Cell adhesion receptors, such as integrins, selectins and
immunoglobulin superfamily (IgSF) members, play pivotal
roles in cell adhesion to target cells and extracellular matrices
(Hynes, 1999). In brain, linkages of cell surface proteins, such
as the adhesion molecules and neurotransmitter receptors, with
cytoskeletal elements play key roles in synapse formation and
synaptic plasticity (Walsh and Doherty, 1997; Shapiro and
The intercellular adhesion molecules, ICAMs, form a major
subgroup within the IgSF and one of their major functions is
to mediate leukocyte adhesion through binding to the
leukocyte-specific ?2-integrins (CD11/CD18) (Larson and
Springer, 1990; Gahmberg, 1997; Gahmberg et al., 1997).
Intercellular adhesion molecule-5 (ICAM-5; telencephalin)
(Yoshihara and Mori, 1994; Yoshihara et al., 1994; Mizuno
et al., 1997) is a cell adhesion molecule expressed in the
somatodendritic membrane of telencephalic neurons in
mammalian brain. It is homologous with the other ICAMs,
although more complex (Gahmberg, 1997; Hayflick et al.,
1998). ICAM-5 contains nine extracellular Ig-like domains, a
short transmembrane (TM) domain and a 64-amino acid
cytoplasmic domain. Like the other ICAMs, it binds to
leukocyte ?2-integrins (CD11a/CD18), and may be an
important regulator of the immune response in the central
nervous system (Tian et al., 1997; Tian et al., 2000a). ICAM-
5 is also involved in hippocampal long-term potentiation,
which regulates memory formation and learning (Sakurai et al.,
1998). In addition, the molecule may have an important
function in synapse formation in the developing brain since the
onset of its expression parallels the dendritic development and
the initiation of synapses (Mori et al., 1987; Oka et al., 1990),
and it has been shown to induce dendritic outgrowth (Tamada
et al., 1998; Tian et al., 2000b) and delay the dendritic spine
maturation (Matsuno et al., 2006). It also has a tendency to
form higher molecular weight forms via homophilic
interactions of its extracellular domains (Tian et al., 2000b).
These multimeric forms might also be complexes of different
proteins including cytoskeletal components as our previous
studies implied (Tian et al., 2000b). A C-terminal 17 amino
acid sequence of ICAM-5 has recently been found to act as a
dendritic sorting signal (Mitsui et al., 2005). H?wever, its
cytoskeletal partners have not been characterized.
?-Actinin is an F-actin-binding and cross-linking protein,
forming linkages between the actin cytoskeleton and the
plasma membrane (Otey and Carpén, 2004). In brain, it is a
major actin-binding protein concentrated in dendritic spines
(Shirao and Sekino, 2001). Several molecules, such as ICAM-
Intercellular adhesion molecule-5 (ICAM-5, telencephalin)
is a dendrite-expressed membrane glycoprotein of
telencephalic neurons in the mammalian brain. By deletion
of the cytoplasmic and membrane-spanning domains of
ICAM-5, we observed that the membrane distribution of
ICAM-5 was determined by the cytoplasmic portion.
Therefore we have characterized the intracellular
associations of ICAM-5 by using a bacterially expressed
encompassing the cytoplasmic part of ICAM-5. One of the
main proteins in the neuronal cell line Paju that bound to
the ICAM-5 cytodomain was ? ?-actinin. ICAM-5 expressed
in transfected Paju cells was found in ? ?-actinin
immunoprecipitates, and ICAM-5 colocalized with ? ?-
actinin both in Paju cells and in dendritic filopodia and
spines of primary hippocampal neurons. We were also able
to coprecipitate ? ?-actinin from rat brain homogenate.
Binding to ? ?-actinin appeared to be mediated mainly
through the N-terminal region of the ICAM-5 cytodomain,
(GST) fusion protein
as the ICAM-5857-861 cytoplasmic peptide (KKGEY)
mediated efficient binding to ? ?-actinin. Surface plasmon
resonance analysis showed that the turnover of the
interaction was rapid. In a mutant cell line, Paju-ICAM-5-
KK/AA, the distribution was altered, which implies the
importance of the lysines in the interaction. Furthermore,
we found that the ICAM-5/? ?-actinin interaction is involved
in neuritic outgrowth and the ICAM-5857-861cytoplasmic
peptide induced morphological changes in Paju-ICAM-5
cells. In summary, these results show that the interaction
between ICAM-5 and ? ?-actinin is mediated through
binding of positively charged amino acids near the
transmembrane domain of ICAM-5, and this interaction
may play an important role in neuronal differentiation.
Supplementary material available online at
Key words: Dendrite, Neuron, Cytoskeleton, Adhesion, ICAM
? ?-Actinin-dependent cytoskeletal anchorage is
important for ICAM-5-mediated neuritic outgrowth
Henrietta Nyman-Huttunen*, Li Tian*, Lin Ning and Carl G. Gahmberg‡
Division of Biochemistry, Faculty of Biosciences, PO Box 56 (Viikinkaari 5), 00014 University of Helsinki, Finland
*These authors contributed equally to this work
‡Author for correspondence (e-mail: email@example.com)
Accepted 10 May 2006
Journal of Cell Science 119, 3057-3066 Published by The Company of Biologists 2006
Journal of Cell Science
1 (Carpén et al., 1992), ICAM-2 (Heiska et al., 1996), NCAM
(Büttner et al., 2003), ?1- and ?2-integrins (Otey et al., 1990;
Otey et al., 1993; Pavalko and LaRoche, 1993), L-selectin
(Pavalko et al., 1995) and glutamate receptors (Wyszynski et
al., 1997), have been shown to bind to ?-actinin. ?-Actinin is
also associated with signalling molecules such as the mitogen-
activated protein kinase kinase kinase and the rho-kinase type
protein kinase N (Christerson et al., 1999; Mukai et al., 1997).
In the present study, we show that ICAM-5 associates with
?-actinin through a stretch of positively charged cytoplasmic
residues close to the membrane in vitro and in vivo. Our results
indicate that the interaction between ICAM-5 and ?-actinin
may be involved in neuritic outgrowth and in maintaining the
Characterization of proteins interacting with the ICAM-5
To identify proteins that associate with the cytoplasmic domain
of ICAM-5 in vitro, we made a glutathione S-transferase
(GST) fusion protein encompassing the cytoplasmic sequence
of ICAM-5 (Fig. 1A). The fusion protein was coupled to
glutathione-Sepharose and used as an affinity matrix. To study
the interacting molecules, we used the human neural crest-
derived Paju cell line (Zhang et al., 1996), which does not
express ICAM-5. Paju cell lysates were incubated with the
affinity matrix and the bound proteins were eluted. To identify
associated cytoskeletal proteins
immunoblotted and analyzed with antisera against different
cytoskeletal proteins. ?-Actinin was identified as one of the
proteins which bound to the GST-ICAM-5 cytoplasmic
domain, and it did not bind to GST alone (Fig. 1B,a,b). Talin
and filamin were also able to bind to the ICAM-5 cytoplasmic
domain (not shown). Antiserum against ezrin or tropomyosin
showed no immunoreactivity (not shown).
Since ?-actinin from the cell lysate bound strongly to the
cytoplasmic domain of ICAM-5 (Fig. 1B,a), we also studied
whether purified ?-actinin would bind to GST-cytoICAM-5.
Purified chicken gizzard ?-actinin was incubated with equal
amounts of GST-cytoICAM-5 and GST alone. Our results
showed that purified ?-actinin was able to bind to the full-
length GST-cytoICAM-5 but not to GST alone, confirming a
direct interaction between the two proteins (Fig. 1B,b).
the samples were
Mapping of ICAM-5 interaction with ?-actinin
To map the binding region for ?-actinin in the cytoplasmic
domain of ICAM-5, a short ICAM-5 cytoplasmic domain
peptide representing amino acids 857-861 (ICAM-5857-861)
from the N-terminus was synthesized and linked to thiopropyl-
Sepharose through a cysteine residue added to its C-terminus
(Fig. 1A). This sequence was chosen because it resembles the
?-actinin-binding sites in ICAM-1 and ICAM-2 having
clustered positively charged amino acids (Carpén et al., 1992;
Heiska et al., 1996). Purified ?-actinin efficiently bound to this
peptide (Fig. 1C,a). As a negative control, a scrambled version
of the same peptide was used. The control peptide showed
some background binding, but was much weaker than that with
the specific peptide. After this, a series of peptides which had
one or both of the lysines substituted either with alanine or
arginine were tested (Fig. 1A). ICAM-5-K857/R and ICAM-5-
K858/R peptides were still capable of binding to ?-actinin
Journal of Cell Science 119 (15)
although more weakly. However, there was no ?-actinin
binding to ICAM-5-K858/A and ICAM-5-K857-K858/A-A
peptides. Binding of the ICAM-5-K857/A peptide was weak
(Fig. 1C,a). This implies that the two lysines are necessary
amino acids for efficient binding to ?-actinin. Talin and filamin
were not able to bind to the ICAM-5857-861 peptide suggesting
that they may not compete with ?-actinin at this site (Fig.
1D,a,b). Taken together, the results provide evidence for a
direct interaction between ICAM-5 and ?-actinin and indicate
that a major binding site for ?-actinin is located within residues
857-861 (Fig. 1C,b).
Surface plasmon resonance (SPR) analysis
To determine the binding kinetics of ?-actinin interaction with
ICAM-5-derived peptides, an SPR analysis with a Biacore
biosensor was performed.
GGGKKGEY was captured on streptavidin-coated flow cells,
and a concentration series of ?-actinin was injected over the
surface. The association and dissociation rates could not be
evaluated from the obtained sensorgrams, owing to the fact that
the interaction was very fast under the current conditions. The
interaction reached the steady state within seconds (not
shown), and binding of ?-actinin to the GGGKKGEY peptide
was detected already with 0.5 ?M of ?-actinin concentration
(Fig. 1E). The interaction was saturated at the highest
concentrations of ?-actinin used (20-40 ?M). The obtained
data fitted well with Sigmaplot-software’s ligand-binding
model, in which we assumed a simple one to one interaction.
The experiments were done in duplicate, and averaged for
calculation of the dissociation
The biotinylated peptide
Co-immunoprecipitation of ICAM-5 with ?-actinin
We then tested whether binding between ICAM-5 and ?-
Fig. 1. (A) A schematic picture of the ICAM-5 molecule with the
amino acid sequence of the cytoplasmic domain and the peptides
used in the ICAM-5–?-actinin interaction studies. The short peptides
contain an additional C-terminal cysteine for coupling purposes. The
peptides used in SPR analysis were biotinylated. PM, plasma
membrane. (B) A GST-ICAM-5 cytodomain fusion protein binds ?-
actinin. (a) A GST-cytoICAM-5 fusion protein immobilized on
glutathione Sepharose beads was incubated with a Paju cell lysate,
and interacting proteins characterized by western blotting, in this
case an anti-?-actinin antibody. (b) Binding of purified ?-actinin to
GST-cytoICAM-5, visualized by blotting. (C) Binding of purified ?-
actinin to membrane proximal ICAM-5 cytoplasmic peptides. (a)
Purified ?-actinin was incubated with the ICAM-5 cytodomain
peptides (described in detail in Materials and Methods and in Fig.
1A) affinity matrices and the eluates were analyzed by SDS-PAGE
together with purified ?-actinin, and immunoblotted with anti-?-
actinin antibody. (b) Based on results shown above, the main ?-
actinin-binding domain in the ICAM-5 cytodomain is located at
residues 857-861 (box). (D) Binding of talin and filamin to ICAM-
5857-861peptide. Paju cell lysates were incubated with the ICAM-5
peptide and the scrambled control peptide, and the eluates were run
on SDS-PAGE and immunoblotted for talin (a) and filamin (b). No
binding was observed. (E) Binding of ?-actinin to GGGKKGEY
peptide as determined by SPR. Binding levels of ?-actinin at steady
state of interaction with GGGKKGEY as response units (RU) after
subtraction of control peptide sensorgrams from each GGGKKGEY
binding sensorgram, respectively.
Journal of Cell Science
Association of ICAM-5 with ?-actinin
actinin took place in vivo. ?-Actinin was immunoprecipitated
from ICAM-5 transfected Paju cell lysates under the conditions
that leave the cytoskeletal interactions relatively intact. Under
such conditions, the cytoskeletal proteins are released from the
insoluble actin filaments by DNase I treatment. ICAM-5
coprecipitated with ?-actinin (Fig. 2A). The apparent
molecular weight of the ?-actinin-associated ICAM-5 was
somewhat higher than the major band in the lysate, indicating
a more mature form of the molecule. The successful
precipitation of ?-actinin was ensured by immunoblotting with
anti-?-actinin antibody (not shown). ICAM-5 was also
precipitated from rat brain homogenates, and ?-actinin could
be detected in the ICAM-5 immunoprecipitate, thus providing
evidence for the in vivo interaction (Fig. 2B,C).
Cellular colocalization of ICAM-5 with ?-actinin and F-
To examine whether ICAM-5, ?-actinin and F-actin colocalize
within cells, Paju cells were transfected with the ICAM-5
cDNAs (Fig. 3) and analyzed by confocal microscopy. As
expected, no ICAM-5 could be detected in Paju-neo cells (Fig.
4A,a). In ICAM-5-transfected cells the full-length ICAM-5
Fig. 1. See previous page for legend.
Journal of Cell Science
was mainly localized to the uropods and cell-cell contact sites
(Fig. 4D,d), where it colocalized with both ?-actinin (Fig. 4F)
and F-actin (Fig. 4f) as indicated by the yellow color in the
To confirm that this colocalization was dependent on the
ICAM-5 cytodomain, cells were also transfected with ICAM-
5-TM cDNAs, in which the cytoplasmic domain of ICAM-5
was deleted, and ICAM-5-GPI cDNAs, in which the
cytoplasmic domain of ICAM-5 was replaced with a
glycophosphatidylinositol anchor (Fig. 3). In both cases,
ICAM-5 showed a patchy uniform distribution throughout the
cell body (Fig. 4G,J,g,j) and only weak colocalization was
observed (Fig. 4I,L,I,l). The patches in Paju-ICAM-5 cells,
where ?-actinin and ICAM-5 colocalized, were significantly
bigger than those in the truncated Paju-ICAM-5-TM (Fig.
4I,i) or Paju-ICAM-5-GPI (Fig. 4J,j) cell lines. In the mutant
colocalization (Fig. 4O,o) was no more seen, indicating the
importance of the lysines in binding to ?-actinin. It also
seemed that the mutant ICAM-5-KK/AA construct caused
much more cell death during the transfection compared to the
other transfections. The cells always grew very slowly and
their morphology was more epithelial cell-like (data not
To study the colocalization in a more natural environment,
we also studied the in vitro cultured rat primary hippocampal
neurons. The hippocampal neurons were cultured in vitro for
7 (Fig. 5A-F) or 14 days (Fig. 5G-L), which represents the
developmental stage of dendritic filopodia and dendritic spine
formation, respectively. ICAM-5 and ?-actinin were
colocalized in the cell soma and dendrites of hippocampal
neurons at both stages (Fig. 5C,L). By close observation at high
line (Fig. 4M,m), the
magnification, we found that both molecules showed a
punctated-pattern of expression along the dendrites. ICAM-5
colocalized with ?-actinin in the filopodia along the apical
dendrites at day 7. The two molecules colocalized in nearly
50% of early dendritic spines already at day 7 (Fig. 5F), and
in nearly 80% of more mature dendritic spines at day 14 (Fig.
5L). Thus, our results suggest that ICAM-5 is associated with
the actin cytoskeleton through binding to ?-actinin.
ICAM-5-cytoskeletal association is involved in neuritic
To study the importance of ICAM-5-cytoskeleton interaction
in neuronal differentiation, we activated Paju-ICAM-5, Paju-
ICAM-5-TM and Paju-ICAM-5-KK/AA cells with 100 nM
phorbol 12,13-dibutyrate (PdBu), and continued the culture for
2-3 days until neurites could be clearly seen. After this, the
cells were fixed, stained for ICAM-5, ?-actinin and actin, and
analyzed by confocal microscopy. The cells containing visible
neurites were chosen for immunofluorescent imaging. In Paju-
ICAM-5 cells, most of the ICAM-5 expression was localized
around the cell body (Fig. 6A,a-d) and at the growth cones of
the neurites (I-IV). Paju-ICAM-5-TM (Fig. 6A,e-h) and Paju-
ICAM-5-KK/AA cells (Fig. 6A,i-l) showed less colocalization.
Truncation (Paju-ICAM-5-TM, average length 36 ?m/neurite,
36.2±11.9 ?m) (Fig. 6B) or mutation of the ICAM-5
cytoplasmic tail (Paju-ICAM-5-K857-K858/A-A, average length
35 ?m/neurite, 35±9.7 ?m) (Fig. 6B) resulted in significantly
shorter neurites compared with the full-length ICAM-5 (Paju-
ICAM-5, average length 73 ?M/neurite, 72.7±22.4 ?m) (Fig.
6B). The statistical analysis showed that the differences in
neuritic length between Paju-ICAM-5 and Paju-ICAM-5-TM
or Paju-ICAM-5-KK/AA cells were significant (P<0.001).
There was no significant difference in the neuritic length
between the Paju-ICAM-5-TM and Paju-ICAM-5-KK/AA
cells (P=0.6). These results indicate that the ?-actinin-
dependent cytoskeletal association is important for ICAM-5-
mediated neuronal differentiation.
Peptide internalization induces morphological changes
in Paju-ICAM-5 cells
The cytoplasmic ICAM-5857-861 and ICAM-5-K857-K858/A-A
peptides were covalently coupled to activated penetratin 1 to
get the peptides internalized into the cells. Penetratin 1 is a 16
Journal of Cell Science 119 (15)
Fig. 2. Co-immunoprecipitation of ICAM-5 with ?-actinin. (A) ?-
Actinin was immunoprecipitated from Paju-ICAM-5 cell lysates with
a specific anti-?-actinin antibody and the precipitates were subjected
to SDS-PAGE and immunoblotted with anti-ICAM-5 antibody to
detect possible co-immunoprecipitation of ICAM-5. The successful
precipitation of ?-actinin was ensured by immunoblotting with anti-
?-actinin antibody (not shown). (B) ICAM-5 was
immunoprecipitated from rat brain homogenates and the precipitates
were run on SDS-PAGE and immunoblotted for ICAM-5 and after
stripping for ?-actinin.
Fig. 3. Schematic presentation of full-length ICAM-5, C-terminal
truncated constructs (ICAM-5-GPI, ICAM-5-TM), and point mutant
(ICAM-5-KK/AA) used in the cell transfections. The asterisks
indicate the sites of point mutations. EC, extracellular; TM;
transmembrane; CP, cytoplasmic part.
Journal of Cell Science
Association of ICAM-5 with ?-actinin
amino acid peptide corresponding to the third helix of the
homeodomain of Antennapedia protein, which is able to
translocate across biological membranes (Prochiantz, 1996;
Derossi et al., 1996). The peptide with the sequence AAGEY
was used as a control. After the incubation, the cells were
washed and visualized. The most dramatic effect occurred with
the cytoplasmic peptide: the cell morphology changed in that
the cells became more round-shaped compared to the normal
Paju cells (Fig. 7A,B). The control peptide had no specific
effect on Paju-ICAM-5 or Paju cells (Fig. 7C,D), which
appeared similar to the cells without any treatment (Fig. 7E,F).
The differences in relative percentage of Paju-ICAM-5 cells
with round-shaped morphology between the cells treated
with ICAM-5857-861 peptide (95.2%), the ICAM-5-K857-K858/
A-A peptide (19.2%) or without treatment (17.3%) were
statistically significant (P<0.001) (supplementary material
In this study, we describe an interaction between the
intercellular adhesion molecule-5 (ICAM-5) and ?-actinin. ?-
Actinin from the Paju cell lysate bound efficiently to a GST
fusion protein encompassing the whole cytoplasmic part of
ICAM-5, as did the purified ?-actinin. Furthermore, ICAM-5
was observed to coprecipitate with ?-actinin under mild
conditions where the cytoskeletal interactions were relatively
By using an immobilized peptide representing the amino
acids 857-861 (ICAM-5857-861, KKGEY) of the ICAM-5
cytodomain as an affinity matrix, we were able to determine
that the main binding site for ?-actinin is in the N-terminal
region of the ICAM-5 cytodomain. The main binding site for
?-actinin, comprising the amino acids KKGEY contains
positively charged basic residues. According to our results the
basic lysines at positions 857 and 858 are important in binding
to ?-actinin. Substitution of the lysines either with neutral
alanine or positively charged arginine markedly reduced the
binding of the peptides to ?-actinin. However, the peptides
ICAM-5-K857/R and ICAM-5-K858/R, which each had one
lysine substituted with arginine showed some binding to
purified ?-actinin indicating that positive charges are beneficial
in the ICAM-5/?-actinin interaction. There was no binding to
the ICAM-5-K858/A peptide indicating that the second lysine
is most important in binding to ?-actinin. SPR analysis
Fig. 4. The distribution of ICAM-5, ?-actinin and F-actin in different transfected Paju cell lines. The Paju-neo (A-C,a-c), Paju-ICAM-5 (D-F,d-
f), Paju-ICAM-5-TM (G-I,g-i), Paju-ICAM-5-GPI (J-L,j-l), and Paju-ICAM-5-KK/AA cells (M-O,m-o) were double-stained for ICAM-5
(green) and ?-actinin (red, left panel) or F-actin (red, right panel) as described in Materials and Methods, and analyzed by confocal microscopy.
The regions where the two proteins colocalize appear in yellow. Bars, 10 ?m.
Journal of Cell Science
3062 Journal of Cell Science 119 (15)
Fig. 6. Effect of ICAM-5 contructs on neuritic outgrowth. (A) The Paju-ICAM-5 (a-d, I-IV), Paju-ICAM-5-TM (e-h) and Paju-ICAM-5-
KK/AA cells (i-l) were activated by 100 nM PdBu to induce neurite outgrowth. The cells were triple-stained for ?-actinin (green), actin (red),
ICAM-5 (magenta), and visualized by confocal microscopy. Concentrations of ICAM-5, ?-actinin and F-actin at cell-anchorage sites (a-d) and
growth cones of neurites (I-IV) in Paju-ICAM-5 cells were seen. Bars, 20 ?m. (B) Histogram displaying neurite outgrowth in PdBu-activated
Paju-ICAM-5, Paju-ICAM-5-TM and Paju-ICAM-5-KK/AA cells (neurite length presented as micrometers). The differences between the
neuritic length in Paju-ICAM-5 cells compared with Paju-ICAM-5-TM and Paju-ICAM-5-KK/AA cells are statistically significant (P<0.001).
Standard deviations are shown.
Fig. 5. Colocalization of ICAM-5 with ?-actinin in
cultured rat hippocampal neurons. The 7-day-old (A-F) or
14-day-old neurons (G-L) were stained for ICAM-5
(green) and ?-actinin (red), and analyzed by confocal
microscopy. Both ICAM-5 and ?-actinin showed a
punctated expression pattern along the dendrites. The
colocalization of the two molecules in apical dendritic
filopodia (indicated by arrows, image F) at day 7, and in
dendritic spines at day 14 (indicated by arrowheads, image
L) are shown under high magnification. Bars, 10 ?m.
Journal of Cell Science
Association of ICAM-5 with ?-actinin
confirmed the interaction between the KKGEY peptide and
The cytoplasmic binding sites for ?-actinin in ICAM-1,
ICAM-2, ICAM-5, L-selectin, ?1- and ?2-integrins show some
resemblance to each other by containing highly positively
charged residues (Table 1). The ?-actinin-binding site in
ICAM-5 (KKGEY) seems to be most similar to that of ICAM-
1 (RKIKK) (Carpén et al., 1992), and both contain two
adjacent basic residues. It appears that positively charged basic
residues are present in many ?-actinin-binding sites of
transmembrane proteins (Otey and Carpén, 2004; Tang et al.,
2001). The crystal structure of the ?-actinin molecule shows
that the central rod domain region is very acidic, and this acidic
surface has been postulated to constitute a potential binding
site for many transmembrane receptors (Tang et al., 2001;
Ylänne et al., 2001). Otherwise, the cytoplasmic domains of
different ICAMs are poorly conserved, in contrast to integrins,
indicating that they have quite distinct functions (Hayflick et
al., 1998). Except for ICAM-5, all the other ICAMs (ICAM-
1-4) are expressed by blood and endothelial cells (Gahmberg,
1997; Gahmberg et al., 1997).
Immunofluorescence studies showed that ICAM-5 was
mainly concentrated at the uropods and cell-cell contact sites
of transfected Paju cells, whereas deletion of the cytoplasmic
tail (ICAM-5-GPI or ICAM-5-TM constructs) caused a more
even but patchy distribution of ICAM-5 on the cell membrane.
?-Actinin colocalized with the intact ICAM-5, and only
weakly with the truncated ICAM-5. In hippocampal neurons,
ICAM-5 and ?-actinin colocalized in the dendritic filopodia
and spines. Strong accumulation of ?-actinin in ICAM-5-
transfected cells may be explained by the homophilic
interaction of ICAM-5 multimers at the cell-cell contact sites
(Tian et al., 2000). The different distribution patterns in the
transfected Paju cells suggest that ?-actinin regulates the
localization of ICAM-5 through the cytoplasmic domain of
ICAM-5. Furthermore, it is possible that ICAM-5 is involved
in the organization of the actin cytoskeleton. ICAM-5 also
partially colocalized with F-actin. This provides further
evidence of an indirect interaction between ICAM-5 and actin
filaments due to ?-actinin crosslinking.
In Paju-ICAM-5-KK/AA cells, there was no clear
colocalization confirming that the lysines are needed for
binding to ?-actinin. Interestingly, when we transfected the
mutant ICAM-5-KK/AA construct into Paju cells, most of the
cells died. The morphology of the transfected cells became
epithelial cell-like, and they grew slowly. Furthermore, when
we treated the Paju-ICAM-5 cells with the ICAM-5857-861
peptide coupled to penetratin, the morphology of the cells
changed dramatically and became rounded. Evidently the
peptide competes with intact ICAM-5 and other relevant
molecules in Paju cells for binding to ?-actinin leading to
disruption of the normal cytoskeleton structure.
The ICAM-5/?-actinin colocalization pattern also changed
quite rapidly after activation with phorbol ester. When the Paju-
ICAM-5 cells started to extend neurites, most of the ICAM-5
and ?-actinin was concentrated at the cell-anchorage sites
around the soma and at the growth cones of the neurites, which
differs from the pattern seen in Paju-ICAM-5-TM and Paju-
ICAM-5-KK/AA cells. The length of the neurites in Paju-
ICAM-5-TM or Paju-ICAM-5-KK/AA cells was shorter than
that of the Paju-ICAM-5 cells. This indicates that the
interaction between ICAM-5 and ?-actinin plays a role in
neuritic outgrowth. As shown previously, ICAM-5 shows
homophilic binding between neurons and strongly stimulates
dendritic outgrowth (Tian et al., 2000b). It is possible that ?-
actinin is involved in the regulation of these phenomena.
Besides, ICAM-5 may also be involved in synapse formation
and plasticity, since F-actin is essential for the development
and maintenance of young synapses (Zhang and Benson,
2001), and ?-actinin is the major actin-binding protein
concentrated in the postsynaptic dendritic spines (Shirao and
Although mutant mice lacking ICAM-5 show no detectable
abnormalities in brain anatomy or in synaptic structures, their
cognition functions were changed (Nakamura et al., 2001).
Interestingly, LTP was enhanced in hippocampal synapses,
suggesting that ICAM-5 may regulate the synaptic stability by
determining the dynamic range of synaptic efficacy (Nakamura
et al., 2001). In synapses, ICAM-5 is expressed around the
postsynaptic dendritic spines and, thus, it may serve as a
structural constriction in synaptic connections by contributing
Fig. 7. Effect of ICAM-5 cytoplasmic peptides on cell morphology.
Paju-ICAM-5 (A,C,E) and Paju (B,D,F) cells were incubated with
penetratin-coupled ICAM-5857-861 and ICAM-5-K857-K858/A-A
peptides at 20 ?M concentrations. After 2 hours of incubation the
cells were washed and bright-field pictures were taken to visualize
the effects of the peptides on cell morphology. Bars, 20 ?m. The
relative percentage of round-shaped Paju-ICAM-5 cells (treated with
ICAM-5857-861peptide) was 95.2%, and the difference is statistically
significant (P<0.001) when compared with the cells treated with
ICAM-5-K857-K858/A-A peptide or without treatment (data not
Table 1. Binding sites for ? ?-actinin in different adhesion
Protein Peptide sequence
Journal of Cell Science
to morphological and functional changes through crosslinking
its counter-receptors with the cytoskeleton (Sakurai et al.,
1998). Furthermore, it has been recently discovered that
ICAM-5 is a negative regulator of spine formation (Matsuno
et al., 2006), and since LTP is associated with structural
changes of formed synapses as well as new spine formation
(Matsuzaki et al., 2004), ICAM-5 may be an essential molecule
in contributing to refinement of functional neural circuits in the
telencephalon, which regulates higher brain functions such as
memory and learning.
A soluble form of ICAM-5 is found in the cerebrospinal
fluid and plasma, and its level increases during inflammation
and other conditions in the brain, such as acute encephalitis
(Lindsberg et al., 2002) and temporal lobe epilepsy
(Rieckmann et al., 1998). Decreased immunoreactivity of
ICAM-5 in the brain of patients with Alzheimer’s disease has
also been reported (Hino et al., 1997). More recent studies have
shown that ICAM-5 binds to presenilins 1 and 2, which are
part of a large ?-secretase complex implicated in Alzheimer’s
disease (Annaert et al., 2001). Furthermore, presenilin 1 seems
to mediate the turnover of ICAM-5 via an autophagic
degradative pathway (Esselens et al., 2004). Cytoskeletal
protein aggregations are one of the pathological hallmarks of
neurodegenerative disorders. Amyloid beta-protein precursor,
actin, tropomyosin, vinculin and ?-actinin have all been
observed in intraneuronal inclusions (Hirano bodies) in
patients of Alzheimer’s disease (Galloway et al., 1987; Maciver
and Harrington, 1995). Therefore, it would be interesting to
study whether ICAM-5 is involved in regulation of ?-secretase
protein complex formation via cytoskeletal association, and
furthermore, whether this is important for the pathogenesis of
Materials and Methods
Paju is a human neural crest-derived cell line (Zhang et al., 1996) that is cultured
in Dulbecco’s modified Eagles Medium (DMEM) containing 10% fetal calf serum,
1% penicillin, streptomycin and L-Glutamine (BioWhittaker). Paju cells were
transfected with pCDM8neo stuffer, and with different ICAM-5 constructs: (1)
complete ICAM-5 cDNA (Paju-ICAM-5), (2) a truncated cDNA that replaced the
transmembrane (TM) and cytoplasmic domains with a glycophosphatidylinositol
(GPI) anchor, site of CD55 [complement decay-accelerating factor (Moran and
Caras, 1991); Paju-ICAM-5-GPI], (3) another truncated construct where the
cytoplasmic domain was deleted (Paju-ICAM-5-TM), (4) the complete ICAM-5
cDNA with amino acids lysine 857 and lysine 858 mutated to alanines (Paju-ICAM-
5-KK/AA). All these cDNA constructs were cloned in the expression vector pEF-
BOS (Tian et al., 1997; Moran and Caras, 1991) (Fig. 3). The control cell line, Paju-
neo, was transfected with the empty pEF-BOS vector and the pCDM8neo stuffer.
Transfections were stably performed with the LipofectamineTM2000 reagent
according to the manufacturer’s protocol (Invitrogen). Cell surface protein
expression was analyzed by FACS and immunofluorescent staining (data not
shown). All the cell lines were cultured as mentioned above, but in the presence of
0.5 mg/ml G418 (Calbiochem-Novabiochem Corporation, CA). All the cDNAs had
been verified by sequencing (DNA synthesis and sequencing laboratory, Institute of
Biotechnology, University of Helsinki).
The polyclonal rabbit anti-serum against the cytoplasmic part of mouse ICAM-5
(Tian et al., 1997) was kindly provided by Y. Yoshihara, Laboratory for
Neurobiology of Synapse, Brain Science Institute/RIKEN, Wako-City, Japan. The
polyclonal antibody against rat ICAM-5, 1000J, and the monoclonal antibodies
(mAbs) 246K and 127E were from ICOS Corporation, Washington, USA. The mAb
against ICAM-5, TL-3, has been described previously (Tian et al., 2000a). The
mAbs against ?-actinin (MAB1682) and filamin (MAB1678) were purchased from
Chemicon International, Temecula, USA. The talin mAb 8d4 and the polyclonal
rabbit anti-?-actinin A2543 were from Sigma-Aldrich, St Louis, MO. The mAb
AT6/172 against ?-actinin was from SeroTec (Oxford, UK), and rabbit anti-mouse
immunoglobulins from Dako A/S, Denmark. A mouse negative IgG control was
purchased from Silenius, Hawthorn, Australia. Sheep anti-mouse and anti-rabbit
IgG horseradish peroxidase conjugates were from Amersham Biosciences,
Buckinghamshire, UK. Rhodamine-phalloidin was from Sigma-Aldrich. Goat anti-
mouse and anti-rabbit linked to fluorophore Alexa488 were from Molecular Probes.
Cy3-conjugated goat anti-rabbit and Cy5-conjugated goat anti-mouse IgG were
from Jackson ImmunoResearch Laboratories.
Peptide synthesis and coupling
Synthetic peptides from the cytoplasmic domain of ICAM-5 were made in the
peptide synthesis unit of the Division of Biochemistry, Faculty of Biosciences,
University of Helsinki, Finland, purified with HPLC, and verified by mass
spectrometry. The synthetic peptides include (1) the sequence encompassing amino
acids 857-861 (ICAM-5857-861) (KKGEY-C), and (2) the same sequence but with
one or both of the lysines substituted either with alanine or arginine: ICAM-5-
K857/R (RKGEY-C), ICAM-5-K858/R (KRGEY-C), ICAM-5-K857-K858/A-A
(AAGEY-C), ICAM-5-K857/A (AKGEY-C), ICAM-5-K858/A (KAGEY-C) (Fig.
1A). The peptide encompassing amino acids 857-861 was also synthesized in
random order as a scrambled control peptide (GKEYK-C). An additional cysteine
was added to the C-terminus of each peptide. The ICAM-5 peptides and the
scrambled control peptide (2.5 mg) were coupled to 0.5 ml of thiopropyl-Sepharose
6B (Amersham Biosciences) and used for peptide affinity chromatography. The
coupling efficiency was 60-90%.
The biotinylated peptide GGGKKGEY (Fig. 1A) was used in surface plasmon
resonance (SPR) analysis. The biotinylated control peptide had the sequence
Surface plasmon resonance analysis
Binding of ?-actinin to control and GGGKKGEY peptides was studied by SPR
analysis in a Biacore 2000TMbiosensor (Biacore AB, Uppsala, Sweden). The flow
cells of an SA (streptavidin) biosensor chip (Biacore AB) were immobilized with
saturating amounts of biotinylated peptides according to the manufacturer’s
instructions. The coupling levels in resonance units (RU) were 330 for control and
159 for GGGKKGEY peptides. A wide range of concentrations of ?-actinin (0.5-
40 ?M) were injected for 2 minutes at a flow rate of 5 ?l/minute over the peptide-
coated flow cells. The dilution series of ?-actinin were made in the HBS system
buffer of the manufacturer (10 mM Hepes, pH 7.4; 150 mM NaCl; 3 mM EDTA
and 0.005% p20). Before dilution, ?-actinin was in a suspension of 2 M (NH4)2SO4
containing 20 mM Tris acetate, pH 7.6, 20 mM NaCl, 0.1 mM EDTA, 15 mM ?-
mercaptoethanol and 1 mM phenylmethylsulfonylfluoride (PMSF). Regeneration of
the flow cells was achieved by washing for 20 minutes with HBS at the flow rate
of 5 ?l/minute. The data were evaluated by subtracting the sensorgram of the flow
cell containing the control peptide from the sensorgrams of flow cells containing
the GGGKKGEY peptide (Biaevaluation 3.1 software, Biacore AB, Uppsala,
Sweden). The binding response of ?-actinin to the peptide at the steady state was
used to evaluate the Kdof the interaction with Sigmaplot 8.0 one site saturation
model (Systat software, Richmond, USA).
Peptide affinity chromatography
5 ?g of chicken gizzard ?-actinin (Sigma-Aldrich) or Paju cell lysates (for talin and
filamin-binding assay) containing 2-3 mg protein were preincubated with blocked
thiopropyl-Sepharose (Amersham Biosciences) without peptide for 1 hour at
4°C under constant agitation. The incubation was further continued with
peptide-coupled Sepharose (described in Peptide synthesis and coupling) for 2 hours
at 4°C. The beads were washed three times with the lysis buffer (50 mM Tris-
HCl, pH 7.4, 60 mM NaCl, 1% TX-100, 10 mM EDTA, 50 mM NaF, 2 mM
phenylmethylsulfonylfluoride, 10 ?g/ml of both aprotinin and leupeptin, 0.1 mM
quercetin and 5 mM iodoacetamide) and bound proteins were eluted with the
Laemmli sample buffer, separated in 4-15% or 8-16% SDS-PAGE gradient gels
(Bio-Rad Laboratories, CA) and detected by immunoblotting. ?-Actinin was
visualized with MAB1682, talin with mAb 8d4, and filamin with MAB1678.
Peptide coupling with penetratin and peptide internalization
An equimolar amount of the ICAM-5857-861 (KKGEY-C) peptide or the mutated
peptide ICAM-5-K857-K858/A-A (AAGEY-C) was added to the activated penetratin
(Qbiogene), and the mixture was incubated for 2 hours at 37°C. After this the
coupled peptides were stored at –70°C.
Paju-ICAM-5 and Paju cells (20?104/well) were grown in 24-well plates for 2
days and incubated for 2 hours at 37°C with the penetratin-coupled peptides
prediluted in culture medium at a final concentration of 20 ?M, and washed with
media. After this the bright-field images were taken under 10? or 20?
magnification (Olympus IX71 inverted research microscope). The relative amount
of cells with changed morphology was quantitated. The cells were counted from
two random fields, and the number of rounded cells was compared with the total
amount of cells, and averaged. 70-80 cells were counted for each treatment.
Glutathione S-transferase (GST) fusion protein constructs
A plasmid construct encoding the ICAM-5 cytoplasmic domain was generated by
PCR (GeneAmp PCR system 9700, Applied Biosystems, Foster City, CA) using
Journal of Cell Science 119 (15)
Journal of Cell Science
Association of ICAM-5 with ?-actinin
appropriate 5? (ATGGATCCCAGTCCACCGCCT) and 3? (ATGAATTCTCACG-
CCGATGTCAG) primers. The represented amino acid sequence was 852-912 for
the whole cytoplasmic part (GST-cytoICAM-5). Human ICAM-5 cDNA in the pEF-
BOS vector was used as the template. All PCR reactions were performed using
HotStarTaq DNA polymerase (Qiagen, CA). The PCR products were cut with
BamHI and EcoRI restriction enzymes (New England Biolabs, Beverly, MA) and
ligated into the pGEX-2TK vector (Amersham Biosciences, Uppsala, Sweden). All
PCR-derived clones were verified by sequencing (DNA synthesis and sequencing
laboratory, Institute of Biotechnology, University of Helsinki).
GST fusion protein interaction assay
The GST-ICAM-5 fusion protein and GST were produced in the E. coli strain BL21
(DE3) (Stratagene, La Jolla, CA) after induction for 4 hours at 37°C with IPTG
(isopropylthiogalactopyranoside, Sigma-Aldrich). The bacteria were sonicated in
1% TX-100 (Sigma-Aldrich), PBS (phosphate-buffered saline: 0.14 M NaCl, 10
mM Na-phosphate, pH 7.4), and after centrifugation (13,000 rpm, 45 minutes) the
supernatants were allowed to bind to glutathione-Sepharose 4B (Amersham
Biosciences). Aliquots of this matrix and of the negative control GST, containing
comparable amounts of the fusion proteins, were used for binding experiments.
Paju cells were lysed at 0°C for 15 minutes in the lysis buffer containing 50 mM
Tris-HCl, pH 7.4, 60 mM NaCl, 1% TX-100, 10 mM EDTA, 50 mM NaF, 2 mM
PMSF, 10 ?g/ml of both aprotinin and leupeptin, 0.1 mM quercetin and 5 mM
iodoacetamide. The lysates were centrifuged in an Eppendorf centrifuge at 4000
rpm for 5 minutes at 4°C. The protein concentration of the lysates was estimated
by the Biuret method (Stoscheck, 1990). Lysates containing 2-3 mg protein or
purified chicken gizzard ?-actinin (5 ?g) were rotated with the ICAM-5 GST fusion
protein and GST affinity matrices at 4°C and washed three times each with 1 ml of
the lysis buffer. The incubation times were overnight for the lysates and 2 hours for
purified ?-actinin. Bound proteins were eluted with the Laemmli sample buffer,
separated in 8% SDS-PAGE or 4-15% gradient gels (Bio-Rad Laboratories, CA)
and detected by immunoblotting. ?-Actinin was visualized with MAB1682.
Coimmunoprecipitation of ICAM-5 with ?-actinin was done by the solid-phase
immunoisolation technique, as described (Stefanova et al., 1993). Briefly, for
indirect solid-phase immunoisolation, rabbit anti-mouse immunoglobulin was
coated on microtiter plates (U-shaped, Nunc A/S, Denmark) and allowed to incubate
at 37°C for 2 hours. After washes with PBS, the mAb AT6/172 against ?-actinin
was added to the wells and incubated at 4°C overnight, excess protein binding sites
were then blocked with 5% BSA. Paju-ICAM-5 cell lysates were treated with
DNaseI (Roche Diagnostics, Indianapolis, IN) for 50 minutes at 22°C prior to
applying to coprecipitations. The cell lysates were applied to the wells and incubated
for 4 hours at 4°C. The wells were washed three times with the same lysis buffer
as used in peptide affinity chromatography. The proteins eluted with the Laemmli
sample buffer were separated using 8% SDS-PAGE gels and visualized by
immunoblotting. ICAM-5 was detected with the polyclonal anti-serum against the
cytoplasmic part of ICAM-5, and ?-actinin with MAB1682.
Brains of adult rats were homogenized in TNF buffer [50 mM Tris-HCl, pH 8.0,
1% Tx-100, 0.5% NP-40, 140 mM NaCl, 1 mM EDTA, NaF, and Na3VO4, 10 ?g/ml
aprotinin and leupeptin, and 1 mM 4-(2-Aminoethyl)benzenesulphonyl fluoride
(AEBSF)]. The homogenates were cleared by centrifugation, and further
preabsorbed with protein G Sepharose (Amersham Biosciences) for 2 hours at 4°C
in rotation. After this, 10 ?g of the mAb 127E against rat ICAM-5 or mIgG were
added, and allowed to incubate overnight at 4°C. After the incubation, protein G
Sepharose was added to the brain lysates and the incubation was continued for 2-4
hours at 4°C. Protein G Sepharose was washed three times with TNF buffer and the
bound proteins were eluted with Laemmli Sample Buffer, and separated by 4-15%
gradient gels (Bio-Rad Laboratories). The eluted proteins were visualized by
30 ?l of eluted fractions from the ICAM-5 peptide affinity chromatographies,
peptide blocking assays, GST fusion protein interaction assays, or
coimmunoprecipitation assays were separated by SDS-PAGE and blotted onto
nitrocellulose membranes. The membranes were blocked using 5% milk powder in
TCN (50 mM Tris-HCl, pH 8.0, 80 mM NaCl, 2 mM CaCl2) containing 0.2% NP-
40. The membranes were reacted with primary and secondary antibodies at
appropriate concentrations in 3% milk powder-TCN/0.2% NP-40 for 1 hour,
respectively. The blots were washed extensively with washing buffer (TCN/0.2%
NP-40 or 0.05% TX-100). The bound antibodies were detected by enhanced
chemiluminescence (Amersham, Buckinghamshire, UK).
Hippocampus was dissected from the brains of 19-day-old rat embryos and treated
with 0.5 mg/ml papain for 10 minutes (Worthington Chemical Corp.) in HBSS
(Gibco BRL). The neurons were washed with HBSS and 0.1?106cells/cover slip
were plated on poly-L-lysine (Sigma-Aldrich)-coated 8-mm coverslips. After
plating, the cells were cultured in Neurobasal medium (Gibco BRL) with 2% B27
supplement (Gibco BRL), 25 ?M L-glutamic acid (Sigma-Aldrich), and 1%
penicillin, streptomycin, and L-glutamine for 1-2 weeks before the costainings. The
cells were permeabilized with 0.2% TX-100 and blocked with 3% BSA. ICAM-5
was detected with anti-rat mAb 127E and 246K, and ?-actinin with the rabbit anti-
?-actinin antibody A2543.
Paju, Paju-ICAM-5, truncated Paju-ICAM-5 (TM and GPI), and Paju-ICAM-5-
KK/AA cells (1?104/well) were grown on coverslips in serum-containing medium
for 3 days in 24-well plates. After paraformaldehyde fixation the cells were
permeabilized with 0.1% TX-100 in PBS, blocked with 3% BSA, and double-
stained for ICAM-5 (mAb TL-3) and ?-actinin (polyclonal rabbit anti-?-actinin
antiserum) or F-actin (rhodamine-phalloidin). For ICAM-5 and ?-actinin staining,
the coverslips were incubated with Alexa488-conjugated goat anti-mouse IgG and
Cy3-conjugated goat anti-rabbit IgG.
Neurite outgrowth assay
Paju-ICAM-5, Paju-ICAM-5-TM and Paju-ICAM-5-KK/AA cells were grown in
serum-containing medium in 24-well plates (1?104/well) on cover slips over night.
Then the cells were activated with 100 nM phorbol 12,13-dibutyrate (PdBu), and
cultured for 2-3 days before visualization with the microscope. After
paraformaldehyde fixation, the cells were permeabilized with 0.1% TX-100 in PB,
blocked with 3% BSA, and triple-stained for ICAM-5, ?-actinin and F-actin. The
secondary antibodies were Cy5-conjugated goat anti-mouse and Alexa488-
conjugated goat anti-rabbit IgG. The fluorescent images were taken with an inverted
confocal microscope (Leica TCS SP2 AOBS) under 63? magnification. The length
of the neurites was measured in 2-3 random fields and averaged. Cells with neurite
length of at least twice the diameter of the cell body were quantitated. 30 neurons
were counted for Paju-ICAM-5 and Paju-ICAM-5-TM cells, and 20 neurons for
For all the quantization, statistical analysis of variance (ANOVA) was used to
compare the different groups in the neurite outgrowth assay and peptide
We thank Heikki Rauvala for providing rat hippocampal neurons,
Y. Yoshihara for polyclonal antisera to ICAM-5, Patrick Kilgannon
for rat ICAM-5 mAbs, Carmela Kantor-Aaltonen for the different
cytoplasmic ICAM-5 peptides, Hilkka Lankinen and Tomas Strandin
for the SPR analysis work, Outi Nikkilä, Leena Kuoppasalmi and
Maria Aatonen for technical assistance, and Yvonne Heinilä for
secretarial help. This study was supported by the Sigrid Jusélius
Foundation, the Academy of Finland, the Finnish Cultural
Foundation, Magnus Ehrnrooth Foundation, and the Finnish Cancer
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Journal of Cell Science 119 (15)
Journal of Cell Science