Chapter 05
Lecture Outline*
Control of Cells by
Chemical Messengers
Eric P. Widmaier
Boston University
Hershel Raff
Medical College of Wisconsin
Kevin T. Strang
University of Wisconsin - Madison
*See PowerPoint Image Slides for all
figures and tables pre-inserted into
PowerPoint without notes.
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
1
Receptors
• Chemical messengers bind to proteins called
receptors.
• Most chemical messengers are water-soluble
and bind to receptors located at the plasma
membrane.
• Some messengers like steroids are lipidsoluble and bind to an intracellular receptor.
2
Transmembrane Receptor Protein
Fig. 5-1
3
Receptor Specificity
Remember that not
all cells express the
same receptors.
This selective
expression leads to
specificity in the
systems.
The response of
individual cells with
the same receptor
also vary based on
the cell type,
intracellular
signaling cascade
coupling, and other
simultaneous signals
being received.
Fig. 5-2
4
Receptor Affinity
Fig. 5-3
5
6
Table 5.1 continued
7
Signal Transduction Pathways
8
Pathways Initiated by Lipid-Soluble
Messengers
Fig. 5-4
9
Pathways Initiated by Lipid-Soluble Messengers
• Critical Points to remember:
1. Lipid messengers can diffuse through the plasma
membrane.
2. They have intracellular receptors.
3. The receptors bind directly to recognized
sequences in the DNA and alter gene
transcription.
4. This is a slower response compared to membrane
receptors, but it is a sustained response.
10
Pathways Initiated by Water-Soluble Messengers
Fig. 5-5
11
Pathways Initiated by Membrane-Bound Receptors
• Critical Points to remember:
1. This is a broad range of receptors: ion channels,
G-protein coupled receptors, receptors with
intrinsic kinase activity, etc.
2. These receptors activate intracellular signaling
cascades that affect cell function.
3. These receptors can activate downstream
mediators which affect DNA transcription but
also have many other effects in the cell.
4. This is a faster response compared to
lipid/steroid receptors, but it is a less sustained
response.
12
Receptors that are Ligand-Gated Ion Channels
• Activation of the receptor by a first messenger (ligand) results
in a conformational change of the receptor so it forms an open
channel through the plasma membrane.
• Because the opening of ion channels has been compared to the
opening of a gate in a fence, these types of channels are known
as ligand-gated ion channels.
• The opening of ligand-gated ion channels in response to
binding of a ligand results in an increase in the net diffusion
across the plasma membrane of one or more types of ions
specific to that channel.
• This often results in a change in the membrane potential.
– Examples: Na+, K+, Ca2+ channels
13
Receptors that Function as Enzymes
• Some receptors ( like the insulin receptor) have intrinsic
enzyme activity.
• Most of these receptors that possess intrinsic enzyme activity
are all protein kinases that specifically phosphorylate the
amino acid tyrosine (receptor tyrosine kinases).
• The typical sequence of events for receptors with intrinsic
tyrosine kinase activity is:
1. The binding of a specific messenger to the receptor
changes the conformation of the receptor so that its
enzymatic portion, located on the cytoplasmic side of the
plasma membrane, is activated.
14
Receptors that Function as Enzymes
2. This results in autophosphorylation of the receptor (the
receptor phosphorylates its own tyrosine groups).
3. The newly created phosphotyrosines on the cytoplasmic
portion of the receptor then serve as docking sites for
cytoplasmic proteins.
4. The bound docking proteins then bind and activate other
proteins, which in turn activate one or more signaling
pathways within the cell.
• The common denominator of these pathways is that they all
involve activation of cytoplasmic proteins by phosphorylation.
15
Signaling
• The number of kinases that mediate these
phosphorylations can be very large, and their
names constitute a veritable alphabet soup—
RAF, MEK, MAPKK, and many others.
• Most of the receptors with intrinsic tyrosine
kinase activity bind ligands that typically
influence cell proliferation and differentiation,
and are often called growth factors.
16
cGMP
• The one major exception to the generalization is Guanylyl
Cyclase.
• Guanylyl cyclase is a receptor that acts to catalyse the
formation, in the cytoplasm, of a molecule known as
cyclic GMP (cGMP).
• In turn, cGMP functions as a second messenger to
activate a protein kinase called cGMP-dependent
protein kinase.
• This kinase phosphorylates specific proteins that then
mediate the cell’s response to the original messenger.
17
cGMP cont’d
• Receptors that function both as ligand-binding molecules
and as guanylyl cyclases are present in high amounts in
the retina of the vertebrate eye, where they are important
for processing visual inputs.
• This signal transduction pathway is used by only a small
number of messengers and should not be confused with
the much more prevalent cAMP system.
• Also, in certain cells, guanylyl cyclase enzymes are
present in the cytoplasm. In these cases, a first
messenger—nitric oxide—diffuses into the cell and
combines with the guanylyl cyclase there to trigger the
formation of cGMP.
18
Receptors that Interact with Cytoplasmic Kinases
• There are several families of cytoplasmic protein kinases:
src, JAKs, etc.
• These receptors do not have intrinsic kinase activity, but
must use a cytoplasmic kinase.
• The binding of a ligand to the receptor causes a
conformational change in the receptor that leads to
activation of the JAK kinase.
• Janus kinases (JAK) are a commonly used cytoplasmic
kinase.
The Janus kinases are a family of 4 kinases that are all tyrosine
kinases. They are differentially expressed among the tissues in
the body.
19
JAK Kinases
• Different receptors associate with different members of the JAK
kinase family, and the different JAK kinases phosphorylate
different target proteins, many of which act as transcription
factors.
• JAK’s traditional targets are the Signal Transducers of Activated
transcription (STATs). However, they have also been shown to
interact with other proteins.
• The result of these pathways is the synthesis of new proteins,
which mediate the cell’s response to the first messenger.
• Signaling by cytokines—proteins secreted by cells of the immune
system that play a critical role in immune defenses—occurs
primarily via receptors linked to JAK kinases.
20
G Protein-Coupled Receptors
• Bound to the inactive receptor is a protein complex
located on the cytosolic surface of the plasma membrane
and belonging to the family of heterotrimeric (containing
three different subunits) proteins known as G proteins.
• All G proteins contain three subunits, called the alpha,
beta and gamma subunits. The alpha subunit can bind
GDP and GTP. The beta and gamma subunits help anchor
the alpha subunit in the membrane.
• The binding of a ligand to the receptor changes the
conformation of the receptor.
21
G Protein-Coupled Receptors
• This activated receptor increases the affinity of the alpha
subunit of the G protein for GTP.
• When bound to GTP, the alpha subunit dissociates from
the beta and gamma subunits.
• This dissociation allows the activated alpha subunit to
link up with still another plasma membrane protein, either
an ion channel or an enzyme.
• These ion channels and enzymes are termed plasma
membrane effector proteins because they mediate the next
steps in the sequence of events leading to the cell’s
response.
22
G Protein-Coupled Receptors
• In essence, then, a G protein serves as a switch to couple a receptor to an ion channel or
to an enzyme in the plasma membrane.
• G proteins can either be stimulatory or inhibitory.
• Once the alpha subunit of the G protein activates its effector protein, a GTP-ase activity
inherent in the alpha subunit cleaves the GTP into GDP plus Pi.
• This cleavage renders the alpha subunit inactive, allowing it to recombine with its beta
and gamma subunits.
• There are several subfamilies of plasma membrane G proteins, each with multiple
distinct members, and a single receptor may be associated with more than one type of G
protein. Moreover, some G proteins may couple to more than one type of plasma
membrane effector protein. In this way, a first-messenger-activated receptor, via its Gprotein couplings, can call into action a variety of plasma membrane effector proteins
such as ion channels and enzymes. These molecules can, in turn, induce a variety of
cellular events.
• To illustrate some of the major points concerning G proteins, plasma membrane effector
proteins, second messengers, and protein kinases, the next two sections describe the two
most important effector protein enzymes regulated by G proteins—adenylyl cyclase and
phospholipase C. In addition, the subsequent portions of the signal transduction
pathways in which they participate are described.
23
G Protein-Coupled Receptors
• There are several subfamilies of plasma membrane G
proteins, each with multiple distinct members, and a
single receptor may be associated with more than one
type of G protein.
Examples: Gs, Gi, Gq, Gα
• Moreover, some G proteins may couple to more than one
type of plasma membrane effector protein.
• G-protein coupled receptors are the most numerous type
of receptor family and have a large variety of signaling
pathways associated with them.
24
Adenylyl Cyclase and Cyclic AMP
• Activation of the receptor by the binding of ligand (for
example, the hormone epinephrine) allows the receptor to
activate its associated G protein (Gs ; “stimulatory”).
• This causes Gs to activate its effector protein, the
membrane enzyme called adenylyl cyclase (also known
as adenylate cyclase).
• The activated adenylyl cyclase catalyzes the conversion
of cytosolic ATP molecules to cyclic 3´,5´-adenosine
monophosphate, or cyclic AMP (cAMP).
• Cyclic AMP then acts as a second messenger.
25
Adenylyl Cyclase and Cyclic AMP
• The action of cAMP eventually terminates when it is
broken down to noncyclic AMP, a reaction catalyzed by
the enzyme cAMP phosphodiesterase.
• Thus, the cellular concentration of cAMP can be changed
either by altering the rate of its messenger-mediated
generation or the rate of its phosphodiesterase-mediated
breakdown.
• Caffeine and theophylline, the active ingredients of coffee
and tea, are widely consumed stimulants that work partly
by inhibiting phosphodiesterase activity, which results in
prolonging the actions of cAMP within a cell.
26
Adenylyl Cyclase and Cyclic AMP
• Inside the cell, cAMP binds to and activates an enzyme
known as cAMP-dependent protein kinase (PKA).
PKA then phosphorylates downstream targets.
• Examples: epinephrine acts via the cAMP pathway on fat
cells to stimulate the breakdown of triglyceride, a process
that is mediated by one particular phosphorylated
enzyme. In the liver, epinephrine acts via cAMP to
stimulate both glycogenolysis and gluconeogenesis,
processes that are mediated by phosphorylated enzymes
that differ from those in fat cells.
27
Gi Proteins
• Not all G proteins stimulate cAMP; some inhibit adenylyl
cyclase.
• This inhibition results in less, rather than more, generation of
cAMP.
• This occurs because these receptors are associated with a
different G protein known as Gi (“inhibitory’’).
• Activation of Gi causes the inhibition of adenylyl cyclase.
The result is to decrease the concentration of cAMP in the
cell and thereby the phosphorylation of key proteins inside
the cell.
28
G-protein Coupled Receptors: cAMP
Fig. 5-6 29
Signal Amplification
Fig. 5-8 30
Actions of cAMP-dependent Kinases
Fig. 5-9 31
Phospholipase C, Diacylglycerol, and
Inositol Trisphosphate
• This system uses the G protein called Gq.
• Activated Gq then activates a plasma membrane effector
enzyme called phospholipase C (PLC).
• This enzyme catalyzes the breakdown of a plasma
membrane phospholipid known as phosphatidylinositol
bisphosphate, abbreviated PIP2, to diacylglycerol
(DAG) and inositol tris-phosphate (IP3).
32
PLC, DAG, IP3
• Both DAG and IP3 then function as second messengers.
• DAG activates a class of protein kinases known
collectively as protein kinase C (PKC), which then
phosphorylate a large number of other proteins, leading
to the cell’s response.
• There are currently 13 known isoforms of PKC which
contribute to the large variety of cellular responses
observed.
33
IP3
• IP3 binds to receptors located on the endoplasmic
reticulum.
• These receptors are ligand-gated Ca2+ channels which when
bound to IP3 open and result in increased cytosolic Ca2+
concentration.
• This increased Ca2+ concentration then continues the
sequence of events leading to the cell’s response.
• One of the actions of Ca2+ is to help activate some forms of
protein kinase C.
34
G-protein Coupled Receptors: DAG & IP3
Fig. 5-10 35
Control of Ion Channels by G Proteins
• An ion channel can be the effector protein for a G
protein and it can be directly or indirectly regulated.
• In direct regulation, the G protein interacts with the
channel without any second messengers being
involved.
• In indirect regulation, you have involvement of second
messengers. Example: PKA phosphorylates a plasma
membrane ion channel, thereby causing it to open.
36
Ca2+ as a Second Messenger
• Ca2+ functions as a second messenger in many pathways.
• Ca2+ can be either increased or decreased cytosolically to elicit
a cellular response (change membrane potential). Ca2+ also has
direct actions on other signaling proteins.
• By means of active-transport systems in the plasma membrane
and cell organelles, Ca2+ is maintained at an extremely low
concentration in the cytosol.
• Consequently, there is always a large electrochemical gradient
favoring diffusion of Ca2+ into the cytosol via Ca2+ channels
found in both the plasma membrane and the endoplasmic
reticulum.
37
Ca2+ as a Second Messenger
• A stimulus to the cell can alter this steady state by
influencing the active-transport systems and/or the ion
channels, resulting in a change in cytosolic Ca2+
concentration.
• There are Ca2+ channels in the plasma membrane that are
opened directly by an electrical stimulus to the membrane
(voltage gated channels).
• Extracellular Ca2+ entering the cell via these channels can, in
certain cells, bind to Ca2+ sensitive channels in the
endoplasmic reticulum and open them (Ca2+ induced Ca2+
release).
38
Ca2+ as a Second Messenger
• Besides the changes to the membrane potential, Ca2+ also acts by
its ability to bind to various cytosolic proteins, altering their
conformation and thereby activating their function.
• One of the most important of these is a protein, found in virtually
all cells, known as calmodulin.
• On binding with Ca2+ calmodulin changes shape, and this allows
calcium-calmodulin to activate or inhibit a large variety of
enzymes and other proteins, many of them protein kinases.
• Other proteins Ca2+ binds include: troponin, nitric oxide synthase,
PYK2.
39
Calmodulin
Fig. 5-11
40
Arachidonic Acid and Eicosanoids
• The eicosanoids are a family of molecules produced from
the polyunsaturated fatty acid arachidonic acid (AA)
which is present in plasma membrane phospholipids.
• The eicosanoids include the cyclic endoperoxides, the
prostaglandins, the thromboxanes, and the leukotrienes.
They are generated in many kinds of cells in response to an
extracellular signal.
• The synthesis of eicosanoids begins when an appropriate
stimulus binds to its receptor and activates phospholipase
A2 (PLA2).
41
Arachidonic Acid and Eicosanoids
• PLA2 splits off AA from the membrane phospholipids, and
the AA can then be metabolized by two pathways.
• One pathway is initiated by an enzyme called
cyclooxygenase (COX) and leads ultimately to formation
of the cyclic endoperoxides, prostaglandins, and
thromboxanes.
• The other pathway is initiated by the enzyme lipoxygenase
and leads to formation of the leukotrienes.
• Within both of these pathways, synthesis of the various
specific eicosanoids is enzyme-mediated.
42
Arachidonic Acid and Eicosanoids
• Each of the major eicosanoid subdivisions contains more
than one member, as indicated by the use of the plural in
referring to them (prostaglandins, for example).
• On the basis of structural differences, the different
molecules within each subdivision are designated by a
letter—for example, PGA and PGE for prostaglandins of
the A and E types—which then may be further
subdivided—for example, PGE2.
43
Arachidonic Acid and Eicosanoids
• Eicosanoids may in some cases act as intracellular messengers,
but more often they are released immediately and act locally
(paracrine and autocrine agents).
• After they act, they are quickly metabolized by local enzymes
to inactive forms. The eicosanoids exert a wide array of
effects, particularly on blood vessels and in inflammation.
• Because AA transduces a signal from a messenger and its
receptor into a cellular response (production and secretion of
eicosanoids), it is sometimes considered a second messenger
as well as a substrate to be converted into other products.
44
Arachidonic Acid and Eicosanoids
• Some of the most commonly used drugs influence the eicosanoid
pathway.
• Aspirin inhibits cyclooxygenase blocking the synthesis of the
endoperoxides, prostaglandins, and thromboxanes.
• It and other drugs that also block cyclooxygenase are collectively
termed nonsteroidal anti-inflammatory drugs (NSAIDs).
• Their major uses are to reduce pain, fever, and inflammation. The
term nonsteroidal distinguishes them from synthetic
corticosteroids (hormones made by the adrenal glands) that are
used in large doses as anti-inflammatory drugs; these steroids
inhibit phospholipase A2 and block the production of all
eicosanoids.
45
AA Pathways
Fig. 5-12
46
47
Cessation of Activity in Signal Transduction Pathways
• Once initiated, signal transduction pathways are eventually shut off
because chronic overstimulation of a cell can in some cases be
detrimental.
• The key event is usually the cessation of receptor activation.
Responses to messengers are transient events that persist only briefly,
and subside when the receptor is no longer bound to the ligand.
• A major way that receptor activation ceases is by a decrease in the
concentration of first messenger molecules in the region of the
receptor.
• This occurs as enzymes in the vicinity metabolize the first messenger,
as the first messenger is taken up by adjacent cells, or as it simply
diffuses away.
48
Cessation of Activity in Signal
Transduction Pathways
• Receptors can be inactivated in at least three other ways:
1. The receptor becomes chemically altered (usually by
phosphorylation), which may lower its affinity for a first
messenger, and so the messenger is released.
2. Phosphorylation of the receptor may prevent further G protein
binding to the receptor.
3. Plasma membrane receptors may be removed when the
combination of first messenger and receptor is taken into the
cell by endocytosis.
49
Interactions of Signal Transduction Pathways
• It is essential to recognize that the pathways do not exist in
isolation but may be active simultaneously in a single cell,
undergoing complex interactions.
• This is possible because a single first messenger may trigger
changes in the activity of more than one pathway and, much more
importantly, because many different first messengers—often
dozens—may simultaneously influence a cell.
• Moreover, a great deal of “cross-talk” can occur at one or more
levels among the various signal transduction pathways. For
example, active molecules generated in the cAMP pathway can
alter the of receptors and signaling molecules generated by other
pathways.
50