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

S100–annexin complexes – biology of conditionalassociation Naofumi Miwa1, Tatsuya Uebi2,* and Satoru Kawamura2,3 1 Department of Physiology, School of Medicine, Toho University, Tokyo, J

S100–annexin complexes – biology of conditional

association

Naofumi Miwa1, Tatsuya Uebi2,* and Satoru Kawamura2,3

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 Ca2+ -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: kawamura@fbs.osaka-u.ac.jp

*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 Ca2+-binding pro-teins, each of which comprises more than 10 members S100 proteins are small, dimeric, EF-hand-type Ca2+-binding proteins that exert both intra-cellular and extraintra-cellular functions Within the cells, S100 proteins regu-late various reactions, including phosphorylation, in response to changes

in the intracellular Ca2+ 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

Ca2+-binding proteins that exhibit Ca2+-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

Ca2+-dependent or Ca2+-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

manner when the intracellular Ca2+ level increases,

and therefore this complex has been postulated to

regulate Ca2+-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)

Ca2+-binding proteins that regulate various

intracellu-lar and extracelluintracellu-lar 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 Ca2+ sensors Each S100 protein has a pair of high-affinity Ca2+ -binding sites, called EF-hand motifs When intracellu-lar Ca2+ concentrations increase after environmental stimuli, for example, S100 proteins can bind to Ca2+

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 A2) 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 Ca2+-binding pro-teins Their Ca2+-binding motifs are different from the EF-hand type described above and are called annexin type or type II [11,12] On Ca2+ binding, annexins can interact with anionic membrane phos-pholipids, making them ‘Ca2+-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 Ca2+-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.

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 Ca2+ 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 Ca2+-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

Ca2+-binding study showed cooperative Ca2+binding

of dicalcin, with an apparent overall dissociation

con-stant (Kd) of 10–20 lm [19] On Ca2+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

Ca2+-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

Ca2+ binding: S100A10 is unable to bind to Ca2+ 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 Ca2+ inde-pendent: these two proteins form a heterotetrameric complex constitutively regardless of the Ca2+ concen-tration Instead of Ca2+, 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

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 Ca2+dependent with a Kdvalue of

2 lm [33], which probably reflects Ca2+ binding to

annexin A2 (S100A10 does not have Ca2+-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-HT1Breceptors [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-HT1B 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 Ca2+ -dependent manner [47], evoking the suggestion that this complex regulates Ca2+-dependent cellular events S100A11 has been shown to bind to annexin A1 at high Ca2+concentrations (1 mm), presumably forming

a heterotetramer [(S100A11)2–(annexin A1)2] [47]

As an individual protein, S100A11 alone binds to

Ca2+ with a Kd value of 8–16 lm [53] and undergoes conformational changes with a half-maximal effec-tive Ca2+ concentration at a similar concentration

( 35 lm) in measurements with fluorescent-labelled

probes [54] Annexin A1 alone binds to Ca2+ with a

Kd value of 20–75 lm, enhancing its binding activity

for phospholipid vesicles [55,56] Although detailed

analysis of the Ca2+ 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 Ca2+

concentra-tion range in which both S100A11 and annexin A1 can

bind to Ca2+

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 Ca2+-dependent

manner [55,56]; (b) S100A11 alone binds to

cytoskele-tal proteins with a Kd value of 3 lm in porcine heart

[53]; (c) S100A11 is also able to interact with annexin

A6 at a high Ca2+ 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

Ca2+(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 Mg2+-ATPase activity in a Ca2+-dependent manner and to regulate the generation of smooth mus-cle force with a Kd 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

Ca2+-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 Ca2+ This S100A6–annexin A11 complex has been shown to bind

to phospholipid vesicles in the presence of Ca2+ (1 mm) [76]

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

Ca2+-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 Ca2+

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 Ca2+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 Ca2+ 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 Ca2+-dependent manner, as revealed

by Ca2+-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

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 Ca2+-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 Ca2+

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 Ca2+, respectively These effective Ca2+

concentrations did not change significantly in the

pres-ence or abspres-ence of dicalcin, and therefore the

differ-ence in the Ca2+ concentration for half-maximal

effects between the two complexes can be attributed to

the different affinity of each annexin for Ca2+

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 Ca2+- 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 Ca2+ 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 Ca2+

concentration by utilizing two annexin subtypes that

cover different Ca2+ concentrations This mechanism may serve to reseal the cilia membrane in response to

a wide range of Ca2+ 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 Ca2+ -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

proteins in order to reveal distinctive phenotypic

changes

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