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Calmodulin: a
prototypical
calcium sensor
David Chin and Anthony R. Means
Calmodulin is the best studied and prototypical example of the
EF-hand family of Ca21-sensing proteins. Changes in
intracellular Ca21 concentration regulate calmodulin in three
distinct ways. First, at the cellular level, by directing its
subcellular distribution. Second, at the molecular level, by
promoting different modes of association with many target
proteins. Third, by directing a variety of conformational states in
calmodulin that result in target-specific activation. The
calmodulin-dependent regulation of protein kinases illustrates the
potential mechanisms by which Ca21-sensing proteins can
recognize and generate affinity and specificity for effectors in a
Ca21-dependent manner.

the Ca21 signal. Hence, separate intracellular loci or

organelles are potentially distinct compartments of
localized Ca21 signalling2 (Fig. 1a). Therefore, Ca21
signals in the nucleus exert different effects from
those generated in the cytoplasm or near the plasma
membrane of the same cell3. Additionally, the
modulation of the amplitude or frequency of Ca21
spikes (AM and FM, respectively) encodes important
signalling information4. This has recently been
illustrated for cases in which an optimal frequency
of intracellular Ca21 oscillations is important for the
expression of different genes5.
Calcium-regulated proteins: calmodulin
How do Ca21 signals produce changes in cell function? The information encoded in transient Ca21
signals is deciphered by various intracellular Ca21binding proteins that convert the signals into a wide
variety of biochemical changes. Some of these
proteins, such as protein kinase C, bind to Ca21 and
are directly regulated in a Ca21-dependent manner.
Other Ca21-binding proteins, however, are intermediaries that couple the Ca21 signals to biochemical
and cellular changes (Fig. 1b). Among this latter
group are a family of proteins that is distinguished
by a structural motif known as the EF hand. An EF
hand consists of an N-terminal helix (the E helix)
immediately followed by a centrally located, Ca21coordinating loop and a C-terminal helix (the
F helix). The three-dimensional arrangement of
these domains is reminiscent of the thumb, index
and middle fingers of a hand, hence the name EF
hand.
These proteins respond to Ca21 in one of two ways
(Fig. 1b). One group (e.g. parvalbumin and calbindin)
do not undergo a significant change in conformation on binding Ca21 and function as Ca21 buffers
or Ca21 transporters. The second group, the Ca21
sensors, undergo a Ca21-induced change in conformation6. The most prominent examples of sensors
include troponin C (a protein dedicated to regulating striated-muscle contraction), the multifunctional Ca21 transducer calmodulin (CaM), the S100
family of proteins and, most recently, the neuronal
myristoylated proteins such as recoverin7.
The molecular and cellular mechanisms underlying the ability of a majority of the Ca21-sensor
proteins to integrate Ca21 signals into specific cellular
responses are not clearly understood. Much of what
we do know about the mechanisms that the sensor
proteins use to transduce Ca21 signals is based on
information gained from CaM, probably the most
intensively studied member of the EF-hand family
of sensors. In the remainder of this article, CaM will
therefore serve as a model or prototype for other
potential Ca21 transducers. A review of some of the
mechanisms responsible for regulating CaM at the
subcellular and molecular levels might reveal valuable
clues as to how Ca21-sensor proteins convert Ca21
signals into cellular function.
CaM is expressed in all eukaryotic cells where it
participates in signalling pathways that regulate
many crucial processes such as growth, proliferation
and movement. It is relatively small (vertebrate CaM

The authors are in

the Dept of
Pharmacology
and Cancer
Biology, Duke
University Medical
Center, Durham,
NC 27710, USA.
E-mail: chin0001@
mc.duke.edu;
means001@
mc.duke.edu

Calcium (as Ca21) is an element that is crucial for

numerous biological functions. In many organisms,
the vast majority of Ca21 is complexed with phosphates to form exo- or endoskeletons that not only
serve as structural scaffolds but also buffer the levels
of Ca21 within extracellular fluids at ~1023 M. By
contrast, the resting concentrations of intracellular
free Ca21 (~1027 M) is 104 times lower than that outside cells, providing the potential for the ready
import of Ca21 into cells, where it can act as a second
messenger.
Various extracellular stimuli promote the movement of Ca21 either from outside the cell (via
plasma-membrane Ca21 channels) or from intracellular
stores into the intracellular milieu (Fig. 1a). The
Ca21 is released in elemental aliquots called sparks,
puffs or waves depending on the extent of the intracellular area covered. This free Ca21 is only briefly
available to act as a cellular signal, however, because
Ca21-binding proteins and Ca21 pumps immediately
combine to sequester and transport it to intracellular
storage sites or outside the cell.
The short pulses of Ca21 exert specific changes in
cellular function depending on their route of entry
into the cell, their local sites of action and, finally, by
their pattern of modulation. The particular membrane channel or intracellular receptor responsible
for the release of Ca21 exerts considerable influence
on the eventual effects of the Ca21 signal1. The mode
of cellular entry also influences the site of action of

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0962-8924/00/$ see front matter 2000 Elsevier Science Ltd. All rights reserved.
PII: S0962-8924(00)01800-6

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(a)

Ca2+

(b)

Ca2+
Amplitude
modulated (AM)

Frequency
modulated (FM)
Ca2+

Ca2+
Nucleus

trends in Cell Biology

Ca2+

FIGURE 2

(b) Ca2+ buffers and

transporters
Ca2+

Effectors

Ca2+ sensors

trends in Cell Biology

FIGURE 1
(a) Sources of intracellular Ca21 signals. Ca21 enters cells via
extracellular plasma-membrane receptors or from intracellular
stores, producing transient local or global changes in its
distribution. The Ca21 oscillations are modulated in their
amplitudes (AM) or frequencies (FM) and are therefore capable
of conveying signalling information in complex ways. (b) EFhand Ca21-binding proteins are classified as buffers/transporters
and sensors. The Ca21 sensors change conformation on binding
Ca21 and transduce changes in cell function by regulating
downstream effectors.

has 148 residues), evolutionarily highly conserved

and comprises four EF hands. The first two EF
hands combine to form a globular N-terminal
domain that is separated by a short flexible linker from
a highly homologous C-terminal domain consisting of
EF hands 3 and 4 (Fig. 2).
Ca21 sensors must be able to detect and respond
to a biologically relevant range of intracellular free
Ca21 concentrations. CaM fits this profile as its
affinity for Ca21 (Kd 5 5 3 1027 M to 5 3 1026 M) falls
within the range of intracellular Ca21 concentrations
exhibited by most cells (1027 M to 1026 M). However,
it has additional discrimination for Ca21, as the Cterminal pair of EF hands has a three- to fivefold
higher affinity for Ca21 than the N-terminal pair of
sites. By contrast, many Ca21-binding proteins with
a considerably higher affinity (Kd ,1027 M) act as
buffers by sequestering excess free Ca21, whereas
Ca21-binding proteins with a considerably lower
affinity (Kd .1025 M) could not act as sensors
because they are unable to detect the range of
changes in intracellular free Ca21 concentrations
that normally occur in cells.
The two domains of CaM adopt different
conformations in the absence or presence of Ca21
trends in CELL BIOLOGY (Vol. 10) August 2000

The Ca21-regulated conformational change in calmodulin. The

main chain structure of Ca21-free (apo) CaM (a) and
Ca214CaM (b) are shown in red with their respective
N-terminal domains on top. Methionine side chains are shown
in purple to denote the location of potential hydrophobic
pockets in each of the two domains. Ca21 binding produces
large changes in the helices in both domains, resulting in the
exposure of several hydrophobic residues.

(Fig. 2). In the absence of Ca21, the N-terminal

domain of the apo-CaM molecule adopts a closed
conformation in which the helices in both EF
hands are packed together. By contrast, still in the
absence of Ca21, the C-terminal domain of apo-CaM
adopts a semiopen conformation in which a partially exposed hydrophobic patch is accessible to
solvent. This might allow the C-terminal domain of
CaM to interact with some target proteins at resting
levels of intracellular free Ca21 (Ref. 8).
In the event of a transient rise in Ca21, the Ca21
ion is coordinated in each Ca21-binding loop of
Ca21CaM by seven, primarily carboxylate, ligands.
The binding of Ca21 leads to substantial alterations
in the interhelical angles within the EF hands in
each domain and dramatically changes the two
domains of CaM to produce more open conformations (Fig. 2). These structural rearrangements in
CaM result in the concerted exposure of hydrophobic
groups in a methionine-rich crevice of each domain
that is distinct from the Ca21-binding loops. The exposure to solvent of these hydrophobic residues is
akin to a Ca21-controlled unfolding of CaM and unleashes considerable free energy. It is this capacity to
convert the Ca21-binding event into biochemical energy that characterizes the Ca21-sensor proteins and
is the basis of their ability to transduce Ca21 signals.
Calmodulin: location, mobility and translocation
Is CaM regulated at the subcellular level, and how
is this related to Ca21 signalling? The concentration
and location of CaM do appear to play an important
role in regulating its biological activity. CaM constitutes at least 0.1% of the total protein present in
cells (1026 M 1025 M) and is expressed at even

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(a)

(b)

(c)

cytokinesis9. Other fluorescently labelled CaM

molecules have provided information on its cellular
mobility and location. Experiments with serumdeprived Swiss 3T3 fibroblasts first indicated that
the majority of CaM was freely diffusible, but the
CaM was then immobilized in response to stimulation
by serum10. However, other studies on unstimulated
smooth-muscle cells showed that most CaM is
bound, possibly to Ca21-independent binding
proteins, at resting concentrations of free Ca21.
In response to a rise in Ca21, CaM exhibits a complex pattern of cellular localization, including a
significant redistribution from the cytosol to the nucleus11. This stimulus-dependent movement of CaM
trends in Cell Biology
to the nucleus and its activation has also been deFIGURE 3
tected in neurons12. CaM has also been seen to
accumulate slowly in the nucleus of hormoneDistribution of calmodulin and tubulin in the mitotic spindle.
(a) A spindle visualized with an anticalmodulin antibody.
treated pancreatic acinar cells13. The mechanism of
(b) A Nomarski image of a mitotic spindle formed by incubation
translocation in smooth-muscle cells apparently
in a Xenopus extract. (c) A spindle visualized with
involves the passive diffusion of CaM into the
an antitubulin antibody.
nucleus, where it might associate with targets in a
Ca21-dependent manner14.
The synchronization between CaM and Ca21 signals
higher levels in rapidly growing cells, especially
those undergoing cell division and differentiation.
is also being explored. A direct relationship between
The local intracellular availability of CaM is likely to
a rise in the levels of intracellular free Ca21 and the
be biologically significant because various CaMCa21-dependent activation of CaM was first observed
dependent effectors are regulated over a wide range
during a response to wound healing in fibroblasts15.
of free CaM concentrations (10212 M 1026 M).
Ca21 oscillations in the secretory granules of panRecent studies of CaM tagged with green-fluorcreatic acinar cells have also been correlated with
escent protein (GFP) show that CaM is found throughoscillations in the local concentration of CaM13.
out the cytosol and nucleus in HeLa cells, although
Recent studies on sea-urchin eggs undergoing mitoit is concentrated around the mitotic apparatus in
sis, however, indicate that the spatial patterns of
cells undergoing mitosis (Fig. 3), especially around
Ca21 are different from those of Ca21-activated
the centrioles and the cytoplasmic furrow during
CaM16. Interestingly, the Ca21-dependent activation
of CaM exhibits a heterogenous distribution pattern in the cells that have
TABLE I SOME RECENT EXAMPLES OF CALMODULIN-REGULATED PROTEINS
been studied, indicating the presence of
discrete populations of CaM. These
Protein
Comments
Ref.
studies emphasize the importance of
temporal and spatial relationships beCabin1
Thymocyte transcriptional regulator
45
tween Ca21 signals and CaM function.
NAP-22

Neuronal substrate of protein kinase C

46

Striatin

Neuronal, associates with phosphatase 2A

47

CAP-19

Neuronal, IQ calmodulin-binding motif

48

EGF-receptor

Human, CaM binds at juxtamembrane

49

MLC phosphatase (targeting subunit)

Participant in muscle contraction/relaxation

50

Connexin 32

Located at gap junctions

51

ChURP

Located in the nucleus

52

High MW protein

Cardiac muscle phosphoprotein

53

Beta-2-glycoprotein

Membrane-associated protein in kidney

54

Retinal proteins

Involved in neuronal synaptic transmission

55

Extracellular proteins

Located in animal body fluids

56

Sperm proteins

Spermatocyte, acrosome reaction

57

Plant proteins

Plasma membrane transporter

58

Yeast proteins

Involved in cell growth and division

59

Phosphatidylinositol 3-kinase

Component in receptor signalling

60

324

Calmodulin: regulation of effectors

An important obstacle to studies on
many Ca21-sensor proteins is the problem of identifying downstream targets.
Biochemical and genetic approaches
have recently started to identify targets
for some of the S100 class of proteins17
and also for members of the myristoylated Ca21 sensors, such as frequenin18.
By contrast, CaM has been known for
some time to regulate several classes of
proteins and enzymes in a Ca21-dependent manner. The binding of target proteins by CaM raises the affinity of CaM
for Ca21 by approximately tenfold19 and
sensitizes the CaMeffector complex to
changes in Ca21 concentrations. Interestingly, many of the most highly characterized effectors (e.g. the CaMdependent adenylyl cyclases, phosphodiesterases, protein kinases and the protein
phosphatase calcineurin) are directly or
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Low [Ca2+]
indirectly involved in protein phosphorylation.
CaM also regulates the activities of the plasmamembrane Ca21 pump, various ion channels, the
ryanodine receptor and isoforms of the inositol
(1,4,5)-trisphosphate receptor. The list of CaM targets is extensive and constantly growing (Table 1).
Are there differences in the mechanisms by which
CaM and other Ca21 transducers regulate their
targets? CaM performs a variety of roles, and CaMbinding proteins can be categorized into at least six
classes based on their modes of regulation in the
presence and absence of Ca21 (Fig. 4). One group of
effectors, which we designate class A, binds essentially irreversibly to CaM irrespective of Ca21. CaM
is thus more appropriately considered a subunit of
these proteins. One example is phosphorylase
kinase, an enzyme that requires denaturing conditions to dissociate CaM but is activated in the
presence of Ca21. Members of a second group of effectors (class B) bind to CaM in the absence of Ca21
(i.e. to the apo-CaM form) but dissociate reversibly
in the presence of Ca21 (Ref. 20). Examples include
proteins such as neuromodulin and neurogranin,
which might serve as intracellular reservoirs for
CaM at resting concentrations of Ca21 but liberate
Ca21-activated CaM in response to a transient Ca21
signal.
A third group of effectors (class C) includes
smooth-muscle myosin-light-chain kinase (MLCK)
and calcineurin. These class-C effectors form lowaffinity, inactive complexes with CaM at low concentrations of Ca21, when CaM is unoccupied or
partially occupied by Ca21 [,2 (mole Ca21) (mole
CaM)21]. At high concentrations of Ca21, these targets engage in a high-affinity complex and are activated by CaM21,22. A fourth class of proteins (class D)
binds to CaM in the presence of Ca21, but, in this
case, CaM inhibits their function. This group includes enzymes such as select members of the Gprotein-receptor kinases23, as well as the inositol
(1,4,5)-trisphosphate receptor type 124.
A fifth group of effectors (class E), such as the
CaM-dependent protein kinases I, II and IV, exhibit
more conventional behaviour and are activated by
Ca21CaM. The class-E targets also exhibit an accessory form of regulation in which CaM binding promotes their regulation (specifically via phosphorylation) by another CaM-regulated kinase (i.e. a
CaM-kinase kinase ), which we designate class F. In
the specific case of the multimeric CaM kinase II,
both the substrate and the catalytic subunits require
CaM binding to promote intermolecular autophosphorylation25. This novel case, in which one CaMdependent protein (class E) is directly regulated by
another CaM-dependent protein (class F), demonstrates the convergence of different CaM-regulated
pathways and is indicative of CaM-signalling
cascades.
The observation that CaM regulates a specific set
of proteins yet engages in different types of Ca21dependent interactions implies that CaM and its
targets both exhibit certain complementary features
that enable CaM recognition but possess other aspects that still allow CaM to discriminate between
trends in CELL BIOLOGY (Vol. 10) August 2000

apo
CaM

+A

apo
CaM

+B

CaM

High [Ca2+]

CaM-A

CaM-A+
B

CaM-B

CaM
+B

+C
CaM-C

CaM-C+

+D

CaM

CaM-D-

CaM-E++
CaM-E+
CaM

+E

+F

CaM-F+
trends in Cell Biology

FIGURE 4
Ca21-dependent functions of various classes of calmodulin-binding (CaM-binding)
proteins. CaM and various classes of targets exist in free or bound states. Target
classes A, B and C are associated with CaM or Ca21-free CaM (apo-CaM) at low
(resting) intracellular free Ca21 concentrations (red). When Ca21 concentrations are
high (green), class B dissociates from CaM, classes D, E and F associate with
CaM, classes A, C, E and F are activated by CaM (1), and class D is inactivated
by CaM (2).

various classes of effectors. As CaM, like many Ca21

sensors, is a relatively small protein, it must therefore use multiple interaction surfaces to accomplish
these ends. These interaction sites enable CaM to
convert the energy provided by Ca21 binding into
effector regulation.
Calmodulineffector coupling: binding and
activation
The Ca21-controlled exposure of hydrophobic
groups in the two domains of CaM releases a considerable amount of biochemical energy, which is
transduced into two separable effects: a change in
the affinity of CaM for the effector and/or an alteration in the effectors function. Studies focusing on
one group of CaM-regulated enzymes in particular,
the CaM-dependent protein kinases, have provided
important insights into some of the mechanisms
underlying these phenomena. A short peptide of
~20 residues that is responsible for binding
Ca21CaM, designated a CaM-binding domain, has
been identified in many CaM-regulated proteins
(Fig. 5a) and in other types of CaM-binding proteins26. The crystal structure of CaM kinase I reveals
that the CaM-binding domain directly interacts
with and sterically obstructs the putative substratebinding sites of the inactive enzyme27. Furthermore,
the N-terminal part of the CaM-binding sequence
loops away from the enzyme, exposing the
hydrophobic side chain of Trp303 to solvent and
providing potential access for Ca21CaM to bind
(Fig. 5b). This proposal is supported by experiments
showing that the mutation of Trp303 to Ser in CaM
kinase I significantly lowered the apparent affinity
of CaM kinase I for Ca21CaM28.

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(a)

Interaction site on calmodulin

CaM C-domain
CaM N-domain

CaM KI

S K W K Q A F N A T A V V R H M R K

CaM KII

R K L K G A I L T T M L A T R N F S

smMLCK

R K W Q K T G H A V R A I G R L S S

skMLCK

R R W K K N F I A V S A A N R F K K

Ca

2+ATPase

CaM KK

I L W F R G L N R I Q T Q I R V V N
P S W T T V I L V K S M L R K R S F

(b)

(c)

Main chain of
Ca2+4CAM
CAM-binding domain
of smMLCK
Conserved tryptophan
Met124 residue

trends in Cell Biology

326

FIGURE 5
(a) Alignment of amino acid sequences from selected
calmodulin-binding (CaM-binding) domains. The enzymes are
CaM kinase I (CaM KI), CaM kinase II (CaM KII), smooth- and
skeletal-muscle myosin-light-chain kinases (smMLCK and
skMLCK), the plasma-membrane Ca21-pumpATPase (Ca21
ATPase) and CaM-kinase kinase (CaM KK). In most cases, a
hydrophobic residue (red) from the corresponding peptides
interacts with the C-terminal domain of Ca21CaM. The boxed
residue in CaM KI is Trp303. The N-terminal domain of
Ca21CaM interacts primarily with the C-terminal half of the
peptides. (For additional information on CaM binding domains,
see http://calcium.oci.utoronto.ca/) (b) Two crystal
structures showing the main chain of Ca214CaM on the left and
CaM kinase I on the right. The N-terminal domain of CaM and
the ATP-binding lobe of CaM kinase I are both positioned on top,
with helices red and sheets green. The Trp303 side chain from
the CaM-binding domain of CaM kinase I (black) extends away
from the enzyme in the direction of CaM. (c) Crystal structure
showing the main chain of Ca214CaM (white) in complex with
the helical CaM-binding domain of smooth-muscle myosin-lightchain kinase (green). CaM wraps around the helix so that the
conserved Trp of the peptide makes contact with Met124 (red)
in the C-terminal domain of CaM.

A homologous hydrophobic residue is conserved

in other CaM kinases (Fig. 5a). In the absence of
detailed information on complexes between CaM
and its intact effectors, spectroscopic and crystallographic studies of Ca21CaM complexed with peptides corresponding to the CaM-binding domains of
four CaM kinases including CaM kinase I show in
each case that this conserved hydrophobic residue
interacts exclusively with the methionine-rich
hydrophobic pocket in the C-terminal domain of
Ca21CaM2932 (Fig. 5c). Recently determined threedimensional structures of Ca21CaM bound to
peptides from the plasma membrane Ca21ATPase
pump and a CaM-kinase kinase also reveal additional
modes of interaction between CaM and these other
CaM-binding peptides33,34. These peptide studies
indicate that the C-terminal domain of Ca21CaM
might confer binding energy on the intact enzymes.
Indeed, this appears to be the case because complementary mutagenesis experiments on the Met
residues of CaM showed that an evolutionarily invariant Met124 in the C-terminal domain of
Ca21CaM that contacts the conserved hydrophobic
residues in several CaM-binding peptides (Fig. 5c) is
necessary for high-affinity binding and activation of
CaM kinase I as well as for three other CaM-dependent
protein kinases35,36.
In contrast to the C-terminal domain of
Ca21CaM, residues in the hydrophobic pocket of
the N-terminal domain of Ca21CaM perform varying functions with different CaM-dependent kinases.
The results from the crystallographic studies show
that hydrophobic residues in the N-terminal domain of Ca21CaM mainly interact with the Cterminal part of the CaM-binding peptides of smoothmuscle MLCK and CaM kinase II, respectively
(Fig. 5a). Progressive C-terminal deletions and
chimeric substitutions in the CaM-binding domain
of smooth-muscle MLCK showed that the C-terminal
half of the CaM-binding domains of these enzymes
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are required for Ca21CaM-dependent activation37,38. Furthermore, deletion studies of CaM
kinase I show that the C-terminal portion of its
CaM-binding sequence confers high affinity for
Ca21CaM, as well as CaM-dependent activity39.
These results complement those from experiments
on CaM showing that the hydrophobic pocket in its
N-terminal domain generates a high-affinity complex with CaM kinase II, activates smooth-muscle
MLCK and combines the functions of high-affinity
binding and activation of CaM kinase I35,40.
In comparison with unbound Ca21CaM, the
Ca21CaMpeptide complexes exhibit a dramatic
contraction enabling CaM to wrap around and sequester the helical CaM-binding peptides (Fig. 5c).
Experiments on hydrophilic residues of CaM show
that charged and polar residues are also required to
activate the smooth-muscle MLCK by promoting
the accessibility of substrate to this particular enzyme. Surprisingly, the hydrophilic residues on
CaM responsible for this effect are originally
separated from each other on both domains of free
Ca21CaM by more than 50 but then form a
stable latch less than 5 apart in the Ca21
CaMpeptide complex41. These polar groups do not
contact the CaM-binding peptide directly, so they
might exert their effects on the intact protein kinase
by interacting with an area distinct from its CaMbinding domain. Indeed neutron- and X-rayscattering studies on the kinase domain of skeletalmuscle MLCK indicate that the CaM-binding
domain is displaced to one side of the enzyme by
the binding of Ca21CaM42. This event could expose
the substrate-binding site of the enzyme and indicates
how CaM might remove an inhibitory CaM-binding
domain away from the kinase domain, thus leading
to enzyme activation.
The preceding mechanistic studies show that the
regulation of enzymes by Ca21CaM is a highly ordered, cooperative and complementary process that
contributes to both the affinity and specificity for
targets. Another surprising outcome is the discovery
that the structures of Ca21CaMpeptide complexes
are relevant to their corresponding enzymes. However,
in addition to the obvious limitation in the use of
peptides to study mechanisms of enzyme activation, there are mounting indications that peptides
might not be entirely suitable for studying other
functions of the full-length target protein. For
example, Ca21 has a considerably higher affinity for
the CaMMLCK-peptide complex than for the corresponding CaMMLCK-enzyme complex19. Also,
mutations in the CaM-binding domain of smoothmuscle MLCK have a significantly greater effect on
CaM binding and activation than the same changes
within the context of the corresponding CaM-binding
peptide40. Differences in the Ca21-dependent interaction of CaM with either the CaM-binding peptide
of skeletal-muscle MLCK or the intact enzyme have
also been observed by small-angle scattering43. One
explanation for the adaptability and the higher
affinity exhibited by the shorter CaM-binding domains is the conformational flexibility inherent in
isolated peptides. By contrast, the relatively fixed
trends in CELL BIOLOGY (Vol. 10) August 2000

conformation of the intact, folded enzymes restricts

their ability to adapt similarly to structural changes
within their CaM-binding domains. It is helpful to
bear these caveats in mind when peptides are used
to model effector function.
Perspective and conclusion
The extensive characterization of CaM provides a
useful precedent for less-well-understood Ca21 sensors. At the subcellular level, the spatial and temporal
coordination between Ca21, CaM and its effectors
are important for channelling all three components
into a productive signalling pathway. The ability of
CaM to integrate Ca21 signals into different cellular
contexts by migrating between various compartments further underscores this point. At the intermolecular level, CaM uses different modes of Ca21dependent interactions, which are responsible
for generating high affinity as well as specificity for
targets. At the submolecular level, the Ca21-triggered
exposure of energy-donating groups on CaM is coupled
to energy-accepting groups on its targets, leading to
changes in Ca21 binding by CaM as well as in the
function of its effectors.
Finally, how are these levels of regulating CaM
related to each other? It is likely that the mobility of
separate pools of CaM derives from the different
interactions between CaM and its targets. Therefore,
some classes of proteins might anchor CaM to specific cellular locations, depending on the stability of
a particular CaMeffector complex in the absence or
presence of a Ca21 signal. The affinity of such complexes is likely to be due to complementary interactions between sites on the target proteins and sites
on CaM that change conformation in response to
Ca21. The Ca21-dependent interactions not only affect the affinity of the complex but also regulate the
activity of effectors. This apparent ability of a
CaMeffector complex to decode Ca21 signals has
been exemplified in a recent study showing that
CaM participates in converting Ca21 oscillations into
changes in the autonomous enzymatic activity of at
least one target, CaM kinase II44. Additional studies
on CaM will lead to a more-complete integration of
its levels of regulation. Meanwhile, it will be interesting to see whether any of the mechanisms exhibited by CaM will be relevant to other Ca21 sensors.
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4 Thomas, A.P. et al. (1996) Spatial and temporal aspects of cellular
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5 Dolmetsch, R.E. et al. (1998) Calcium oscillations increase the efficiency
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6 Ikura, M. (1996) Calcium binding and conformational response in
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7 Braunewell, K-H. and Gundelfinger, E.D. (1999) Intracellular neuronal
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reviews

Acknowledgements
Our research
cited in this
review was
funded by NIH
grants HD-07503
and GM-33976
to ARM. We
thank the
members of the
Means
laboratory for
stimulating
discussions.

328

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