Gerhard Krauss
Biochemistry of Signal Transduction and Regulation(3rd
Edition) ISBN: 3-527-30591-2
LOGO
Intracellular Messenger
Substances: “Second
Messengers”
授課老師: 褚俊傑副教授 (生物科技系暨研究所)
聯絡電話: 0986-581835
電子信箱: jjchuu@mail.stut.edu.tw
Second Messengers inside the cell
Many different kinds of molecules can serve as second messengers. The signal, or ligand,
binding to a membrane receptor leads to the production of second messengers inside the cell.
The original signal usually doesn't enter the cell. The small molecule "cAMP" was the initial
second messenger to be identified. Other examples of second messengers include NO, IP3,
and DAG. The figure below shows an example of the production of second messengers.
Outline
 6.1 General Functions of Intracellular
Messenger Substances
 6.2 cAMP
 6.3 cGMP
 6.4 Metabolism of Inositol Phospholipids and
Inositol Phosphates
 6.5 Inositol 1,4,5-Triphosphate and Release of
Ca2+
 6.6 Phosphatidyl Inositol Phosphates and PI3Kinase
 6.7 Ca2+ as a Signal Molecule
 6.8 Diacylglycerol as a Signal Molecule
 6.9 Other Lipid Messengers
6.1 General Functions of Intracellular Messenger
Substances
 Extracellular signals are registered by membrane receptors
and conducted into the cell via cascades of coupled reactions.
The first steps of signal transmission often take place in close
association with the membrane, before the signal is
conducted into the cell interior. The cell uses mainly two
mechanisms for transmission of signals at the cytosolic side
of the membrane and in the cell interior. Signal transmission
may be mediated by a protein-protein interaction.
 The proteins involved may be receptors, proteins with adaptor
function alone, or enzymes. Signals may also be transmitted
with the help of low-molecular-weight messenger substances.
These are known as “second messengers”. The intracellular
messenger substances are formed or released by specific
enzyme reactions during the process of signal transduction,
and serve as effectors, with which the activity of proteins
further in the sequence is regulated (Fig. 6.1).
Fig. 6.1 Function and formation of intracellular messenger substances in signaling pathways.
Starting from the activated receptor, effector proteins next in sequence are activated that
create an intracellular signal in the form of diffusible messenger substances. The hydrophilic
messenger substances diffuse to target proteins in the cytosol and activate these for signal
transmission further. Hydrophobic messenger substances, in contrast, remain in the cell
membrane and diffuse at the level of the cell membrane to membrane-localized target
proteins. PK: protein kinase; S: substrate of the protein kinase.
6.1 General Functions of Intracellular Messenger
Substances
 The most important “second messengers” are
– hydrophilic, cytosolic:
cAMP, cGMP
inositol phosphates
Ca2+
– hydrophobic, membrane-associated:
diacylglycerol
phosphatidyl inositol phosphates.
6.2 cAMP
 3’-5’-cyclic AMP is a central intracellular “second
messenger” that influences many cellular functions,
such as gluconeogenesis, glycolysis, lipogenesis,
muscle contraction, membrane secretion, learning
processes, ion transport, differentiation, growth
control and apoptosis.
6.2 cAMP
 cAMP-gated Ion Channels
An important function of cAMP is the regulation of ion
passage through cAMP-gated ion channels. cAMP binds
to cytoplasmic structural elements of these ion channels
and regulates their open state.
An example is the cAMP-regulated Ca2+ passage
through cation channels. cAMP also performs this
function during the perception of smell in mammals.
6.2 cAMP
 Protein Kinase A
The majority of the biological effects of cAMP are
mediated by the activation of protein kinases. Protein
kinases regulated by cAMP are classified as protein
kinase A. The mechanism of activation of protein kinases
of type A by cAMP is schematically represented in Fig.
6.2.
In the absence of cAMP, protein kinase A exists as a
tetramer, composed of two regulatory (R) and two
catalytic (C) subunits. In the tetrameric R2C2 form,
protein kinase A is inactive since the catalytic center of
the C subunit is blocked by the R subunit.
Fig. 6.2 Regulation of protein kinase A via cAMP. Protein kinase A is a tetrameric
enyzme composed of two catalytic subunits (C) and two regulatory subunits (R). In
the R2C2 form, protein kinase A is inactive. Binding of cAMP to R leads to
dissociation of the tetrameric enyzme into the R2 form with bound cAMP and free C
subunits. In the free form, C is active and catalyzes the phosphorylation of
substrate proteins (S) at Ser/Thr residues.
6.3 cGMP
 Like cAMP, 3’-5’-cGMP is widespread as an
intracellular messenger substance. Analogous
to cAMP, cGMP is formed by catalysis via
guanylyl cyclase from GTP.
 Although the guanylyl cyclases catalyze a
similar reaction as the adenylyl cyclases, the
two enzyme classes differ considerably in
structure and mechanism of activation. The
guanylyl cyclases can be divided into three
groups according to the number of
transmembrane segments. One group contains
enzymes that do not contain a transmembrane
segment and are referred to as soluble guanylyl
cyclases.
 A second group contains one transmembrane
segment. The members of this group are
directly regulated by extracellular ligands and
therefore have receptor function. A third group
with more than two transmembrane segments
is only poorly characterized, and its ligands or
mechanism of activation is not yet known.
6.3 cGMP
 Guanylyl Cyclases with a Single Transmembrane
Segment
The guanylyl cyclases with a single transmembrane segment
function as receptors that contain an extracellular ligandbinding domain and various intracellular domains that are
required for the ligand-regulated activation of the enzyme
(Fig. 6.3) . As ligands for the guanylyl cyclase receptors,
peptides with vasodilatory properties like the atrial natriuretic
peptide have been identified. The receptor-type guanylyl
cyclases are therefore also termed natriuretic peptide
receptors, NPR.
The receptors exist in a homodimeric transmembrane form,
and its intracellular guanylyl cyclase domain is activated by
peptide binding to the extracellular domains. A complicated
series of reactions follow activation, which include
phosphorylation of an intracellular kinase-homology domain,
ATP binding and finally activation of cGMP synthesis.
Fig. 6.3 Model of the domain
structure of the natriuretic peptide
receptor NPR, a receptor type
guanylyl cyclase. NPR is a
dimeric transmembrane receptor
which spans the membrane with
two transmembrane elements.
The
extracytosolic
domain
comprises the ligand binding site
and contains several disulfide
bridges. The cytosolic part is
composed of a kinase homology
domain
with
multiple
phosphorylation sites, an ATP
binding site of unknown function
and the catalytic guanylyl cyclase
domain.
6.3 cGMP
 Soluble Guanylyl Cyclases
The soluble guanylyl cyclases exist as heterodimers and
are regulated by the second messenger NO. A heme
group that confers NO-sensitivity is bound at the Nterminus of these enzymes. NO binding to the heme
group results in activation of the guanylyl cyclase
activity. The second messenger function of cGMP is
directed towards three targets:
* cGMP-dependent protein kinases
* Cation channels
* cAMP-specific phosphodiesterases
6.4 Metabolism of Inositol Phospholipids and Inositol
Phosphates
 Inositol-containing phospholipids of the plasma membrane are
the starting compounds for the formation of various lowmolecular-weight inositol messengers in response to various
intra-and extracellular signals. These messengers include the
central second messengers diacylglycerol and inositol
trisphosphate as well as membrane bound phosphatidyl
inositol phosphates.
 The plasma membrane contains the phospholipid
phosphatidyl inositol (PtdIns), in which the phosphate group is
esterified with a cyclic alcohol, myo-D-inositol (Fig. 6.4).
Starting from PtdIns, a series of enzymatic transformations
lead to the generation of a diverse number of second
messengers. PtdIns is first phosphorylated by specific kinases
at the 4’ and 5’ positions of the inositol residue, leading to the
formation
of
phosphatidyl
inositol-4,5-bisphosphate
[PtdIns(4,5)P2].
Fig.
6.4
Formation
of
diacylglycerol, Ins(1,4,5)P3 and
PtdIns(3,4,5)P3.
PL-C:
phospholipase of type C: PI3kinase: phosphatidyl inositol-3’kinase.
6.4 Metabolism of Inositol Phospholipids and Inositol
Phosphates
 Inositol
Phosphates
and
Regulation
of
Phospholipase C
Phospholipase C, which occurs in different subtypes in
the cell, is a key enzyme of phosphatide inositol
metabolism. Two central signaling pathways regulate
phospholipase C activity of the cell in a positive way (Fig.
6.5).
Phospholipases of type Cβ (PL-Cβ) are activated by G
proteins and are thus linked into signal paths starting
from G protein-coupled receptors. Phospholipases of
type γ (PL-Cγ), in contrast, are activated by
transmembrane receptors with intrinsic or associated
tyrosine kinase activity.
Fig. 6.5 Formation and function of
diacylglycerol and Ins(1,4,5)P3.
Formation of diacylglycerol (DAG)
and Ins(1,4,5)P3 is subject to
regulation by two central signaling
pathways, which start from
transmembrane receptors with
intrinsic or associated tyrosine
kinase activity or from G-proteincoupled receptors. DAG activates
protein kinase C (PKC), which has
a regulatory effect on cell
proliferation, via phosphorylation
of substrate proteins. Ins(1,4,5)P3
binds to corresponding receptors
(InsP3-R) and induces release of
Ca2+ from internal stores. The
membrane association of DAG,
PtdIns(3,4)P2 and PL-C is not
shown here, for clarity.
6.4 Metabolism of Inositol Phospholipids and Inositol
Phosphates
 Metabolic Cycle of Inositol Phosphate
The inositol phosphates are linked into a metabolic cycle (Fig.
6.6) in which they can be degraded and regenerated. Via
these pathways, the cell has the ability to replenish stores of
inositol phosphate derivatives, according to demand. PtdIns
may be regenerated from diacylglycerol via the intermediate
levels of phosphatidic acid and CDP-glycerol. Regeneration of
PtdIns in the inositol cycle is of particular importance in the
vision process in Drosophil.
In Drosophila, InsP3 serves as a messenger during perception
of light. On incidence of light, InsP3 is formed from PtdInsP2.
It has been shown that CDP diacylglycerol synthase, which
supplies CDP diacylglycerol for the resynthesis of PtIns (see
Fig. 6.6) has an essential role in light perception in Drosophila
Fig. 6.6 Metabolic cycle of regeneration of PtdIns(4,5)P2
6.5 Inositol 1,4,5-Triphosphate and Release of
Ca2+
 6.5.1 Release of Ca2+ from Ca2+ Storage
 6.5.2 Influx of Ca2+ from the Extracellular Region
 6.5.3 Removal and Storage of Ca2+
 6.5.4 Temporal and Spatial Changes in Ca2+
Concentration
6.5 Inositol 1,4,5-Triphosphate and Release of Ca2+
 The primary signal function of Ins(1,4,5)P3 is the mobilization
of Ca2+ from storage organelles. Ca2+ is a ubiquitous signaling
molecule whose signaling function is activated by its release
from intracellular stores or through Ca2+ -entry channels from
the extracellular side. A multitude of second messengers has
been shown to induce an increase of intracellular Ca2+.
 The free Ca2+ concentration is subject to strict regulation, and
targeted increase of Ca2+ is a universal means of controlling a
vast array of metabolic and physiological reactions. Many
processes are involved in Ca2+ regulation (Fig. 6.7), allowing
the cell to shape Ca2+ signals in the dimensions of space,
time and amplitude. Fig. 6.7 gives an overview of the main
pathways leading to an increase or decrease of intracellular
calcium.
Fig. 6.7 Paths for increase and reduction of cytosolic Ca2+ concentration. Influx of Ca2+ from the
extracellular space takes place via Ca2+ channels; the open state of these is controlled by binding of ligand
L or by a change in the membrane potential (V). According to the type of ion channel, the ligand may bind
from the cytosolic or the extracellular side to the ion channel protein. The entering Ca2+ binds to InsP3
receptors on the membrane of Ca2+ storage organelles and induces, together with InsP3, their opening.
Ca2+ flows out of the storage organelle into the cytosol via the ion channel of the InsP3 receptor. Transport
of Ca2+ back into the storage organelles takes place with the help of ATP-dependent Ca2+ transporters.
6.5.1 Release of Ca2+ from Ca2+ Storage
 Mobilization of Ca2+ from the Ca2+ stores of the
endoplasmic reticulum takes place with the help of Ca2+
channels, of which two types stand out: the InsP3
receptors and the ryanodin receptors. Both are ligandgated Ca2+ channels, in which receptor and ion channel
form a structural unit.
 The InsP3 receptors and ryanodin receptors are localized
in the endoplasmic and sarcoplasmic reticulum,
respectively, and may be opened during the process of
signal transduction (Fig. 6.8).
Fig. 6.8 Tetrameric Ca2+ channels and control of Ca2+ release. a) A change in the membrane potential (V)
induces a conformational change in the dihydropyridine receptor of skeletal muscle; this is transmitted as a
signal to the structurally coupled ryanodin receptor. Opening of the Ca2+ channel takes place and efflux of
Ca2+ from the sarcoplasmic reticulum into the cytosol occurs. b) In cardiac muscle, the release of Ca2+
takes place by a Ca2+ -induced mechanism. A potential change V induces opening of voltage-gated Ca2+
channels. Ca2+ passes through, which serves as the trigger for release of Ca2+ from Ca2+ storage
organelles by binding to ryanodin receptors on the surface of the storage organelles. c) Membraneassociated signaling pathways are activated by ligands and lead, via activated receptor and phospholipase
C (PL-C) to formation of InsP3 and to release of Ca2+ from storage organelles.
6.5.1 Release of Ca2+ from Ca2+ Storage
 The InsP3 Receptor
Binding of InsP3 to the InsP3 receptor leads to opening
of the receptor channel, so that stored Ca2+ can flow into
the cytosol. The InsP3 receptor is a transmembrane
protein, probably with two transmembrane domains in
the vicinity of the C terminus.
The active receptor is composed of four identical
subunits. It is assumed that the Ca2+ channel is formed
by the C-terminal transmembrane element and that the
binding site for InsP3 is localized in the large cytoplasmic
region of the receptor. Opening of the InsP3 receptor is
subject to complex regulation involving Ca2+, Mg2+ and
ATP, in addition to InsP3.
6.5.1 Release of Ca2+ from Ca2+ Storage
 Ryanodin Receptor and Cyclic ADP Ribose
The ryanodin receptor takes its name from its stimulation
by the plant alkaloid ryanodin. In all, it has a similar
composition to the InsP3 receptor and is involved in Ca2+
signal conduction in many excitatory cells. In some cell
types (including cardiac muscle cells and pancreatic
cells), another “second messenger”, the cyclic ADPribose (Fig. 6.9), is involved in opening the ryanodin
receptors .
The cADP-ribose is formed from NADP by an enzymatic
pathway with the help of an ADP-ribosyl cyclase.
Fig. 6.9 Reactions of ADP ribosyl cyclase. Structures of NADP, nicotinic acid
adenine dinucleotide phosphate (NAADP) and cyclic ADP-ribose phosphate
(cADPRP). ADP-ribosyl cyclase, in base exchange mode, can catalyze
replacement of the nicotinamide
group of NADP (yellow) with nicotinic acid to generate NAADP. ADP-ribosyl cyclase
can also catalyze cyclization of NADP to cADPRP.
6.5.1 Release of Ca2+ from Ca2+ Storage
 Tool Kit for Ca2+ Release
Overall, multiple pathways can be used for mobilising Ca2+
from the internal stores. A Ca2+ signaling ‘toolkit’ is available
from which cells can select specific components to activate
the internal Ca2+ stores and to generate a variety of different
Ca2+ signals that suit their physiology. In summary, the
following pathways can induce Ca2+ release from internal
stores (Fig 6.10):
* Ca2+-induced Ca2+ release from ryanodine receptors caused by influx of
Ca2+ through voltage-operated Ca2+ channels on the plasma
membrane.
* Cyclic ADP-ribose-evoked Ca2+ release.
* NAADP-evoked Ca2+ release
* InsP3-evoked Ca2+ release.
* Ca2+ release by interaction of InsP3 receptors with calcium binding
proteins
* Ca2+ release triggered by sphingolipids or leukotriene B4
2+
Fig. 6.10 Tools for Ca2+ release. The figure illustrates the major pathways for mobilising Ca2+
from internal stores. 1, Ca2+ induced Ca2+ release from ryanodine receptors (RyR) caused by
the influx of Ca2+ through voltage- or ligand-gated channels on the outer cell membrane. This
release may be also triggered by direct interaction of the channel with RYR. 2, PLC/InsP3
evoked release of Ca2+ from InsP3 receptors or ryanodine receptors. 3, cyclic ADP-ribose
(cADPR)- evoked Ca2+ release. 4, nicotinic acid adenine dinucleotide phosphate (NAADP)
evoked Ca2+ release. 5, Ca2+ release evoked by sphingosine. 6, Ca2+ release from
mitochondria.
6.5.2 Influx of Ca2+ from the Extracellular Region
 The main Ca2+ influx channels are
* Voltage-gated channels are opened by a depolarization or
change in membrane potential.
* Ligand-gated channels are activated by binding of an
agonist to the exctracellular domain of the channel. Examples
are provided by the acetylcholine receptor and the N-methylD-asparate receptor.
* Mechanically activated channels are present on many cell
types and respond to mechanical stress.
 In addition we know of Ca2+ channels that are controlled by
Ga proteins and Ca2+ channels that are gated by
sphingolipids.
6.5.3 Removal and Storage of Ca2+
 The cytosolic Ca2+ concentration is generally only temporarily
and is often only locally increased during stimulation of cells.
 The cell possesses efficient Ca2+ transport systems, which
can rapidly transport Ca2+ back into the extracellular region or
into the storage organelles. Ca2+-ATPases, in particular, are
involved in draining the cytosol of Ca2+ back into the
extracellular region.
 The Ca2+-ATPases perform active transport of Ca2+ against its
concentration gradient, using the hydrolysis of ATP as an
energy source. Other transport systems in the plasma
membrane exchange Na+ ions for Ca2+.
 These Na+-Ca2+ exchange proteins are located especially in
muscle cells and in neurons. Ca2+-ATPases, which can fill the
empty Ca2+ storage, are also located in the membrane of the
endoplasmic reticulum.
6.6 Phosphatidyl Inositol Phosphates and PI3Kinase
 6.6.1 PI3-Kinases
 6.6.2 The Messenger Substance PtdIns(3,4,5)P3
 6.6.3 Akt Kinase and PtdIns(3,4,5)P3 Signaling
 6.6.4 Functions of PtIns(4,5)P3
6.6 Phosphatidyl Inositol Phosphates and PI3Kinase
 Several metabolic pathways lead from phosphatidyl inositol to
compounds with “second messenger” character. One main
pathway, the formation of diacylglycerol and Ins(1,4,5)P3 from
PtdIns(4,5)P2, has already been described in Section 6.4 and
Fig. 6.4. Other major compounds of regulatory importance
can be formed by phosphorylation at the 3’ position of the
inositol part of PtdIns.
 The reaction is catalyzed by a class of enzymes known as
phosphatidyl inositide 3-kinases (PI3-kinases). The PI3kinases phosphorylate various phosphatidyl inositol
compounds at the 3’ position. A major substrate is
PtdIns(4,5)P2, which is converted by PI3-kinase into
PtdIns(3,4,5)P3. This compound has an important function as
an intracellular messenger. PtdIns(3,4,5)P3 binds to PH
domains of various signaling proteins promoting their
membrane association. An overview of the function and
regulation of PI3-kinase. is given in Fig. 6.11.
Fig. 6.11 Pathways of PI3-kinase
activation.
PI3-kinase
can
be
activated by growth factor receptors,
either by direct interaction or via the
Ras protein. Another way of PI3kinase activation uses the bcsubunits of heterotrimeric G proteins
liberated upon activation of G proteincoupled receptors, GPCR.
The product of the PI3-kinase
reaction is PtdIns(3,4,5)P3 which
binds to PH domains of various
signaling proteins promoting their
membrane association and activation.
Overall, activation of PI3-kinase
stimulates
cell
growth
and
proliferation and inhibits apoptosis. A
suppressing effect is exerted by the
tumor suppressor PTEN which
hydrolyzes and thus inactivates
PtdIns(3,4,5)P3.
6.6.1 PI3-Kinases
 Many observations indicate that PI3-kinase functions as
a signal protein that receives signals on the cytoplasmic
side of the cell membrane and transmits them further,
although its primary role is to produce membranelocalized messenger substances.
 PI3-kinase is activated via three pathways (see Fig.
6.11).
6.6.1 PI3-Kinases
 Interaction with activated receptor tyrosine kinases
The SH2 domain of the p85 subunit mediates an
interaction with tyrosine residues on signal proteins
involved in transduction of growth-regulating signals.
Thus, binding of the PI3-kinase to tyrosine phosphate
residues of the activated PDGF receptor is observed.
Another binding partner is the insulin receptor substrate
(IRS).
In both cases, it is assumed that the binding of the SH2
domain of p85 to the tyrosine residue of the signal
protein serves to target the PI3-kinase to its membranelocalized substrate. Furthermore, binding of p85 to
phosphotyrosine residues of activated receptors appears
to be accompanied by an allosteric activation of the
catalytic subunit (Fig. 6.12).
Fig. 6.12
(A) Activation of PKB (also known as Akt
kinase)
by
membrane
translocation.
PtdIns(3,4,5)P3 generated in response to
growth factor stimulation serves as a binding
site for the PH domains of PDK1 and PKB.
Membrane translocation is accompanied by
release of an autoinhibition leading to
activation of PDK1 and PKB kinase activities.
Full
activation
of
PKB
requires
phosphorylation by PDK1. Activated PKB
phosphorylates a variety of target proteins
that prevent apoptotic death (Bad) and
regulate transcription (forkhead transcription
factors, FKHR1) and other metabolic
processes.
(B) Activation by a conformational change.
Binding of the SH2 domains of p85, the
regulatory subunit of PI-3 kinase to pTyr sites
on
activated
receptors
releases
an
autoinhibitory constraint that stimulates the
catalytic domain (p110). PI-3 kinase catalyzes
the phosphorylation of the 3’ positions of the
inositol ring of PtdIns(4)P and PtdIns(4,5)P2
to generate PtdIns(3,4)P2 and PtdIns(3,4,5)P3,
respectively.
6.6.1 PI3-Kinases
 Activation in the Ras pathway
The PI3-kinase has also been identified as a part of the Ras
signaling pathway. Signals originating from transmembrane
receptors can be transmitted from the Ras protein to PI3kinase. In this case, the PI3-kinase acts as the effector
molecule of the Ras protein.
 Activation by the Gβγ dimer
Gβγ dimers directly activate the PI3-kinase β and γ subtypes.
In this way, a variety of extracellular signals can be
transmitted via G protein-coupled receptors and G proteins to
PI3-kinase and its effectors.
6.6.2 The Messenger Substance PtdIns(3,4,5)P3
 The products of the PI3-kinase reaction are different
phosphoinositide derivatives phosphorylated at the 3
position, of which PtdIns(3,4,5)P3 has the greatest
regulatory importance. PtIns(3,4,5)P3, like cAMP, has the
function of a messenger substance that activates
effector molecules in the sequence for further signal
conduction.
 The concentration of PtdIns(3,4,5)P3 in the cell depends
both on the rates of synthesis by PI3-kinases and the
rates of hydrolysis of its phosphate residues. Several
inositol polyphosphate phosphatases have been
identified that remove the phosphates at position 3 or 5
of the inositol moiety.
6.6.3 Akt Kinase and PtdIns(3,4,5)P3 Signaling
 PtdIns(3,4,5)P3 formed by PI3-kinase regulates the
activity of a series of protein kinases, including the
Ser/Thr-specific Akt kinases, protein kinase C enzymes,
and the tyrosine-specific Tec kinases. Only the regulation
of Akt kinase will be discussed in the following.
 The first target protein of PtdIns(3,4,5)P3 to be
characterized was Akt kinase, also known as protein
kinase B (PKB). Akt kinase is a Ser/Thr-specfic protein
kinase which regulates multiple biological processes
including
glucose
metabolism,
apoptosis,
gene
expression, and cellular proliferation.
 The signaling pathway for Akt kinase shown in Fig. 6.12
illustrates the central role of PI3-kinase and
PtdIns(3,4,5)P3 in growth factor controlled signal paths
that lead from the cell membrane into the cytosol and the
nucleus.
6.7 Ca2+ as a Signal Molecule
6.7.1 Calmodulin as a Ca2+ Receptor
6.7.2 Target Proteins of Ca2+ /Calmodulin
6.7.3 Other Ca2+ Receptors
6.7 Ca2+ as a Signal Molecule
 Ca2+ is acentral signal molecule of the cell. Following a
hormonalor electrical stimulation, an increase in cytosolic
Ca2+ occurs, leading to initiation of other reactions in the
cell.
 As outlined above, this increase is limited in time and in
space and allows the formation of a variety of differently
shaped Ca2+ signals. Examples of Ca2+-dependent
reactions are numerous and affect many important
processes of the organism, including
– muscle contraction
– vision process
– cell proliferation
– secretion
– cell motility, formation of the cytoskeleton
6.7 Ca2+ as a Signal Molecule
 Ca2+ signals in the form of temporally and spatially
variable changes in Ca2+ concentration serve as
elements of intracellular signal conduction in many
signaling pathways. Three main paths for increase in
Ca2+ concentration stand out (Table 6.1):
– G-protein-mediated signaling pathways
– signaling pathways involving receptor tyrosine
kinases
– influx of Ca2+ via voltage- or ligand-gated Ca2+
channels.
Tab. 6.1 Receptors of the plasma membrane that mediate increase of intracellular
Ca2+.
6.7 Ca2+ as a Signal Molecule
 Direct activation of proteins
Many proteins have a specific binding site for Ca2+, and
their activity is directly dependent on Ca2+ binding. The
available Ca2+ concentration thus directly controls the
activity of these proteins (see Table 6.2).
Tab. 6.2 Ca2+ binding proteins.
6.7 Ca2+ as a Signal Molecule
 Binding to Ca2+ receptors
Another central mechanism of signal transduction via
Ca2+ is its binding to Ca2+ -binding proteins also known
as Ca2+ receptors. The receptor proteins function as
regulatory proteins that couple the Ca2+ signal to other
signaling proteins.
The Ca2+ receptors are Ca2+ sensors that activate target
proteins in response to changes in Ca2+ concentration.
Increases in Ca2+ above the concentration of the resting
state (ca. 10–7M) lead to specific binding of Ca2+ to Ca2+binding sites on the receptor and concomitant
conformational changes that modulate the interaction
with downstream target proteins.
6.7.1 Calmodulin as a Ca2+ Receptor
 The most widespread Ca2+ receptor is calmodulin.
Calmodulin is a small protein of ca. 150 amino. The
structure of the Ca2+ /calmodulin complex has two
globular domains that are separated by a long ahelical section (Fig. 6.13).
 Both globular domains have two binding sites for
Ca2+. Ca2+ is bound via a characteristic helix-loophelix structure, also known as an EF structure.
Similar EF structures are found in many, but not all,
Ca2+ -binding proteins.
Fig. 6.13 Comparison of different Ca2+/Calmodulin structures (from Hoeflich and Ikura,
2002). The figure illustrates the different conformations of calmodulin when bound to
target protein kinases. Calmodulin is shown in yellow and calcium ions are depicted in
blue. The interaction with the calmodulin binding domain of the protein kinases is
mediated by short helices shown in green and blue. CaM-CaMKII: Ca2+/calmodulindependent protein kinase II; CaM-CaMKK: Ca2+/calmodulin-dependent protein kinase
kinase; CaM-MLCK: Ca2+/calmodulin-dependent myosin light chain kinase; CaM-EF:
Ca2+/calmodulin-dependent edema factor, an adenylyl cyclase, from Bacillus anthracis.
6.7.1 Calmodulin as a Ca2+ Receptor
 From the structures of the substrates and their
complexes with Ca2+ /calmodulin, two main mechanisms
of substrate activation have emerged (Fig. 6.14).
 By one mechanism an autoinhibitory element is
displaced from the active site of the target enzyme
relieving autoinhibition.
 Another protein activation mechanism of Ca2+
/calmodulin uses a remodeling of the active site of the
target protein.
Fig. 6.14 Mechanisms of
activation
of
target
proteins
by
Ca2+/
calmodulin (after Hoeflich
and Ikura, 2002).
A)
Binding
of
Ca2+/calmodulin relieves
autoinhibition
(CaMkinases, calcineurin).
B)
Ca2+/calmodulin
remodels the active site
inducing
an
active
conformation
(anthrax
adenylyl cyclase).
C)
Ca2+/calmodulininduced dimerization of
K+-channels.
AID:
autoinhibition domain.
6.7.2 Target Proteins of Ca2+ /Calmodulin
 The Ca2+ /calmodulin complex is a
signal molecule that is involved in
many
signal
transduction
pathways. Ca2+ /calmodulin is
involved, e. g., in regulation of
proliferation, mitosis, neuronal
signal
transduction,
muscle
contraction
and
glucose
metabolism.
 Different calmodulin subtypes are
known which regulate different
target
proteins.
The
best
characterized target proteins are
the
calmodulin-dependent
adenylyl
cyclases,
phosphodiesterases, the protein
phosphatase calcineurin, protein
kinases like the CaM kinases, and
the myosin light chain kinase
6.8 Diacylglycerol as a Signal Molecule
 During cleavage of PtInsP2 by phospholipase C, two
signal molecules are formed, InsP2 and diacylglycerol.
Whilst InsP2 acts as a diffusible signal molecule in the
cytosol after cleavage, the hydrophobic diacylglycerol
remains in the membrane. Diacylglycerol can be
produced by different pathways, and it has at least two
functions (Fig. 6.15).
 Diacylglycerol is an important source for the release of
arachidonic acid, from which biosynthesis of
prostaglandins takes place. The glycerine portion of the
inositol phosphatide is often esterifed in the 2’ position
with arachidonic acid; arachidonic acid is cleaved off by
the action of phospholipases of type A2.
Fig. 6.15 Formation and function of diacylglycerol. The figure schematically shows two
main pathways for formation of diacylglycerol (DAG). DAG can be formed from
PtdInsP2 by the action of phospholipase C (PLC). Another pathway starts from
phosphatidyl choline. Phospholipase D (PL-D) converts phosphatidyl choline to
phosphatidic acid (Ptd), and the action of phosphatases results in DAG. Arachidonic
acid, the starting point of biosynthesis of prostaglandins and other intracellular and
extracellular messenger substances, can be cleaved from DAG. PKC: protein kinase C;
PtdIns: phosphatidyl inositol.
6.9 Other Lipid Messengers
 Ceramide
Ceramide is a lipophilic messenger that regulates diverse
signaling pathways involving apoptosis, stress response, cell
senescence, and differentiation. For the most part, ceramide’s
effects are antagonistic to cell growth and survival. The
starting point for the formation of ceramide is sphingomyelin,
which occurs especially in the outer layer of the plasma
membrane. Ceramide is produced from sphingomyelin by the
action of the enzyme sphingomyelinase (Fig. 6.16).
Sphingomyelinase has similar cleavage specificity to
phospholipase C, in that it cleaves an alcohol-phosphate
bond. Activation of sphingomyelinase is observed in response
to diverse stress challenges including irradiation, exposure to
DNA-damaging agents or treatment with pro-apoptotic ligands
like tumor necrosis factor a (TNFα). Because of these
properties, ceramide is a potent apoptogenic agent.
Fig. 6.16 Formation and
function of the messenger
substance ceramide. The
starting point for the
synthesis of ceramide is
sphingomyelin, which is
converted
to
phosphocholine
and
ceramide by the action of a
sphingomyelinase.
Sphingomyelinase
is
activated via a pathway
starting
from
tumor
necrosis factor α (TNFα)
and its receptor. Ceramide
serves as an activator of
protein kinases and protein
phosphatases. R1: fatty
acid side chain.
6.10 The NO Signaling Molecule
 6.10.1 Reactivity and Stability of NO
 6.10.2 Synthesis of NO
 6.10.3 Physiological Functions and Attack Points of
NO
6.10 The NO Signaling Molecule
 The biological importance of
nitrogen monoxide (NO) as a
messenger substance was
originally
recognized
in
connection with contraction
and relaxation of blood
vessels.
 In the meantime, it has
become clear that NO is a
universal
messenger
substance that is found in
nearly all living cells. NO
takes part in intercellular and
intracellular com-munication
in
higher
and
lower
eucaryotes and it is also
found in bacteria and in plant
cells.
6.10.1 Reactivity and Stability of NO
 NO is a radical that is water soluble and can cross
membranes fairly freely by diffusion. Because of its
radical nature, NO has a lifetime in aqueous solution
of only ca. 4 s. Important reaction partners of NO in
biological systems are oxygen O2, the O2–radical and
transition metals in free or complex form, e. g. Fe2+ in
heme.
 Furthermore, NO readily reacts with nucleophilic
centers in peptides and proteins, in particular with
the SH groups of Cys residues (Fig. 6.17).
Fig. 6.17 Reactions of NO in biological systems. NO reacts in biological
systems primarily with O2, with the superoxide anion O2-and with transition
metals (Me). The products of the reaction, -NOx, metal -NO adducts (Me-NO)
and peroxynitrite (OONO-) react further by nitrosylation of nucleophilic
centers. In the cell, these are especially–SH (or thiolate-S-) groups of peptides
and proteins (RS-).
6.10.2 Synthesis of NO
 NO is formed enzymatically from arginine with the
help of NO synthase, producing citrulline (Fig. 6.18).
 Citrulline and arginine are intermediates of the urea
cycle, and arginine can be regenerated from
citrulline by urea cycle enzymes.
Fig. 6.18 Biosynthesis of NO. The starting point of NO synthesis is arginine.
Arginine is converted by NO synthase, together with O2 and NADP, to NO and
citrulline. Arginine can be regenerated from citrulline via reactions of the urea
cycle.
6.10.3 Physiological Functions and Attack Points
of NO
 Toxic Action of NO and Nitrosative Stress
When NO is produced in excess amounts and in a less
than regulated fashion, nonspecific reactions with
various cell constituents including proteins, lipids and
DNA are observed. This situation has been termed
nitrosative stress in analogy to oxidative stress caused
by the generation of reactive oxygen species, ROS.
Nitration, nitrosation and oxidation of proteins, lipids and
DNA can occur under these conditions and can lead to
damage of cellular functions and eventually to cell death.
6.10.3 Physiological Functions and Attack Points
of NO
 Regulatory Function of NO
NO produced in a regulated way by enzymatic synthesis
is involved in the control of a wide array of cellular
functions including relaxation of blood vessels,
neurotransmission, cellular immune response and
apoptosis. Because of its high reactivity, NO can interact
and react with many effector proteins.
In Table 6.3, some important bioregulatory proteins are
summarized, for which direct modification by NO has
been shown. Two target proteins should be mentioned,
in particular:
Tab. 6.3 Regulatory attack points of NO. Proteins are included for which a direct
regulation by NO is assumed (according to Stammler, 1994). Direct evidence of
regulatory nitrosylation has only been shown for hemoglobin, however.
6.10.3 Physiological Functions and Attack Points
of NO
 NO-sensitive Guanylyl Cyclase
The first cellular target of NO to be identified was a
specific isoform of guanylyl cyclase. Stimulation of NO
synthase leads to activation of a cytoplasmic NO
sensitive guanylyl cyclase. Activation is achieved by
binding of NO to a heme group of the enzyme. The
associated increase in the cGMP level has multiple
consequences.
The cGMP can stimulate cGMP-dependent protein
kinases; it can also open cGMP controlled ion channels.
As a consequence, an increase in the intracellular Ca2+
concentration takes place and a Ca2+ signal is produced.
NO can influence both protein phosphorylation and
InsP3/diacylglycerol and Ca2+ metabolism by this
mechanism and activate a broad palette of biochemical
reactions in the cell.
6.10.3 Physiological Functions and Attack Points of
NO
 S-Nitrosylation of Hemoglobin
Hemoglobin was the first protein for which a regulatory
action of S-nitrosylation was clearly shown (Fig. 6.19).
Hemoglobin (Hb) is a tetramer, composed of two α and
two β chains. In man, each chain has a heme system,
and the β chains have a reactive cysteine group (Cys93).
The Hb may bind NO at two sites. Firstly, NO can bind to
the Fe(II) of the heme grouping; secondly, NO can
accumulate at Cys93 of the β chain by forming an Snitrosyl.
Fig. 6.19 Scheme of the function of
nitroso-hemoglobin. NO synthase is
activated by a stimulatory signal (e. g.
a Ca2+ signal) and NO is formed. The
NO is transferred by direct or indirect
means
to
hemoglobin
in
the
erythrocytes. NO can bind to
hemoglobin as a Fe-NO complex with
the heme, and it can exist as a Snitroso derivative of Cys93 of the β
subunit (Cys93β). Hemoglobin-bound
NO can be transferred to the anion
exchange protein AE1, forming SNOAE1. This can transfer NO activity out
of the red blood cell and into the
vessel wall. In this form, and
transferred to low molecular weight
SH compounds such as glutathione
(GSH) or free cysteine (Cys). The
resulting nitrosyl compounds CysSNO and G-SNO can diffuse to target
proteins and pass the NO signal on to
these. The figure does not show the
complex regulation of NO compounds
LOGO