Tài liệu Báo cáo khoa học: S100–annexin complexes – biology of conditional association doc

REVIEW ARTICLE
S100–annexin complexes – biology of conditional
association
Naofumi Miwa
1
, Tatsuya Uebi
2,
* and Satoru Kawamura
2,3
1 Department of Physiology, School of Medicine, Toho University, Tokyo, Japan
2 Graduate School of Frontier Biosciences, Osaka University, Japan
3 Department of Biology, Graduate School of Science, Osaka University, Japan
Introduction
The interaction between S100 and annexin proteins
was initially identified in porcine intestinal brush bor-
der-derived membranes, as a complex formed between
S100A10 and annexin A2. Annexin A2 (previously
named p36 or calpactin I, etc.) is a substrate of src-
related viral tyrosine kinase [1,2], which raises the
possibility that this complex may be involved in
cancer-related pathology. The complex of S100A10
and annexin A2 (S100A10–annexin A2 complex) has
been found to bind to cytoskeletal components and
to colocalize in submembranous compartments [3],
suggesting that this complex may play a role in sub-
cellular vesicle organization to exert its biological
function.
Following these findings, another S100 member,
S100A11 (originally named S100C or calgizzarin), was
found to interact with annexin A1 in a Ca
2+

-depen-
dent manner, with additional evidence showing that
this complex also binds to cytoskeletal components,
such as tubulin and vimentin. Unlike the interaction
between S100A10 and annexin A2, the interaction
between S100A11 and annexin A1 occurs in a temporal
Keywords
annexin; calcium; colocalization;
comprehensive interaction; dicalcin;
EF-hand; liposome; membrane trafficking;
phospholipid; S100
Correspondence
S. Kawamura, Graduate School of Frontier
Biosciences, Osaka University, Yamada-oka
1–3, Suita, Osaka 565-0871, Japan
Fax: +81 6 6879 4614
Tel: +81 6 6879 4610
E-mail:
*Present address
Laboratory of Cell Signal and Metabolism,
National Institute of Biomedical Innovation,
Osaka, Japan
(Received 17 June 2008, revised 7 August
2008, accepted 22 August 2008)
doi:10.1111/j.1742-4658.2008.06653.x
S100 proteins and annexins both constitute groups of Ca
2+
-binding pro-
teins, each of which comprises more than 10 members. S100 proteins are
small, dimeric, EF-hand-type Ca

2+
-binding proteins that exert both intra-
cellular and extracellular functions. Within the cells, S100 proteins regu-
late various reactions, including phosphorylation, in response to changes
in the intracellular Ca
2+
concentration. Although S100 proteins are
known to be associated with many diseases, exact pathological contribu-
tions have not been proven in detail. Annexins are non-EF-hand-type
Ca
2+
-binding proteins that exhibit Ca
2+
-dependent binding to phospho-
lipids and membranes in various tissues. Annexins bring different mem-
branes into proximity and assist them to fuse, and therefore are believed
to play a role in membrane trafficking and organization. Several S100
proteins and annexins are known to interact with each other in either a
Ca
2+
-dependent or Ca
2+
-independent manner, and form complexes that
exhibit biological activities. This review focuses on the interaction
between S100 proteins and annexins, and the possible biological roles of
these complexes. Recent studies have shown that S100–annexin complexes
have a role in the differentiation of gonad cells and neurological disor-
ders, such as depression. These complexes regulate the organization of
membranes and vesicles, and thereby may participate in the appropriate
disposition of membrane-associated proteins, including ion channels

and ⁄ or receptors.
FEBS Journal 275 (2008) 4945–4955 ª 2008 The Authors Journal compilation ª 2008 FEBS 4945
manner when the intracellular Ca
2+
level increases,
and therefore this complex has been postulated to
regulate Ca
2+
-dependent membrane organization dur-
ing vesiculation or internalization.
To date, several other pairs of S100 proteins and
annexins have been reported (Table 1), and it seems
timely to view these pairs as constituents of a broad
system of S100–annexin complexes. In this system,
some S100 proteins are able to bind to several ann-
exins. The host (for example, S100 protein) and its
binding partner (an annexin protein) can be deter-
mined by their subcellular distributions and temporal
expression patterns in each tissue. In this review, after
a brief description of S100 proteins, annexins and our
recently characterized dicalcin, an S100-like protein,
we review several well-characterized S100–annexin
complexes to obtain an understanding of the diver-
gence of the physiological roles of the different com-
plexes. The structural basis of complex formation is
reviewed in the accompanying article by Rintala-
Dempsey et al. [4].
Proteins
S100 proteins
S100 proteins form a family of small (10–14 kDa)

Ca
2+
-binding proteins that regulate various intracellu-
lar and extracellular processes. Increased levels of S100
proteins have been reported to be associated with a
number of diseases. Originally, S100A1 (originally
named S100a) and S100B (S100b) were isolated in
bovine brain as proteins soluble in 100% (saturated)
ammonium sulfate at neutral pH [5]. To date, 20 S100
genes have been identified exclusively in vertebrates,
including humans, with most of the S100 genes clus-
tered on human chromosome 1q21 (S100A1–
S100A16), whereas no S100 genes have been detected
in invertebrates [6]. S100 proteins are known to exist
as homo- ⁄ heterodimeric functional units in various tis-
sues, including brain, lung and heart. An important
feature of S100 proteins is their role as Ca
2+
sensors.
Each S100 protein has a pair of high-affinity Ca
2+
-
binding sites, called EF-hand motifs. When intracellu-
lar Ca
2+
concentrations increase after environmental
stimuli, for example, S100 proteins can bind to Ca
2+
via EF-hand motifs and undergo large conformational
changes. These changes induce the exposure of a

hydrophobic patch at the surface of these molecules
and assist them to interact with their target proteins,
including enzymes (e.g. kinase, phospholipase A
2
) and
cytoskeletal proteins (e.g. actin). In this way, S100
proteins transduce environmental signals to intracellu-
lar activities to regulate cell proliferation, differentia-
tion, etc. [7,8]. Some S100 members are secreted
from cells through undefined exocytotic machinery,
exerting extracellular actions, such as anti-apoptosis
and anti-coagulation, through their receptors on the
surface of the plasma membrane. A number of tar-
gets have been reported to date [9], and, for several
S100 members, genetically engineered animals have
been produced to study the functional role(s) of S100
proteins [10].
Annexins
Annexins are another family of Ca
2+
-binding pro-
teins. Their Ca
2+
-binding motifs are different from
the EF-hand type described above and are called
annexin type or type II [11,12]. On Ca
2+
binding,
annexins can interact with anionic membrane phos-
pholipids, making them ‘Ca

2+
-dependent phospho-
lipid-binding proteins’. Annexins were first identified
from several sources and were given different names
(e.g. lipocortin, calpactin, etc.). Later, these proteins
were given a new family name of ‘annexin’, because
the major property of this family is to ‘annex’ cellu-
lar membranes in a Ca
2+
-dependent manner [13].
Annexins are distributed in various species from
humans to plants, and, to date, the vertebrate annex-
Table 1. Complex formation between S100 proteins and annexins. An S100–annexin complex is formed as indicated by the reference
numbers.
Annexin A1 Annexin A2 Annexin A5 Annexin A6 Annexin A11
S100A1 [82,83]
S100A4 [61]
S100A6 [60] [88] [66,70,71,74]
S100A10 [3,22–46]
S100A11 [47,51,52,57–59,63] [58] [62]
S100A12 [89]
S100B [82,83]
Biology of S100–annexin complexes N. Miwa et al.
4946 FEBS Journal 275 (2008) 4945–4955 ª 2008 The Authors Journal compilation ª 2008 FEBS
ins, which have been most extensively studied, com-
prise up to 12 members [14]. Annexins are expressed
widely in many tissues, but their localization varies:
some are present intracellularly and others are local-
ized at the plasma membrane. Most annexins consist
of an individually unique N-terminal domain and a

fairly conserved C-terminal core that contains either
four or eight repeating units of approximately 70
amino acids. It is believed that the annexin C-terminal
core is a module that mediates both Ca
2+
and mem-
brane binding. Annexins interact with many targets
and exert various biological functions, including regu-
lation of membrane aggregation and membrane traf-
ficking. They also have extracellular functions, for
example, in anti-inflammation and anti-coagulation
[11,12]. Although a few annexins have been analysed
in knockout animals [14,15], their phenotypes are
subtle, so that their exact physiological functions
remain elusive.
Dicalcin
Dicalcin, an S100-like Ca
2+
-binding protein formerly
called p26olf, was originally identified in frog
(Rana catesbeiana) olfactory epithelium [16]. After the
original identification, however, this protein was also
found in other tissues, including lung and spleen.
Although detailed structural analysis (i.e. crystallo-
graphic study) has not been carried out, sequence
alignment and molecular modelling have suggested
that dicalcin consists of two S100-like regions aligned
in tandem (each region has approximately 50% iden-
tity to the sequence of chick S100A11), and possibly
adopts a remarkably similar conformation to that of a

homodimeric form of S100B [17,18]. As all other S100
members, except calbindin, form a homo- or hetero-
dimer in solution to exert their biological functions,
dicalcin may substitute the function(s) of S100 proteins
in the form of a monomer. Based on this consider-
ation, we gave it a mnemonic name: ‘dimer form of
S100 calcium-binding protein’. Our quantitative
Ca
2+
-binding study showed cooperative Ca
2+
binding
of dicalcin, with an apparent overall dissociation con-
stant (K
d
) of 10–20 lm [19]. On Ca
2+
binding, dicalcin
interacts with a set of annexin members in both the
olfactory and respiratory cilia [20], as well as with
several other olfactory cilia proteins, including
b-adrenergic receptor-like protein, which has not yet
been cloned [21]. Through interactions with annex-
ins, dicalcin enhances liposome aggregation in a
Ca
2+
-dependent manner, which suggests that dicalcin
plays a role in membrane-associated events in the
olfactory and respiratory cilia (see below).
S100–annexin complexes

S100A10–annexin A2 complex
Distribution
The mRNA expression of S100A10 and annexin A2
has been shown in various mouse tissues, and both are
expressed coincidentally at high levels in lung, intestine
and thymus [22]. On the basis of an immunohisto-
chemical colocalization study [3], both S100A10 and
annexin A2 were found in the following sites: (a) brush
border in porcine intestine; (b) glomerular cells inclu-
ding mesangial cells and endothelial cells in porcine
kidney; (c) endothelial cells in porcine brain; and
(d) fibroblasts in bovine heart. Within these cells, both
proteins were mainly localized to endosomes and at
the plasma membrane [23–25].
Properties of interaction
S100A10 (alternatively called p11) and annexin A2 are
known to exist as a heterotetramer [(S100A10)
2
–(ann-
exin A2)
2
] in a membrane fraction [26]. The S100A10-
binding site in annexin A2 is considered to reside in
N-terminal residues (Val3, Ile6, Leu7, Leu10) based on
cosedimentation and gel filtration experiments using
truncated annexin mutants [27,28]. S100A10 is an
exceptional protein amongst S100 members in terms of
Ca
2+
binding: S100A10 is unable to bind to Ca

2+
because of a mutation within its EF-hand motifs.
Three amino acid residues are lost in the N-terminal
EF-hand motif and crucial amino acids are substituted
in the C-terminal motif [29]. As a consequence, the
association of S100A10 and annexin A2 is Ca
2+
inde-
pendent: these two proteins form a heterotetrameric
complex constitutively regardless of the Ca
2+
concen-
tration. Instead of Ca
2+
, post-translational modifica-
tions of annexin A2 have regulatory effects on the
association with S100A10: N-acetylation of annexin A2
is necessary for this association [30,31] and protein
kinase C-mediated phosphorylation decreases the affin-
ity of annexin A2 for S100A10 [32].
Binding targets of the complex
In an S100A10–annexin A2 complex, an S100A10
dimer resides in the centre of the complex, intercon-
necting two annexin A2 molecules [26]. Annexins in
the outer position of this complex preferentially bind
to anionic phospholipids, such as phosphatidylinositol
4,5-bisphosphate, which is enriched in lipid rafts in the
plasma membrane. Because S100A10 has the ability
to bind to cytoskeletal proteins, such as actin, this
N. Miwa et al. Biology of S100–annexin complexes

FEBS Journal 275 (2008) 4945–4955 ª 2008 The Authors Journal compilation ª 2008 FEBS 4947
complex can link membranes and ⁄ or vesicles to cyto-
skeletal proteins to regulate membrane organization.
This association of an S100–annexin A2 complex with
lipid membranes is Ca
2+
dependent with a K
d
value of
2 lm [33], which probably reflects Ca
2+
binding to
annexin A2 (S100A10 does not have Ca
2+
-binding
ability). The S100–annexin A2 complex has also been
shown to interact with membrane-related proteins.
They include certain types of sodium channel [34],
potassium channels [35,36], transient receptor potential
channels [37] and serotonin 5-HT
1B
receptors [38].
The molecular topology of this complex in the
membrane-bound state has been postulated from two
scenarios derived from different experimental
approaches. Cryoelectron microscopy has suggested
that each annexin A2 molecule in the outer position of
the complex binds to one membrane, and therefore the
tetrameric complex links two different membranes [39].
By contrast, scanning force microscopy has suggested

that two annexin A2 molecules bind to the same mem-
brane [40]. In the latter case, the S100A10 dimer
resides in a relatively outer position of the complex
away from the membrane, and thereby interacts with
other proteins (e.g. cytosolic portion of channels or
receptors), enabling them to be associated with or
incorporated into the membranes that are bound by
annexin A2 molecules.
In addition to the intracellular targets described
above, the S100A10–annexin A2 complex has been
shown to bind to tissue-type plasminogen activator in
the extracellular space and to act as a functional recep-
tor to produce plasminogen from tissue-type plasmino-
gen activator [41]. However, the exact binding
character remains a matter of debate [42].
Biological roles
Several studies using knockout animals have suggested
the biological roles of this complex [43]. Foulkes et al.
[44] have demonstrated that S100A10
– ⁄ –
mice show
deficient nociception, which may be attributed to a
severe decrease in the sodium current. Svenningsson
et al. [38] have found that S100A10
) ⁄ )
mice exhibit
a depression-like phenotype with reduced responses
to 5-HT
1B
agonists; this suggests that the lack of

this complex causes a depressive disorder. Recently,
this group has also shown that S100A10 has an
inhibitory role on some abnormal behaviors caused
by l-3,4-dihydroxyphenylalanine administration to an
animal model of Parkinsonism [45]. The identification
of the targets of the S100A10–annexin A2 complex
(see above) led to the suggestion that this complex
functions as a guiding molecule of channels and ⁄ or
receptors from the endoplasmic reticulum to the
Golgi and ⁄ or internalized vesicle to the plasma mem-
brane. The deficits in these knockout animals may
be attributed to the improper association with or
incorporation into the plasma membrane of these
channels and ⁄ or receptors.
Another possible biological role of this complex is
related to fibrin homeostasis. In the normal blood
vessel, fibrin is not deposited and arterial thrombin is
cleared after injury. However, S100A10
) ⁄ )
knockout
animals show a displaced deposition of fibrin in the
microvasculature and incomplete clearance of arterial
thrombin [46]; this may be caused by the loss of the
S100A10–annexin A2 complex on the outer surface of
the plasma membrane of the endothelial cells.
S100A11–annexin A1 complex
Distribution
The association of S100A11 (previously known as
S100C or calgizzarin) with annexin A1 was initially
found during the search for targets of annexin A1, a

prototype of annexin that has attracted considerable
interest because of its involvement in cell growth and
differentiation [47]. S100A11 mRNA is distributed
in almost all human tissues. It is highly expressed in
muscle, heart and bladder [48,49]. Annexin A1 is
also widely expressed in many tissues, including lung,
kidney and spleen [50]. Within the cells, annexin A1 is
localized mostly in the cytosol, except for its presence
within nuclei of the human respiratory epithelium [50].
Although the subcellular colocalization of these two
proteins in vivo has not been studied in detail, ectopi-
cally expressed S100A11 has been shown to colocalize
with intrinsic annexin A1 on the early endosomal
membranes of fibroblastic BHK cells [51]. Biochemical
studies have shown that S100A11 and annexin A1 are
both present in the cornified envelope preparation of
human keratinocytes [52].
Properties of interaction
In contrast with the interaction between S100A10 and
annexin A2, S100A11 binds to annexin A1 in a Ca
2+
-
dependent manner [47], evoking the suggestion that
this complex regulates Ca
2+
-dependent cellular events.
S100A11 has been shown to bind to annexin A1 at
high Ca
2+
concentrations (1 mm), presumably forming

a heterotetramer [(S100A11)
2
–(annexin A1)
2
] [47].
As an individual protein, S100A11 alone binds to
Ca
2+
with a K
d
value of 8–16 lm [53] and undergoes
conformational changes with a half-maximal effec-
tive Ca
2+
concentration at a similar concentration
Biology of S100–annexin complexes N. Miwa et al.
4948 FEBS Journal 275 (2008) 4945–4955 ª 2008 The Authors Journal compilation ª 2008 FEBS
( 35 lm) in measurements with fluorescent-labelled
probes [54]. Annexin A1 alone binds to Ca
2+
with a
K
d
value of 20–75 lm, enhancing its binding activity
for phospholipid vesicles [55,56]. Although detailed
analysis of the Ca
2+
concentration required for the
association of S100A11 with annexin A1 has not yet
been carried out, these two proteins have been hypo-

thesized to associate within a similar Ca
2+
concentra-
tion range in which both S100A11 and annexin A1 can
bind to Ca
2+
.
The S100A11-binding site in annexin A1 is consid-
ered to reside in the N-terminal residues, as revealed
by experiments similar to those used for the identifica-
tion of the S100A10-binding site in annexin A2
[47,57,58]. With regard to the specificity of S100A11
binding to annexin members, a previous study using
fluorescent-labelled peptides has shown that S100A11
interacts specifically with the annexin A1 N-terminal
domain and does not interact with the corresponding
N-terminal domain of annexin A2 [59]. However, a
recent study using annexin A2 peptides has shown that
S100A11 also interacts with the N-terminal domain of
annexin A2 [58], consistent with the finding that ann-
exin A2 shows broad binding specificity to other S100
members (e.g. S100A4 and S100A6) [60,61]. Binding of
S100A11 to both annexins A1 and A2 suggests possi-
ble multifunctional roles of S100A11 in the regulation
of membrane trafficking and ⁄ or organization.
Binding targets and roles of the complex
In contrast with the detailed structural analysis of the
S100A11–annexin A1 complex, the cellular targets and
functions of this complex have not been studied in
detail. Potential targets of this complex may be phos-

pholipids and cytoskeletal proteins based on the con-
sideration of the following reports: (a) annexin A1
alone binds to lipid membranes in a Ca
2+
-dependent
manner [55,56]; (b) S100A11 alone binds to cytoskele-
tal proteins with a K
d
value of 3 lm in porcine heart
[53]; (c) S100A11 is also able to interact with annexin
A6 at a high Ca
2+
concentration (1 mm), and this
S100A11–annexin A6 complex binds to native lipo-
somes derived from rat vascular smooth muscle as well
as phosphatidylserine liposomes in the presence of
Ca
2+
(200 lm) [62].
With regard to a potential biological role(s) of the
S100A11–annexin A1 complex, Robinson et al. [52]
have reported that S100A11 and annexin A1 are colo-
calized beneath the plasma membrane during the final
stages of epidermal keratinocyte differentiation, indi-
cating that this complex may be involved in the forma-
tion of the cornified envelope in human keratinocytes.
A biochemical study has shown that S100A11 sup-
presses the phosphorylation of annexin A1 by protein
kinase C, resulting in a decrease in the aggregation of
phospholipid vesicles [63]. This result also suggests a

role for the S100A11–annexin A1 complex in the regu-
lation of membrane organization.
S100A11 has been shown to inhibit actin-activated
myosin Mg
2+
-ATPase activity in a Ca
2+
-dependent
manner and to regulate the generation of smooth mus-
cle force with a K
d
value of 50 lm [64]. In smooth
muscle, however, annexin A1 is not expressed abun-
dantly [50], and therefore the S100A11–annexin A1
complex may not be involved in this biological effect.
S100A6–annexin A11 complex
Distribution
Both S100A6 (formally called calcyclin) and annexin
A11 have been studied to investigate their involvement
in cell cycle regulation and cancer biology, because the
expression levels of these proteins are high in malig-
nant tumours [65,66].
S100A6 is expressed in smooth muscle cells, epithe-
lial cells and fibroblasts in almost all mammalian
tissues, including intestine, kidney [67,68] and brain
[69]. Within these cells, S100A6 is expressed at the
plasma membrane and the nuclear envelope in embry-
onic pig testis-derived ST cell lines, as well as human
skin and embryonic mouse testis [66,70,71]. The
expression level of S100A6 is elevated in a number of

malignant tumours, such as acute myeloid leukaemia,
neuroblastoma and melanoma cell lines [72,73], with
peak expression between the G0 and S phases of the
cell cycle [68,74,75].
Annexin A11 is also widely distributed in the nucle-
oplasm in many cultured cell lines. The subcellular
distribution of annexin A11 is altered during the cell
cycle: it shows a dynamic and biphasic interaction with
the nuclear envelope, first during envelope breakdown
and second during its reassembly [66].
Properties of interaction and targets of the complex
Ca
2+
-dependent interaction of S100A6 and annexin
A11 was initially found in biochemical S100A6 affinity
chromatography [76]. However, our knowledge of
this interaction (e.g. binding property and molecular
target of the complex) is still limited. S100A6 has been
shown to bind to the N-terminus (Gln49–Thr62) of
annexin A11 at approximately 200 lm Ca
2+
. This
S100A6–annexin A11 complex has been shown to bind
to phospholipid vesicles in the presence of Ca
2+
(1 mm) [76].
N. Miwa et al. Biology of S100–annexin complexes
FEBS Journal 275 (2008) 4945–4955 ª 2008 The Authors Journal compilation ª 2008 FEBS 4949
Biological roles
S100A6 was originally identified as a cDNA clone for

which cognate RNA was growth regulated [65], and
subsequently purified as a protein [77,78]. S100A6 has
been shown to interact with the nuclear envelope in a
Ca
2+
-dependent manner, as does annexin A11, and
subsequently both were found to be colocalized in pro-
liferating cells during certain stages in the cell cycle
[66,70]. In epidermoid carcinoma A431 cells and vas-
cular smooth muscle cells, an increase in the Ca
2+
concentration, especially during the prophase, leads to
the translocation of annexin A11 from the nucleus to
the nuclear envelope, where it is colocalized with
S100A6 [66], suggesting a role of this complex in cell
cycle regulation. In addition, S100A6 and annexin A11
have been shown to be colocalized in mouse gonad
during an important period for male–female deter-
mination, suggesting that this complex plays a role in
cell stage-specific events that trigger a cascade for sex
determination [71].
S100A1–annexin A6 and S100B–annexin A6
complexes
Distribution
S100A1 is expressed in a variety of tissues, including
the nervous system, skeletal muscle, heart, kidney and
fat [79]. S100B is abundant in the nervous system,
testis, fat, skin and cartilage [80]. Annexin A6 is
expressed as two isoforms, a long form (annexin A6-1)
and a short form (annexin A6-2), determined by alter-

native splicing [81]. Both isoforms prevail in a variety
of tissues, including kidney, heart and skeletal muscle,
with predominant expression of annexin A6-1 [81].
S100A1, S100B and annexin A6 have been shown to
colocalize in the sarcolemma, the membranes of the
sarcoplasmic reticulum and transverse tubules in avian
skeletal muscle cells [82].
Properties of interaction and targets of the complex
A biochemical study using fluorescent-labelled proteins
has shown that both S100A1 and S100B interact with
annexin A6 at high Ca
2+
concentrations (100 lm) [83],
and both the N-terminal domain and the C-terminal
core of annexin A6 bind to S100 proteins. The target
molecules and cellular structures of these two complexes
have not been identified. Although several combinations
of S100 proteins and annexins are known to bind to lipo-
somes (see above), the S100A1–annexin A6 and S100B–
annexin A6 complexes showed no apparent interactions
with liposomes in a cosedimentation assay [84].
Biological roles of the complexes
Both S100A1 and S100B alone have been shown to
hamper the assembly of glial fibrillary acidic protein
and desmin, and to inhibit the formation of intermedi-
ate filaments in vitro [85,86]. However, this inhibitory
effect was lost when the C-terminal core, but not the
N-terminal domain, of annexin A6 was added [83].
The molecular mechanism of this effect is, however,
unknown, and therefore it is not certain whether this

effect is brought about by a ‘passive’ decrease in the
amount of effective S100 protein as a result of its
adsorption to annexin A6, or by an ‘active’ action
mediated by a target molecule(s) of the complex. Alter-
natively, these complexes have been suggested to play
a role in the regulation of Ca
2+
fluxes in skeletal
muscle cells by affecting a ryanodine receptor in the
sarcoplasmic reticulum [82].
Dicalcin–annexin complex
Distribution
Dicalcin is expressed in a variety of frog tissues [16].
In the olfactory and respiratory epithelium, dicalcin
and annexins A1, A2 and A5 are all localized in the
cilia of these tissues [20]; furthermore, all four proteins
are colocalized in the same cilia. Western analysis
using a Chaps-solubilized cilia membrane fraction indi-
cated that the ratio of the content of annexins and
dicalcin were A1 : A2 : A5 : dicalcin = 1 :  0.42 :
 0.54 :  1.9, and this estimated content of dicalcin
seems to be sufficient to interact with all members of
annexins expressed in the cilia [20].
Properties of interaction and targets of the complex
Dicalcin and annexins (annexins A1, A2 and A5) form
a complex in a Ca
2+
-dependent manner, as revealed
by Ca
2+

-dependent binding of annexins to dicalcin-
conjugated Sepharose. Although other S100 members
have been shown to bind to the N-terminus of annex-
ins (see above), dicalcin binds to N-terminal truncated
annexins, indicating that the C-terminal core alone is
capable of binding to dicalcin [20]. Indeed, each of the
frog annexins A1, A2 and A5 has at least a few puta-
tive S100-binding motifs in the C-terminal core: for
example, in annexin A2, the consensus sequence
FXFFXXF (where F denotes a hydrophobic residue
and X is any amino acid; [62]) can be found in L54–
V60 and L257–I263 [20]. However, a recent study has
shown that full-length annexin A2 possesses an approx-
imately four- to five-fold increased capacity for binding
to dicalcin-conjugated Sepharose, relative to that of
Biology of S100–annexin complexes N. Miwa et al.
4950 FEBS Journal 275 (2008) 4945–4955 ª 2008 The Authors Journal compilation ª 2008 FEBS
N-terminal truncated annexin A2 (T. Uebi, N. Miwa
and S. Kawamura, unpublished results), indicating the
involvement of the N-terminus of annexin A2 in its
binding to dicalcin. The binding affinity of the N-termi-
nus or the core domain has not yet been determined.
The binding of dicalcin–annexins to liposomes has
been examined. As shown above, annexins A1 and A2,
by themselves, exhibit activities to induce liposome
aggregation in a Ca
2+
-dependent manner. Remark-
ably, dicalcin enhances this liposome aggregation activ-
ity of annexin A1 and A2, but shows little effect on

the activity of annexin A5 [20]. As our assay mixture
contained only dicalcin, annexins and liposomes, the
dicalcin–annexin A1 and dicalcin–annexin A2 com-
plexes are likely to bind directly to liposomes and to
enhance liposome aggregation. The effective Ca
2+
concentration for liposome aggregation depends on
which annexin binds to dicalcin. Half-maximal effects
with dicalcin–annexin A1 and dicalcin–annexin A2
complexes were observed at approximately 30 lm
and < 5 lm Ca
2+
, respectively. These effective Ca
2+
concentrations did not change significantly in the pres-
ence or absence of dicalcin, and therefore the differ-
ence in the Ca
2+
concentration for half-maximal
effects between the two complexes can be attributed to
the different affinity of each annexin for Ca
2+
.
As described above, dicalcin probably binds to two
molecules of annexin. To determine whether dicalcin
binds to two of the same subtype of annexin or to two
different subtypes, we measured Ca
2+
- and dicalcin-
dependent liposome aggregation in the presence of a

mixture of annexins of different subtypes. The profile
of liposome aggregation was simply the sum of the
results obtained with a single subtype of annexin, sug-
gesting that dicalcin tends to bind to the same subtype
of annexin, even in the presence of different subtypes
in a mixture.
Biological roles of the complexes
Dicalcin and annexins are colocalized in olfactory and
respiratory cilia which are motile. Motile cells are
often subject to mechanical stress and damage [87]. In
addition, olfactory cilia are exposed to environmental
chemicals, microorganisms and viruses, so that the cilia
membrane is often likely to be damaged and disrupted.
Therefore, the cytoplasmic Ca
2+
concentration at the
disrupted site may increase in a variable manner
according to the severity of the damage, and some-
times increase even to the extracellular level (a few
mm). Dicalcin–annexin complexes are able to regulate
membrane aggregation within a wide range of Ca
2+
concentration by utilizing two annexin subtypes that
cover different Ca
2+
concentrations. This mechanism
may serve to reseal the cilia membrane in response to
a wide range of Ca
2+
increases caused by disruption

of these membranes [20]. In this sense, dicalcin–
annexin complexes in the olfactory and respiratory
cilia may be a typical example of a system in which
different subtypes of family proteins act in a comple-
mentary manner to cover a wide range of changes in
intracellular conditions.
In addition to annexins, dicalcin has been shown to
interact with several olfactory cilia proteins in a Ca
2+
-
dependent manner [21]. One possible candidate is
olfactory b-adrenergic receptor kinase-like protein.
Considering the possible role of annexins in membrane
organization, we hypothesize that the dicalcin–annexin
complex could bind to a protein, such as b-adrenergic
receptor kinase-like protein, to incorporate or associate
the protein into the membranes, as is postulated for
the S100A10–annexin A2 complex (see above).
Other S100–annexin complexes
Although the number of reports is limited, other S100–
annexin complexes have been reported: S100A4–annexin
A2 [61], S100A6–annexin A2 [60], S100A6–annexin A6
[88], S100A11–annexin A2 [58], S100A11–annexin A6
[62] and S100A12–annexin A5 [89] (see Table 1).
Future perspectives
As discussed above, various pairing of S100 and ann-
exins may be an intrinsic and conventional mechanism
of the S-100 annexin system to function in a variety of
tissues. The participants of these complexes are likely
to be determined by their spatial and temporal distri-

bution patterns in cells. By switching partners, an
S100–annexin complex may exhibit tissue- and cell
stage-specific biological actions, such as the regulation
of cell cycle and membrane traffic. Our current knowl-
edge of this system is still fragmentary, and the exact
molecular mechanisms remain unknown. For a better
understanding of the S100–annexin system, further
investigations are certainly required. As shown in this
review, some S100–annexin pairs exhibit broad binding
specificity. These proteins may interact with a less
favourable member protein in the absence of their
most favourable partner, and this complex may possi-
bly substitute for the function of the complex of the
most favourable pair. This may be the reason why
only subtle changes are observed in the phenotype of
knockout animals of S100 proteins and annexins.
Therefore, there is a need to generate multiple knock-
out animals deficient in several S100 and ⁄ or annexin
N. Miwa et al. Biology of S100–annexin complexes
FEBS Journal 275 (2008) 4945–4955 ª 2008 The Authors Journal compilation ª 2008 FEBS 4951
proteins in order to reveal distinctive phenotypic
changes.
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