(Received for publication, June 5, 1995; and in revised form, October 20, 1995)
From the
C-CAM is a cell adhesion molecule belonging to the
immunoglobulin supergene family and is known to mediate
calcium-independent homophilic cell-cell binding. Two major isoforms,
C-CAM1 and C-CAM2, which differ in their cytoplasmic domains, have been
identified. Previous investigations have demonstrated that both
cytoplasmic domains can bind calmodulin in a calcium-dependent
reaction. In this investigation, peptides corresponding to the
cytoplasmic domains of C-CAM were synthesized on cellulose membranes
and used to map the binding sites for I-labeled
calmodulin. Both C-CAM1 and C-CAM2 had one strong calmodulin-binding
site in the membrane-proximal region. These binding regions were
conserved in C-CAM from rat, mouse, and man. In addition, C-CAM1 from
rat and mouse contained a weaker binding site in the distal region of
the cytoplasmic domain. Biosensor experiments were performed to
determine rate and equilibrium constants of the C-CAM/calmodulin
interaction. An association rate constant of 3.3
10
M
s
and two dissociation
rate constants of 2.2
10
and 3.1
10
s
were determined. These
correspond to equilibrium dissociation constants of 6.7
10
and 9.4
10
M, respectively. In dot-blot binding experiments, it was
found that binding of calmodulin causes a down-regulation of the
homophilic self-association of C-CAM. This suggests that calmodulin can
regulate the functional activity of C-CAM.
Cell adhesion molecules (CAMs) ()are important for
the organization and integrity of tissues in multicellular
organisms(1, 2) . Recently, several CAMs have also
been demonstrated to be active in transmembrane
signaling(3, 4, 5) . The spatiotemporal
expression of many CAMs during development, as well as during various
physiological and pathological processes in adult organisms, is highly
dynamic(1, 2, 6) , which implies that their
expression and functional activities are strictly regulated. It has
been demonstrated that growth factors, cytokines, hormones, and
homeodomain proteins regulate the expression of various
CAMs(7, 8, 9, 10) . It is also well
documented that the functional activity of some adhesion proteins can
be regulated by phosphorylations of their cytoplasmic
domains(3) . Several CAMs also interact with other cytoplasmic
proteins that influence their
function(2, 11, 12, 13) . However,
for most adhesion proteins, our knowledge of interacting cytoplasmic
components, their mode of functional regulation, and their
participation in signal transduction pathways (14) is still
very scanty. Identification and characterization of cytoplasmic
components that interact with transmembrane cell adhesion proteins are
therefore of great importance as a first step toward understanding how
these proteins are regulated.
C-CAM is a cell adhesion molecule that originally was identified in adult rat hepatocytes(15) . It is also expressed in several epithelial, vessel endothelial, and hematopoietic cells(16) . The homologous molecules in mouse and man are known as Bgp and BGP, respectively (17, 18) . C-CAM/Bgp/BGP are members of the carcinoembryonic antigen gene family, which belongs to the immunoglobulin gene superfamily(19, 20, 21, 22) . Different isoforms of C-CAM exist as a function of alternative splicing and different glycosylation(21, 23, 24) . The two major isoforms differ in the length of their cytoplasmic domains, C-CAM1 having a cytoplasmic domain of 75 amino acid residues, whereas that of C-CAM2 only consists of 14 amino acid residues.
C-CAM can mediate cell-cell adhesion via calcium-independent homophilic binding(25) . Based on expression in insect cells, it has been reported that the C-CAM1 cytoplasmic domain is necessary for cell adhesion activity(26) , but expression studies in other cell types have recently demonstrated that both C-CAM1 and C-CAM2 are effective adhesion molecules(27) . In addition to cell adhesion, other activities have also been described for C-CAM. C-CAM1 has been described as an ecto-ATPase (21) and a bile salt transporter(28) . In the mouse, one of the allelic variants of Bgp functions as the receptor for mouse hepatitis virus(29) . Moreover, C-CAM1 can suppress tumorigenicity of prostate cancer cells(30) . Whether this activity depends on its adhesive properties or some other function is not known.
We have previously reported that the C-CAM cytodomain binds to the intracellular calcium-regulated protein calmodulin(31, 32) . Calmodulin is a major regulator of enzyme activities and cytoskeletal functions in eukaryotic cells. High concentrations of calmodulin are found in submembranous regions coinciding with sites of C-CAM enrichment (33, 34, 35, 36) , suggesting that it might regulate C-CAM activities. In this investigation, we have used synthetic peptides to determine the binding sites in C-CAM for calmodulin and biosensor technology to determine kinetic and equilibrium binding constants of the C-CAM/calmodulin interaction. We also present evidence indicating that calmodulin regulates the homophilic binding activity of C-CAM.
Binding experiments were performed at
25 °C by injecting 35-40-µl portions of various
concentrations of C-CAM (0.6-63 nM) dissolved in 10
mM Tris-HCl, pH 7.4, 0.15 M NaCl, 0.1% octyl
glucoside, and 1 mM CaCl or 2 mM EGTA at
a continuous flow maintained at 5 or 10 µl/min. In some
experiments, calmodulin (0.5 µM) was included in the flow
buffer during the dissociation phase to prevent rebinding of C-CAM to
the sensor surface. The sensor chip was regenerated by washing with
reaction buffer. C-CAM was purified from rat liver, and its
concentration was determined by radioimmunoassay as described
previously(43) .
Evaluation and calculation of binding
parameters were done according to the manual provided by Pharmacia
Biosensor AB, using the following relationship: dR/dt = kCR
- k
R, k
= k
C + k
, where k
is the association rate constant, k
is the dissociation rate constant, k
is an apparent first-order rate constant, R is the binding expressed in relative units, and R
is the maximum binding that theoretically can
be obtained(44) . Plots of dR/dt versus relative response (R) gave linear relationships. The
negative values of the slopes (k
) at each
concentration of C-CAM were replotted against the C-CAM concentration (C). This gave a straight line, from the slope of which the
association rate constant (k
) could be determined.
The equilibrium dissociation constant K
was
determined from the equilibrium binding levels (R
) by Scatchard plot analysis using the
following relationship: R
/C = K
R
- K
R
, where K
is the equilibrium association constant (K
= 1/K
). K
was
also calculated from the relationship between the rate constants (K
= k
/k
).
Figure 1:
Calmodulin binding to C-CAM1
cytoplasmic domain peptides. Cellulose membranes containing 67
individual decapeptides covering the cytoplasmic domain of C-CAM1 were
incubated with I-labeled calmodulin (1 µg/ml, 2
10
cpm/µg of protein). Calmodulin binding was
detected by autoradiography (A) or
-radiation counting (B). A, the peptide numbers (given for the first and
last peptide in each row) correspond to the sequences shown in B. B, the amino acid sequence of the long cytoplasmic
domain of C-CAM1 is shown in single letter code. TM,
transmembrane sequence. Sixty-seven overlapping decapeptides staggered
by one amino acid were synthesized as indicated. The peptides are
numbered from the amino terminus. The amounts of calmodulin bound to
peptides 1-10, 46-58, and 63-67 (the sequences of
which are indicated by horizontal lines) are given as cpm
values. Each value represents the mean of three individually
synthesized peptides. Peptides 11-45 and 59-62 showed no
binding activity (see also A).
Figure 2:
Calmodulin binding to C-CAM2 cytoplasmic
domain peptides. Six overlapping decapeptides covering the short
cytoplasmic domain of C-CAM2 were synthesized and subjected to I-calmodulin binding. For further details, see the legend
to Fig. 1. TM, transmembrane
sequence.
Since the coupling efficiency might differ for different amino acids in the peptide synthesis, it was important to investigate if the observed differences in calmodulin binding between the different peptides reflected differences in peptide content on the filters rather than sequence-specific differences. Analyses of the amino acid contents in the different spots on the filters showed no correlation between the amount of peptide in each spot and the degree of bound calmodulin (data not shown). Thus, the different calmodulin binding activities are sequence-related.
To investigate if the mouse and human homologues of rat C-CAM, Bgp and BGP, respectively, also would bind calmodulin, peptides corresponding to the cytoplasmic domains of Bgp and BGP were synthesized and tested. The membrane-proximal regions of both the long and short isoforms of Bgp and BGP bound calmodulin in a similar manner as the corresponding regions of C-CAM (Fig. 3). The distal region of the long cytoplasmic domain of Bgp showed weak binding, whereas that of BGP showed no binding (data not shown).
Figure 3:
Calmodulin binding to BGP and Bgp
cytoplasmic domain peptides. Homologous peptides corresponding to the
membrane-proximal portions of the long and short cytoplasmic domains of
C-CAM, BGP, and Bgp were synthesized and subjected to I-calmodulin binding. Binding was detected by
autoradiography. The amino acid sequences (shown in single letter code)
represent homologous regions of C-CAM, BGP, and
Bgp.
Figure 4:
Calcium
dependence of calmodulin binding to C-CAM peptides. Calmodulin binding
to SPOT peptides was determined at calcium concentrations
ranging from 10
to 10
M.
The peptide sequences are shown. The binding level obtained at the
highest calcium concentration tested in each experimental series was
set to 100% for each peptide. The level of binding obtained at other
calcium concentrations is given as the percent of this level. A, calmodulin binding to the membrane-proximal C-CAM1 peptide; B, calmodulin binding to three different C-CAM1 peptides
(
,
, and
), one C-CAM2 peptide (
), and one
peptide from the calmodulin-binding region of myosin light chain kinase
(
).
To identify amino acid residues
that are needed for calmodulin binding, we systematically changed all
amino acids in the peptides, one by one, to an alanine residue. The
mutations that gave the largest changes in binding efficiency are shown
in Table 1. Mutation of lysine or arginine to alanine gave the
strongest effects; the largest decrease in binding was observed when
both the arginine and lysine residues were changed simultaneously.
Furthermore, peptides in which the arginine and lysine residues were
retained, but all the other amino acid residues were changed to alanine
residues, only bound calmodulin to 40% of the binding level of the
unmodified peptides (data not shown). When the peptide sequences were
altered so that neighboring residues were reversed in sequence, the
levels of bound calmodulin changed markedly (data not shown). This
suggests that the arginine and lysine residues are necessary, but not
sufficient, for calmodulin binding to the cytoplasmic domains of C-CAM.
Figure 5: Binding kinetics of C-CAM to immobilized calmodulin. Representative overlaid sensograms illustrating the real-time binding of C-CAM at various concentrations (0.6, 3.2, 6.4, and 12.7 nM, from bottom to top) to calmodulin immobilized on a biosensor chip are shown. The inset shows dissociation, with and without 0.5 µM calmodulin included in the buffer during the dissociation phase, after injection of 12.7 nM C-CAM. Arrows indicate phases of association (A), equilibrium (B), and dissociation (C). RU, relative units.
As seen
in Fig. 5, a significant rebinding of C-CAM to the
calmodulin-containing surface occurred, which could be prevented by
inclusion of 0.5 µM calmodulin in the flow buffer during
the dissociation phase. Under these conditions, analysis of the
dissociation curves revealed two independent dissociation reactions
with rate constants (k) of 2.2
10
and 3.1
10
s
, respectively.
From the association and
dissociation rate constants, two equilibrium dissociation constants of
6.7 10
and 9.4
10
M were calculated from the relationship K
= k
/k
. The
existence of two classes of binding sites was confirmed by Scatchard
analysis of the binding data obtained at the equilibrium phase (phase B in Fig. 5). Equilibrium dissociation constants
of 3.8
10
and 1.8
10
M, respectively, were calculated from
the slopes of the straight lines drawn through the data points at the
two extremes of the Scatchard plot, as shown in Fig. 6. This is
in reasonable agreement with the K
values obtained
from the kinetic data. However, it is obvious from Fig. 6that
the slopes of the lines, due to the limited number of data points, do
not correspond exactly to the limiting slopes of the smooth curve to
which the experimental values were fitted. Thus, the calculated value
of the larger K
(3.8
10
M) is too small, and that of the smaller K
(1.8
10
M) is too large.
Accordingly, there is good agreement between the kinetic and
equilibrium analyses.
Figure 6:
Scatchard plot analysis. The equilibrium
binding of C-CAM to the calmodulin biosensor chip was evaluated by
Scatchard plot analysis. C-CAM concentrations ranged from 0.6 to 12.7
nM. Relative responses expressed as equilibrium binding values (R) and C-CAM concentrations (C) were
plotted as indicated. The broken line represents a calculated
curve fit of the experimental values. The reciprocal of the slopes of
the solid lines gave values for the dissociation constants (K
) of 3.8
10
and 1.8
10
M. RU,
relative units.
The Scatchard data could not be used to
calculate the stoichiometry of the C-CAMcalmodulin complex since
it was not known how much of the surface-bound calmodulin was available
for interaction with C-CAM. Since calmodulin is a much smaller molecule
than C-CAM, it penetrates the hydrogel of the sensor chip more
effectively and accordingly is withdrawn from binding interactions with
C-CAM. Furthermore, some of the calmodulin might be inactivated as a
result of the covalent coupling to the hydrogel.
Figure 7:
Calmodulin binding to liver membranes. The
influence of calcium ions and antibodies against C-CAM on binding of I-calmodulin to rat liver plasma membrane was determined. Bar 1, 10
M Ca
,
no antibodies; bar 2, 10
M Ca
, no antibodies; bar 3,
10
M Ca
, nonimmune IgG
(500 µg/ml); bar 4, 10
M
Ca
, anti-C-CAM IgG (500 µg/ml); bar 5,
10
M Ca
, anti-C-CAM IgG
(500 µg/ml).
Figure 8:
Influence of calmodulin on the
self-association of C-CAM. Various amounts of purified C-CAM were
spotted onto a nitrocellulose membrane that was incubated with I-labeled C-CAM in the presence or absence of 1 µg/ml
calmodulin (CaM). The incubations were performed in the
presence of 1 mM Ca
or 2 mM EGTA as
indicated.
In a previous study, we demonstrated that the cytoplasmic domains of both C-CAM1 and C-CAM2 could bind calmodulin(32) . The data were compatible with two binding sites in C-CAM1 and one binding site in C-CAM2. In the present investigation, we have mapped the calmodulin-binding sites in the cytoplasmic domains of C-CAM1 and C-CAM2 more closely, utilizing synthetic peptides attached to a cellulose filter. The membrane-proximal regions of both C-CAM isoforms bound calmodulin effectively; a weaker binding site was also found in the distal portion of the C-CAM1 cytoplasmic domain. All peptide sequences, as well as C-CAM intercalated in plasma membranes, bound calmodulin in a strictly calcium-dependent manner. Biosensor analyses demonstrated that two classes of binding interactions with different dissociation rates existed. We could also demonstrate that calmodulin binding caused a down-regulation of the self-association of C-CAM.
Attached peptides, directly synthesized on cellulose membranes, have
several advantages over soluble, conventionally synthesized peptides in
ligand binding studies. They allow screening of large sequences in a
rapid and relatively inexpensive way. Furthermore, binding of various
ligands to the cellulose membranes is easy to determine. With soluble
peptides, a different monitoring assay has to be used. One approach
that has been used for calmodulin is to determine the change in
fluorescence intensity of dansyl-labeled calmodulin that occurs, due to
conformational changes, when large peptides (20 amino acids or more)
bind(47) . We tried this approach with a soluble decapeptide
corresponding to the cytoplasmic domain of C-CAM2. However, we did not
observe any change in the fluorescence intensity, probably because a
decapeptide is too short to induce the conformational change of the
calmodulin. On the other hand, a decapeptide corresponding to the
identified calmodulin-binding sequence of myosin light chain kinase,
synthesized by the SPOT method, bound calmodulin with the
expected calcium dependence. These results clearly demonstrate the
superiority of membrane-bound peptides to soluble peptides for
determination of binding sequences for various ligands.
Binding experiments with mutated C-CAM peptides showed that the positively charged amino acids arginine and lysine are important for calmodulin binding. That amino acids other than arginine and lysine are also important for calmodulin binding was clearly demonstrated by the fact that the binding activities of two overlapping peptides, FLYSRKTGGS and AYFLYSRKTG, differed 5-fold even though both contained the two basic residues. These results are in good agreement with the characteristics of binding sequences in other calmodulin-binding proteins(48) .
Mouse Bgp and human BGP, homologous molecules to rat C-CAM, were
also able to bind calmodulin. For mouse Bgp, the binding is not
surprising as mouse Bgp and rat C-CAM show a high degree of sequence
identity in their cytoplasmic domains. The only difference between the
short isoforms is that a threonine residue in the rat molecule is a
serine residue in the mouse molecule. The short form of mouse Bgp bound
calmodulin similarly compared with the corresponding sequence of
C-CAM2. The long cytoplasmic domain of Bgp, the sequence of which is
84% identical (93% conserved) to the corresponding part of C-CAM1,
exhibited the same two calmodulin-binding regions as C-CAM1. The human
BGP cytoplasmic sequences are not conserved to the same extent. The
overall sequence identity between the long cytoplasmic domains of the
human and rat proteins is only 60% (83% conserved), and the similarity
is even less in the region corresponding to the short cytoplasmic
domain (50% identity and 71% conserved). These differences
notwithstanding, calmodulin bound effectively also to sequences
corresponding to the membrane-proximal portion of both cytoplasmic
domain variants of human BGP. This can be explained if BGP and C-CAM
adopt an -helical conformation, which makes the distribution of
positively charged and hydrophobic amino acids strikingly similar in
the two molecules. Calmodulin generally seems to bind to
-helical
stretches(46, 49) . Thus, it seems as if the ability
to bind calmodulin to the short isoform and to the membrane-proximal
part of the long isoform has been conserved in spite of the relatively
large sequence difference. Unlike the rat and mouse proteins, peptides
from the distal part of the long isoform of human BGP did not bind
calmodulin. Although we cannot exclude the possibility that longer
peptides would reveal distal binding sites in the human cytodomain,
this result suggests that the proximal binding site may be more
significant than the other ones in the native proteins.
The
activation of calmodulin in vivo is caused by an increased
intracellular calcium concentration(50) , which leads to
binding to its target proteins(45) . Therefore, the finding
that calmodulin binding to the synthetic C-CAM peptides was regulated
by variations of the calcium concentration in the physiological range
(10 to 10
M) supports
the notion that the interaction is of physiological importance.
The
biosensor analyses revealed both a weak (K = 6.7
10
M) and a
strong (K
= 9.4
10
M) binding interaction between
calmodulin and intact rat liver C-CAM, which contains both C-CAM1 and
C-CAM2. Thus, the two classes of binding sites might correspond to the
membrane-proximal and membrane-distal binding sites that were
discovered by the peptide binding experiments. Alternatively, the two
classes of binding sites might reflect the presence of different
oligomeric forms of C-CAM, which has been found to occur as both
monomers and dimers in the plasma membrane. (
)Whereas both
classes of binding sites had the same rate of association, their
dissociation rates differed. The association rate was relatively fast,
which might be of physiological importance because it would allow a
rapid association of calmodulin with C-CAM in response to an increased
intracellular calcium concentration.
Because we could demonstrate
that calmodulin significantly reduced the self-association of C-CAM and
because we earlier have demonstrated that the extracellular domain of
C-CAM does not bind calmodulin(31) , it seems reasonable to
assume that calmodulin can regulate the adhesive binding activity of
C-CAM and/or the supramolecular organization of C-CAM within the plasma
membrane. Presently, we do not know if calmodulin affects trans-binding (i.e. C-CAM-mediated binding between
opposing membranes) or cis-binding (i.e. binding
between C-CAM molecules within the same membrane) between different
C-CAM molecules. In any case, it seems plausible that ligand/receptor
interactions at the cell surface, which trigger a transient increase in
the intracellular concentration of Ca, can lead to
increased binding of calmodulin to the cytoplasmic domains of C-CAM.
This could then lead to reduced homophilic C-CAM binding, which could
be either between opposed membranes or between C-CAM molecules within
the same membrane.