(sarcoplasmic reticulum).
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Current views of ankyrin function are based on co-localization and
biochemical interactions of ankyrin with other proteins. Ankyrin associates
with a variety of membrane proteins including ion channels
(Na+/K+ ATPase, H+/K+ ATPase,
anion exchangers AE 1-3, voltage-sensitive Na+ channels,
Na+/Ca2+ exchanger), calcium-release channels [ryanodine
receptor, inositol (1,4,5)-trisphosphate receptor], cell adhesion molecules
[CD44, L1CAMs (L1, NgCAM, neurofascin, LAD-1, NrCAM, neuroglian)], as well as
cytoplasmic proteins, including clathrin and tubulin
(Bennett and Baines, 2001).
Many of these interactions are mediated by ANK repeats within the MBD,
although the Na+/K+ ATPase and
H+/K+ ATPase associate at least in part with the
spectrin-binding domain. Finally, ankyrin phosphorylation is important for
regulating the affinity of ankyrin for specific proteins, including
spectrin.
ANK repeats are 33-residue motifs involved in protein recognition and are
found in more than 325 unrelated human proteins [>5800 repeats in more than
1400 predicted proteins in the non-redundant sequence database
(http://smart.embl-heidelberg.de
)], including tankyrase, p53-binding protein (53BP2), transcriptional
regulators GABPß and NF-B inhibitory protein IkB
, and the
TRP family of ion channels (Sedgwick and
Smerdon, 1999
). ANK repeats fold into stacks of antiparallel
-helices interconnected by exposed loops arranged perpendicular to the
-helices. The specificity for ANK-repeatprotein interactions is
likely to be conferred by non-conserved residues that flank each ankyrin
repeat, located at the tips of exposed loops. Ankyrin MBDs with 24 ANK repeats
are multivalent and can accommodate multiple protein interactions; thus, they
may assemble multiprotein complexes at specific cellular sites.
Physiological roles of ankyrin-G and ankyrin-B in targeting proteins to
specialized membrane domains have been demonstrated by gene-knockout studies
in mice. Mice with cerebellar-specific loss of ankyrin-G display coordinate
loss of voltage-gated Na+ channels, ß-IV spectrin and
neurofascin at the plasma membrane of axon initial segments, decreased ability
of Purkinje neurons to fire action potentials, and progressive ataxia
(Jenkins and Bennett, 2001;
Zhou et al., 1998
).
Ankyrin-B-null mice die at birth with multisystem disorders including
degeneration of long axon tracts, myopathy and degeneration of the thymus
(Tuvia et al., 1999
).
Ankyrin-B-/- cardiomyocytes display downregulation and mis-sorting
of calcium-release channels [ryanodine and inositol (1,4,5)-trisphosphate
receptors] within the endoplasmic reticulum in cardiomyocytes that can be
rescued by transfection with cDNA encoding ankyrin-B. Both ankyrin-G and
ankyrin-R are expressed in cardiomyoctes, but cannot compensate for loss of
ankyrin-B. Rescue studies with ankyrin-B/G chimeras have identified the
C-terminal domain of ankyrin-B as the defining domain in specifying ankyrin-B
activity (Mohler et al., 2002
).
A working hypothesis to explain the cellular basis for these phenotypes is
that ankyrins play roles as chaperones or guides that direct vesicle transport
of a variety of ion channels to sites in the plasma membrane as well as the
endoplasmic reticulum.
Ankyrins have been implicated in human disease
(Bennett and Baines, 2001).
Hereditary spherocytosis results from decreased expression and/or mutated
forms of ankyrin-R. A similar model (nb/nb) is observed in mice due
to a near loss of ankyrin-R (210 kDa) in multiple cell types, which causes
anemia, degeneration of a set of Purkinje neurons and cerebellar dysfunction.
In addition, ankyrin-B has been mapped to human chromosome 4q25-27, the
linkage site for human type 4 long QT syndrome
(Schott et al., 1995
). This
disorder results in bradycardia and can cause cardiac arrhythmias leading to
loss of consciousness or sudden death. As ankyrin-B is highly expressed in
cardiac tissue, further examination of both ankyrin-B+/- and
ankyrin-B-/- mice may reveal a similar cardiac phenotype. More
generally, ankyrin mutations could result in improper localization and,
therefore, activity, of ion channels identified with ankyrin-binding domains
and thus result in a variety of functional `channelopathies'.
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References |
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Bennett, V. and Baines, A. J. (2001). Spectrin
and ankyrin-based pathways: metazoan inventions for integrating cells into
tissues. Physiol. Rev.
81,1353
-1392.
Jenkins, S. M. and Bennett, V. (2001).
Ankyrin-G coordinates assembly of the spectrin-based membrane skeleton,
voltage-gated sodium channels, and L1 CAMs at Purkinje neuron initial
segments. J. Cell Biol.
155,739
-746.
Mohler, P. J., Gramolini, A. O. and Bennett, V. (2002). The ankyrin-B C-terminal domain determines activity of ankyrin-B/G chimeras in rescue of abnormal inositol 1,4,5-trisphosphate-and ryanodine receptor distribution in ankyrin-B (-/-) neonatal cardiomyocytes. J. Biol. Chem. 7,7 .
Schott, J. J., Charpentier, F., Peltier, S., Foley, P., Drouin, E., Bouhour, J. B., Donnelly, P., Vergnaud, G., Bachner, L., Moisan, J. P. et al. (1995). Mapping of a gene for long QT syndrome to chromosome 4q25-27. Am. J. Hum. Genet. 57,1114 -1122.[Medline]
Sedgwick, S. G. and Smerdon, S. J. (1999). The ankyrin repeat: a diversity of interactions on a common structural framework. Trends Biochem. Sci. 24,311 -316.[Medline]
Tuvia, S., Buhusi, M., Davis, L., Reedy, M. and Bennett, V.
(1999). Ankyrin-B is required for intracellular sorting of
structurally diverse Ca2+ homeostasis proteins. J. Cell
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-1008.
Zhou, D., Lambert, S., Malen, P. L., Carpenter, S., Boland, L.
M. and Bennett, V. (1998). AnkyrinG is required for
clustering of voltage-gated Na channels at axon initial segments and for
normal action potential firing. J. Cell Biol.
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