For Isha GPCRs

13.
3 • G Protein–Coupled Receptors That Activate or Inhibit Adenylyl Cyclase 545
in turn, regulate the activities of enzymes and nonenzy- (GPCRs) contain seven membrane-spanning regions with
matic proteins. their N-terminal segment on the exoplasmic face and their
C-terminal segment on the cytosolic face of the plasma
■ Conserved proteins that act in many signal-transduction
membrane (Figure 13-10). The GPCR family includes recep-
pathways include monomeric and trimeric G proteins (see
tors for numerous hormones and neurotransmitters, light-
Figure 13-8) and protein kinases and phosphatases.
activated receptors (rhodopsins) in the eye, and literally
■ Cytosolic proteins that contain multiple PDZ or other thousands of odorant receptors in the mammalian nose.
protein-binding domains cluster receptors and other pro-
teins within the plasma membrane, as occurs in post-
synaptic cells (see Figure 13-9).
NH3+
■ Many receptors and signal-transduction proteins cluster E1 E2 E3 E4
in caveolin-containing lipid rafts. Such clustering may fa- Exterior
cilitate interaction between signaling proteins, thus en-
hancing signal transduction.
H1 H2 H3 H4 H5 H6 H7
■ Rapid termination of signaling once a particular ligand
is withdrawn and receptor desensitization at high ligand
concentrations or after prolonged exposure help cells re- Cytosol COO
−
C1 C2 C4
spond appropriately under different circumstances.
C3 G protein
interaction
13.3 G Protein–Coupled ▲ FIGURE 13-10 Schematic diagram of the general

structure of G protein–coupled receptors. All receptors of
Receptors That Activate this type have the same orientation in the membrane and
or Inhibit Adenylyl Cyclase contain seven transmembrane
-helical regions (H1–H7), four
extracellular segments (E1–E4), and four cytosolic segments
We now turn our attention to the very large group of cell- (C1–C4). The carboxyl-terminal segment (C4), the C3 loop, and,
surface receptors that are coupled to signal-transducing in some receptors, also the C2 loop are involved in interactions
trimeric G proteins. All G protein–coupled receptors with a coupled trimeric G protein.
TABLE 13-1 Major Classes of Mammalian Trimeric G Proteins and Their Effectors*
G
Class Associated Effector 2nd Messenger Receptor Examples
Gs
Adenylyl cyclase cAMP (increased) -Adrenergic (epinephrine)
receptor; receptors for
glucagon, serotonin,
vasopressin
Gi
Adenylyl cyclase cAMP (decreased)
1-Adrenergic receptor
K channel (G activates Change in membrane Muscarinic acetylcholine
effector) potential receptor
Golf
Adenylyl cyclase cAMP (increased) Odorant receptors in nose
Gq
Phospholipase C IP3, DAG (increased)
2-Adrenergic receptor
Go
Phospholipase C IP3, DAG (increased) Acetylcholine receptor in
endothelial cells
Gt
cGMP phosphodiesterase cGMP (decreased) Rhodopsin (light receptor) in
rod cells
*A given G
subclass may be associated with more than one effector protein. To date, only one major Gs
has been identified, but multiple Gq
and
Gi
proteins have been described. Effector proteins commonly are regulated by G
but in some cases by G or the combined action of G
and G .
IP3 inositol 1,4,5-trisphosphate; DAG 1,2-diacylglycerol.
SOURCES: See L. Birnbaumer, 1992, Cell 71:1069; Z. Farfel et al., 1999, New Eng. J. Med. 340:1012; and K. Pierce et al., 2002, Nature Rev. Mol.
Cell Biol. 3:639.
546 CHAPTER 13 • Signaling at the Cell Surface
The signal-transducing G proteins contain three subunits Hormone

designated
, , and . During intracellular signaling the
Exterior
and subunits remain bound together and are usually re-
Trimeric Gs protein
ferred to as the G subunit. The G
subunit is a GTPase Inactive Inactive
receptor
switch protein that alternates between an active (on) state Cytosol
effector
with bound GTP and an inactive (off) state with bound GDP Gα
G
(see Figure 13-8). Stimulation of a coupled receptor causes Gγ Gβ D
RESTING
activation of the G protein, which in turn modulates the ac- STATE
P
tivity of an associated effector protein. Although the effec-
tor protein most commonly is activated by G
·GTP, in some
cases it is inhibited. Moreover, depending on the cell and lig-
and, the G subunit, rather than G
·GTP, may transduce the Active
signal to the effector protein. In addition, the activity of sev- receptor
Gβγ
eral different effector proteins is controlled by different Gα
GPCR-ligand complexes. All effector proteins, however, are G
D
either membrane-bound ion channels or enzymes that cat- P
alyze formation of second messengers (e.g., cAMP, DAG, 1 Binding of hormone induces a conformational
change in receptor
and IP3). These variations on the theme of GPCR signaling
arise because multiple G proteins are encoded in eukaryotic
genomes. The human genome, for example, encodes 27 dif-
ferent G
, 5 G, and 13 G subunits. So far as is known, the
different G subunits function similarly. Table 13-1 sum-
marizes the functions of the major classes of G proteins with Gβγ
Gα
different G
subunits. G
In this section, we first discuss how GPCR signals are D
P
transduced to an effector protein, a process that is similar for Activated receptor binds to Gα subunit
all receptors of this type. Then we focus on pathways in 2
which cAMP is the second messenger, using the epinephrine-
stimulated degradation of glycogen as an example.
The G Subunit of G Proteins Cycles Gβγ Gα

Between Active and Inactive Forms G
T
Figure 13-11 illustrates how G protein–coupled receptors P
transduce signals from extracellular hormones to associated 3 Binding induces conformational change in Gα; bound GDP
effector proteins. Both the G
and G subunits are linked to dissociates and is replaced by GTP; Gα dissociates from Gβγ
the membrane by covalently attached lipids. In the resting
state, when no ligand is bound to the receptor, the G
sub-
unit is bound to GDP and complexed with G . Binding of
the normal hormonal ligand (e.g., epinephrine) or an ago- Active
effector
Gβγ Gα
G
T
P
FIGURE 13-11 Operational model for ligand-induced 4 Hormone dissociates from receptor; Gα binds to effector,
activation of effector proteins associated with G protein– activating it
coupled receptors. The G
and G subunits of trimeric G proteins
are tethered to the membrane by covalently attached lipid
molecules (wiggly black lines). Following ligand binding, dissociation
of the G protein, and exchange of GDP with GTP (steps 1 – 3 ),
the free G
·GTP binds to and activates an effector protein (step 4 ).
Gβγ
Hydrolysis of GTP terminates signaling and leads to reassembly Gα
of the trimeric form, returning the system to the resting state G
D
(step 5 ). Binding of another ligand molecule causes repetition of P
the cycle. In some pathways, the effector protein is activated by 5 Hydrolysis of GTP to GDP causes Gα to dissociate
the free G subunit. from effector and reassociate with Gβγ
13.3 • G Protein–Coupled Receptors That Activate or Inhibit Adenylyl Cyclase 547
(a) (b)
cAMP
Chemoattractant cAMP
Video: Chemotaxis of a Single Dictyostelium Cell to the
MEDIA CONNECTIONS
Fluorescence of yellow light
cAMP
(fraction of maximum)
Inactive Active
added
receptor receptor
YFP Gβγ YFP Gβγ Gα 1.0
Gα
G G
CFP D T
Fluorescence P P
CFP
527 nm 0.8
Fluorescence
(yellow)
energy
transfer Excitation light Fluorescence
440 nm 490 nm Excitation light
(cyan) 440 nm 0 15 30 45 60
Time (s)
▲ EXPERIMENTAL FIGURE 13-12 Receptor-mediated acteristic of YFP. However, if ligand binding leads to
activation of coupled G proteins occurs within a few seconds dissociation of the G
and G subunits, then fluorescence
of ligand binding in living cells. The amoeba Dictyostelium energy transfer cannot occur. In this case, irradiation of
discoideum was transfected with genes encoding two fusion cells at 440 nm causes emission of 490-nm light (cyan)
proteins: a G
fused to cyan fluorescent protein (CFP), a mutant characteristic of CFP (right). (b) Plot of the emission of
form of green fluorescent protein (GFP), and a G fused to an- yellow light (527 nm) from a single transfected amoeba
other GFP variant, yellow fluorescent protein (YFP). CFP normally cell before and after addition of cyclic AMP (arrows), the
fluoresces 490-nm light; YFP, 527-nm light. (a) When CFP and YFP extracellular ligand for the GPCR in these cells. The drop
are nearby, as in the resting G
·G complex, fluorescence energy in fluorescence, which results from the dissociation of
transfer can occur between CFP and YFP (left). As a result, irradi- the G
-CFP fusion protein from the G -YFP fusion
ation of resting cells with 440-nm light (which directly excites protein, occurs within seconds of cAMP addition. [Adapted
CFP but not YFP) causes emission of 527-nm (yellow) light, char- from C. Janetopoulos et al., 2001, Science 291:2408.]
nist (e.g., isoproterenol) to the receptor changes its confor- occurs with GTP. That is because once the GDP bound to G
mation, causing it to bind to the G

subunit in such a way is displaced by the nonhydrolyzable GTP analog, it remains
that GDP is displaced from G
and GTP becomes bound. permanently bound to G
. Because this complex is as func-
Thus the activated ligand-bound receptor functions as a GEF tional as the normal G
·GTP complex in activating the ef-
for the G
subunit (see Figure 3-29). fector protein, the effector remains permanently active.
Once the exchange of nucleotides has occurred, the The GPCR-mediated dissociation of trimeric G proteins
G
·GTP complex dissociates from the G subunit, but both recently has been detected in living cells. These studies have
remain anchored in the membrane. In most cases, G
·GTP exploited the phenomenon of fluorescence energy transfer,
then interacts with and activates an associated effector pro- which can change the wavelength of emitted fluorescence
tein, as depicted in Figure 13-11. This activation is short- when two fluorescent proteins interact. Figure 13-12 shows
lived, however, because GTP bound to G
is hydrolyzed to how this experimental approach has demonstrated the
GDP in seconds, catalyzed by a GTPase enzyme that is an in- dissociation of the G
·G complex within a few seconds of
trinsic part of the G
subunit. The resulting G
·GDP quickly ligand addition, providing further evidence for the model of
reassociates with G , thus terminating effector activation. In G protein cycling. This general experimental protocol can
many cases, a protein termed RGS (regulator of G protein be used to follow the formation and dissociation of other
signaling) accelerates GTP hydrolysis by the G
subunit, re- protein-protein complexes in living cells.
ducing the time during which the effector remains activated.
Early evidence supporting the model shown in Figure
Epinephrine Binds to Several Different
13-11 came from studies with compounds that can bind to
G
subunits as well as GTP does, but cannot be hydrolyzed G Protein–Coupled Receptors
by the intrinsic GTPase. In these compounds the P–O–P Epinephrine is particularly important in mediating the body’s
phosphodiester linkage connecting the and phosphates of response to stress, such as fright or heavy exercise, when all
GTP is replaced by a nonhydrolyzable P–CH2–P or P–NH–P tissues have an increased need to catabolize glucose and fatty
linkage. Addition of such a GTP analog to a plasma- acids to produce ATP. These principal metabolic fuels can
membrane preparation in the presence of the natural ligand be supplied to the blood in seconds by the rapid breakdown
or an agonist for a particular receptor results in a much of glycogen to glucose in the liver (glycogenolysis) and of tri-
longer-lived activation of the associated effector protein than acylglycerols to fatty acids in adipose cells (lipolysis).
In mammals, the liberation of glucose and fatty acids can lease of bound GDP, thus locking Gi
in the inactive state.
be triggered by binding of epinephrine (or norepinephrine) to This inactivation of Gi leads to an increase in cAMP in ep-
-adrenergic receptors on the surface of hepatic (liver) and adi- ithelial cells of the airways, promoting loss of fluids and elec-
pose cells. Epinephrine bound to -adrenergic receptors on trolytes and mucus secretion. ❚
heart muscle cells increases the contraction rate, which in-
creases the blood supply to the tissues. In contrast, epinephrine
Critical Functional Domains in Receptors and
stimulation of -adrenergic receptors on smooth muscle cells
of the intestine causes them to relax. Another type of epineph- Coupled G Proteins Have Been Identified
rine receptor, the 2-adrenergic receptor, is found on smooth As noted already, all G protein–coupled receptors contain seven
muscle cells lining the blood vessels in the intestinal tract, skin, transmembrane
helices and presumably have a similar three-
and kidneys. Binding of epinephrine to these receptors causes dimensional structure. Studies with chimeric adrenergic recep-
the arteries to constrict, cutting off circulation to these periph- tors, like those outlined in Figure 13-13, suggest that the long
eral organs. These diverse effects of epinephrine are directed to C3 loop between
helices 5 and 6 is important for interactions
a common end: supplying energy for the rapid movement of between a receptor and its coupled G protein. Presumably, lig-
major locomotor muscles in response to bodily stress. and binding causes these helices to move relative to each other.
Although all epinephrine receptors are G protein– As a result, the conformation of the C3 loop connecting these
coupled receptors, the different types are coupled to different G two helices changes in a way that allows the loop to bind and
proteins. Thus in addition to their physiological importance,
these receptors are of interest because they trigger different in-
Effect on
tracellular signal-transduction pathways. Both subtypes of -
adenylyl cyclase
adrenergic receptors, termed 1 and 2, are coupled to a Exterior NH3+
stimulatory G protein (Gs ) that activates the membrane-bound
enzyme adenylyl cyclase (see Table 13-1). Once activated, 1 2 3 4 5 6 7 Inhibits (binds Gi)
adenylyl cyclase catalyzes synthesis of the second messenger Cytosol
cAMP. That binding of epinephrine to -adrenergic receptors COO−
induces a rise in cAMP has been demonstrated in functional α 2-Adrenergic receptor (wild type)
expression assays like that depicted in Figure 13-6. When
cloned cDNA encoding the -adrenergic receptor is transfected NH3+
into receptor-negative cells, the transfected cells accumulate
cAMP in response to epinephrine stimulation. Similar experi- 1 2 3 4 5 6 7 Activates (binds Gs)
ments in which mutant receptors are expressed have helped to
define the functions of specific amino acids in binding hor- COO−
mones and activating different G proteins. β 2-Adrenergic receptor (wild type)
The two subtypes of
-adrenergic receptors,
1 and
2,
are coupled to different G proteins. The
1-adrenergic recep- NH3+
tor is coupled to a Gi protein that inhibits adenylyl cyclase,
the same effector enzyme associated with -adrenergic recep- 1 2 3 4 5 6 7 Activates (binds Gs)
tors. In contrast, the Gq protein coupled to the
2-adrenergic
receptor activates a different effector enzyme that generates COO−
different second messengers (see Section 13.5). Chimeric receptor 1
Some bacterial toxins contain a subunit that pene- NH3+

trates the plasma membrane of cells and catalyzes
a chemical modification of Gs
·GTP that prevents 1 2 3 4 5 6 7 Inhibits (binds Gi)
hydrolysis of bound GTP to GDP. As a result, Gs
remains
in the active state, continuously activating adenylyl cyclase in COO−
the absence of hormonal stimulation. Cholera toxin pro- Chimeric receptor 2
duced by the bacterium Vibrio cholera and enterotoxins pro-
duced by certain strains of E. coli act in this way on intestinal
NH3+
epithelial cells. The resulting excessive rise in intracellular
cAMP leads to the loss of electrolytes and water into the in- 1 2 3 4 5 6 7 CONCLUSION
testinal lumen, producing the watery diarrhea characteristic
of infection by these bacteria.
COO−
Bordetella pertussis, a bacterium that commonly infects
Region determining specificity of G protein binding
the respiratory tract, is the cause of whooping cough. Per- (compare chimeras 1 and 2)
tussis toxin catalyzes a modification of Gi
that prevents re-
activate the transducing G

subunit. Specific regions within the (a)
Adenylyl cyclase
C3 loop are thought to assume a unique three-dimensional Exterior
structure in all receptors that bind the same G protein (e.g., Gs
or Gi). Other evidence indicates that the C2 loop, joining hel-
ices 3 and 4, also contributes to the interaction of some recep-
tors with a G protein and that residues in at least four
transmembrane helices participate in ligand binding.
Cytosol COO−
X-ray crystallographic analysis has pinpointed the regions +
in Gs
·GTP that interact with adenylyl cyclase. This enzyme is NH3
Catalytic
a multipass transmembrane protein with two large cytosolic domains
segments containing the catalytic domains (Figure 13-14a).
Because such transmembrane proteins are notoriously diffi- (b)
cult to crystallize, scientists prepared two protein fragments

3 – 5
encompassing the catalytic domains of adenylyl cyclase and
allowed them to associate in the presence of Gs
·GTP and
forskolin, which stabilizes the catalytic adenylyl cyclase frag-
ments in their active conformations. The complex that
formed was catalytically active and showed pharmacological
GTP
and biochemical properties similar to those of intact full-
length adenylyl cyclase. In this complex, two regions of
Gs
·GTP, the switch II helix and the
3-5 loop, contact the Switch II
Forskolin
adenylyl cyclase fragments (Figure 13-14b). Recall that switch
II is one of the segments of a G protein whose conformation
Adenylyl cyclase
is different in the GTP-bound and GDP-bound states (see Fig- Gs
catalytic fragments
ure 13-8). The GTP-induced conformation of Gs
that favors
its dissociation from G is precisely the conformation essen- ▲ FIGURE 13-14 Structure of mammalian adenylyl cyclases
tial for binding of Gs
to adenylyl cyclase. Other studies in- and their interaction with Gs
·GTP. (a) Schematic diagram of
dicate that Gi
binds to a different region of adenylyl cyclase, mammalian adenylyl cyclases. The membrane-bound enzyme
accounting for its different effect on the effector. contains two similar catalytic domains on the cytosolic face of
To understand how binding of Gs
·GTP promotes adeny- the membrane and two integral membrane domains, each of
lyl cyclase activity, scientists will first have to solve the struc- which is thought to contain six transmembrane
helices.
ture of the adenylyl cyclase catalytic domains in their (b) Three-dimensional structure of Gs
·GTP complexed with two
unactivated conformations (i.e., in the absence of bound fragments encompassing the catalytic domain of adenylyl cyclase
determined by x-ray crystallography. The
3-5 loop and the helix
Gs
·GTP). One hypothesis is that binding of the switch II
in the switch II region (blue) of Gs
·GTP interact simultaneously
helix to a cleft in one catalytic domain of adenylyl cyclase
with a specific region of adenylyl cyclase. The darker-colored
leads to rotation of the other catalytic domain. This rotation
portion of Gs
is the GTPase domain, which is similar in structure
is proposed to lead to a stabilization of the transition state, to Ras (see Figure 13-8); the lighter portion is a helical domain.
thereby stimulating catalytic activity. The two adenylyl cyclase fragments are shown in orange and
yellow. Forskolin (green) locks the cyclase fragments in their active
conformations. [Part (a) see W.-J. Tang and A. G. Gilman, 1992, Cell 70:869;
EXPERIMENTAL FIGURE 13-13 Studies with chimeric part (b) adapted from J. J. G. Tesmer et al., 1997, Science 278:1907.]
adrenergic receptors identify the long C3 loop as critical
to interaction with G proteins. Xenopus oocytes were
microinjected with mRNA encoding a wild-type
2-adrenergic,
2-adrenergic, or chimeric
- receptor. Although Xenopus Adenylyl Cyclase Is Stimulated and Inhibited
oocytes do not normally express adrenergic receptors, they by Different Receptor-Ligand Complexes
do express G proteins that can couple to the foreign receptors
The versatile trimeric G proteins enable different receptor-
expressed on the surface of microinjected oocytes. The adenylyl
hormone complexes to modulate the activity of the same ef-
cyclase activity of the injected cells in the presence of
epinephrine agonists was determined and indicated whether
fector protein. In the liver, for instance, glucagon and
the adrenergic receptor bound to the stimulatory (Gs) or epinephrine bind to different receptors, but both receptors
inhibitory (Gi) type of oocyte G protein. Comparison of chimeric interact with and activate the same Gs, which activates
receptor 1, which interacts with Gs, and chimeric receptor 2, adenylyl cyclase, thereby triggering the same metabolic re-
which interacts with Gi, shows that the G protein specificity is sponses. Activation of adenylyl cyclase, and thus the cAMP
determined primarily by the source of the cytosol-facing C3 loop level, is proportional to the total concentration of Gs
·GTP
(yellow) between
helices 5 and 6. [See B. Kobilka et al., 1988, resulting from binding of both hormones to their respective
Science 240:1310.] receptors.
Stimulatory Epinephrine Inhibitory PGE1

hormone Glucagon hormone Adenosine
ACTH
Exterior Ac
tiv
Adenylyl
atio cyclase of E
n of it ion
E (E) Inhib
Cytosol Gβγ Gβγ
Gsα Giα
G G
Receptor for D D Receptor for
P P
stimulatory inhibitory
hormone Stimulatory Inhibitory hormone
G protein cAMP G protein
complex complex
▲ FIGURE 13-15 Hormone-induced activation and receptors differ. Ligand-stimulated formation of active G
·GTP
inhibition of adenylyl cyclase in adipose cells. Ligand binding complexes occurs by the same mechanism in both Gs and
to Gs-coupled receptors causes activation of adenylyl cyclase, Gi proteins (see Figure 13-11). However, Gs
·GTP and Gi
·GTP
whereas ligand binding to Gi-coupled receptors causes inhibition interact differently with adenylyl cyclase, so that one stimulates
of the enzyme. The G subunit in both stimulatory and inhibitory and the other inhibits its catalytic activity. [See A. G. Gilman,
G proteins is identical; the G
subunits and their corresponding 1984, Cell 36:577.]
Positive and negative regulation of adenylyl cyclase activ- Most mammalian cells express receptors coupled to Gs
ity occurs in some cell types, providing fine-tuned control of protein. Stimulation of these receptors by various hormones
the cAMP level. For example, stimulation of adipose cells by leads to activation of PKA, but the resulting cellular response
epinephrine, glucagon, or ACTH activates adenylyl cyclase, depends on the particular PKA isoform and on the PKA sub-
whereas prostaglandin PGE1 or adenosine inhibits the en- strates expressed by the cell. For instance, the effects of epi-
zyme (Figure 13-15). The receptors for PGE1 and adenosine nephrine on glycogen metabolism, which are mediated via
interact with inhibitory Gi, which contains the same and cAMP and PKA, are confined mainly to liver and muscle
subunits as stimulatory Gs but a different
subunit (Gi
). In cells, which express enzymes for making and degrading
response to binding of an inhibitory ligand to its receptor, the glycogen. In adipose cells, epinephrine-induced activation of
associated Gi protein releases its bound GDP and binds GTP; PKA promotes phosphorylation and activation of the phos-
the active Gi
·GTP complex then dissociates from G and in- pholipase that catalyzes hydrolysis of stored triglycerides to
hibits (rather than stimulates) adenylyl cyclase. yield free fatty acids and glycerol. These fatty acids are re-
leased into the blood and taken up as an energy source by
cells in other tissues such as the kidney, heart, and muscles.
cAMP-Activated Protein Kinase A Mediates Likewise, stimulation of G protein–coupled receptors on
Various Responses in Different Cells ovarian cells by certain pituitary hormones leads to activa-
In multicellular animals virtually all the diverse effects of tion of PKA, which in turn promotes synthesis of two steroid
cAMP are mediated through protein kinase A (PKA), also hormones, estrogen and progesterone, crucial to the devel-
called cAMP-dependent protein kinase. As discussed in opment of female sex characteristics.
Chapter 3, inactive PKA is a tetramer consisting of two reg- Although PKA acts on different substrates in different
ulatory (R) subunits and two catalytic (C) subunits. Each R types of cells, it always phosphorylates a serine or threonine
subunit has two distinct cAMP-binding sites; binding of residue that occurs within the same sequence motif: X-Arg-
cAMP to both sites in an R subunit leads to release of the (Arg/Lys)-X-(Ser/Thr)-, where X denotes any amino acid
associated C subunit, unmasking its catalytic site and acti- and denotes a hydrophobic amino acid. Other serine/
vating its kinase activity (see Figure 3-27a). Binding of cAMP threonine kinases phosphorylate target residues within dif-
by an R subunit occurs in a cooperative fashion; that is, ferent sequence motifs.
binding of the first cAMP molecule lowers the Kd for binding
of the second. Thus small changes in the level of cytosolic
Glycogen Metabolism Is Regulated by Hormone-
cAMP can cause proportionately large changes in the
amount of dissociated C subunits and, hence, in kinase ac- Induced Activation of Protein Kinase A
tivity. Rapid activation of an enzyme by hormone-triggered The first cAMP-mediated cellular response to be discov-
dissociation of an inhibitor is a common feature of various ered—the release of glucose from glycogen—occurs in mus-
signaling pathways. cle and liver cells stimulated by epinephrine or other
hormones whose receptors are coupled to Gs protein. This contain a phosphatase that hydrolyzes glucose 6-phosphate
response exemplifies how activation of PKA can coordinate to glucose, which is exported from these cells in part by a
the activity of a group of intracellular enzymes toward a glucose transporter (GLUT2) in the plasma membrane
common purpose. (Chapter 7). Thus glycogen stores in the liver are primarily
Glycogen, a large glucose polymer, is the major storage broken down to glucose, which is immediately released into
form of glucose in animals. Like all biopolymers, glycogen the blood and transported to other tissues, particularly the
is synthesized by one set of enzymes and degraded by an- muscles and brain.
other (Figure 13-16). Three enzymes convert glucose into The epinephrine-stimulated increase in cAMP and sub-
uridine diphosphoglucose (UDP-glucose), the primary inter- sequent activation of PKA enhance the conversion of glyco-
mediate in glycogen synthesis. The glucose residue of UDP- gen to glucose 1-phosphate in two ways: by inhibiting
glucose is transferred by glycogen synthase to the free glycogen synthesis and by stimulating glycogen degradation
hydroxyl group on carbon 4 of a glucose residue at the end (Figure 13-17a). PKA phosphorylates and thus inactivates
of a growing glycogen chain. Degradation of glycogen in- glycogen synthase, the enzyme that synthesizes glycogen.
volves the stepwise removal of glucose residues from the PKA promotes glycogen degradation indirectly by phospho-
same end by a phosphorolysis reaction, catalyzed by glyco- rylating and thus activating an intermediate kinase, glycogen
gen phosphorylase, yielding glucose 1-phosphate. phosphorylase kinase (GPK), that in turn phosphorylates
In both muscle and liver cells, glucose 1-phosphate pro- and activates glycogen phosphorylase, the enzyme that de-
duced from glycogen is converted to glucose 6-phosphate. grades glycogen. The entire process is reversed when epi-
In muscle cells, this metabolite enters the glycolytic pathway nephrine is removed and the level of cAMP drops,
and is metabolized to generate ATP for use in powering mus- inactivating PKA. This reversal is mediated by phosphopro-
cle contraction (Chapter 8). Unlike muscle cells, liver cells tein phosphatase, which removes the phosphate residues
HOCH2 HOCH2 HOCH2

O O O
4 1
O O 4 1
OH OH OH
HO O P O P O Uridine HO O O...

OH O O OH OH
UDP-glucose Glycogen (n residues)
Glycogen
synthase
HOCH2 HOCH2 HOCH2

O O O
O O 4 1 4 1
OH OH OH

O P O P O Uridine HO O O O...
O O OH OH OH
UDP Glycogen (n 1 residues)
Pi Glycogen
phosphorylase
HOCH2 HOCH2 HOCH2

O O O
O
OH OH OH
HO O P O HO O O...

OH O OH OH
Glucose 1-phosphate Glycogen (n residues)
▲ FIGURE 13-16 Synthesis and degradation of glycogen. glycogen is catalyzed by glycogen phosphorylase. Because two
Incorporation of glucose from UDP-glucose into glycogen is different enzymes catalyze the formation and degradation of
catalyzed by glycogen synthase. Removal of glucose units from glycogen, the two reactions can be independently regulated.
(a) Increased cAMP

Stimulation of Inhibition of
glycogen breakdown PKA glycogen synthesis
(active)
GPK GPK P GS GS P Inhibition of

phosphoprotein
phosphatase
GP GP P
IP IP P
PP
Glycogen + n Pi n Glucose 1-phosphate IP P
(active)
PP
(b) Decreased cAMP (inactive)
Inhibition of Stimulation of
glycogen breakdown PP glycogen synthesis
(active)
Abbreviations:
PKA Protein kinase A

GPK P GPK GS P GS PP Phosphoprotein phosphatase
GPK Glycogen phosphorylase kinase
GP Glycogen phosphorylase
GS Glycogen synthase
!P Inhibitor of phosphoprotein
GP P GP
UDP-glucose Glycogen + UDP phosphatase
▲ FIGURE 13-17 Regulation of glycogen metabolism (PP). Binding of the phosphorylated inhibitor to PP prevents
by cAMP in liver and muscle cells. Active enzymes are this phosphatase from dephosphorylating the activated enzymes
highlighted in darker shades; inactive forms, in lighter shades. in the kinase cascade or the inactive glycogen synthase.
(a) An increase in cytosolic cAMP activates PKA, which (b) A decrease in cAMP inactivates PKA, leading to release of
inhibits glycogen synthesis directly and promotes glycogen the active form of phosphoprotein phosphatase. The action of
degradation via a protein kinase cascade. At high cAMP, PKA this enzyme promotes glycogen synthesis and inhibits glycogen
also phosphorylates an inhibitor of phosphoprotein phosphatase degradation.
from the inactive form of glycogen synthase, thereby acti- Signal Amplification Commonly Occurs
vating it, and from the active forms of glycogen phosphory- Downstream from Cell-Surface Receptors
lase kinase and glycogen phosphorylase, thereby inactivating
them (Figure 13-17b). The cellular responses induced by G protein–coupled recep-
Phosphoprotein phosphatase itself is regulated by PKA. tors that activate adenylyl cyclase may require tens of thou-
Activated PKA phosphorylates an inhibitor of phosphopro- sands or even millions of cAMP molecules per cell. Thus the
tein phosphatase; the phosphorylated inhibitor then binds hormone signal must be amplified in order to generate suffi-
to phosphoprotein phosphatase, inhibiting its activity (see cient second messenger from the few thousand receptors for
Figure 13-17a). At low cAMP levels, when PKA is inactive, a particular hormone present on a cell. Signal amplification
the inhibitor is not phosphorylated and phosphoprotein is possible because both receptors and G proteins can dif-
phosphatase is active. As a result, the synthesis of glycogen fuse rapidly in the plasma membrane. A single receptor-
by glycogen synthase is enhanced and the degradation of hormone complex causes conversion of up to 100 inactive
glycogen by glycogen phosphorylase is inhibited. Gs molecules to the active form. Each active Gs
·GTP, in
Epinephrine-induced glycogenolysis thus exhibits dual turn, probably activates a single adenylyl cyclase molecule,
regulation: activation of the enzymes catalyzing glycogen which then catalyzes synthesis of many cAMP molecules dur-
degradation and inhibition of enzymes promoting glycogen ing the time Gs
·GTP is bound to it. Although the exact ex-
synthesis. Such coordinate regulation of stimulatory and in- tent of this amplification is difficult to measure, binding of
hibitory pathways provides an efficient mechanism for a single hormone molecule to one receptor molecule can re-
achieving a particular cellular response and is a common sult in the synthesis of at least several hundred cAMP mole-
phenomenon in regulatory biology. cules before the receptor-hormone complex dissociates and
activation of adenylyl cyclase ceases. Similar amplification Although such a cascade may seem overcomplicated, it
probably occurs in signaling from receptors coupled to other not only greatly amplifies an external signal but also allows
G proteins and some other types of receptors whose activa- an entire group of enzyme-catalyzed reactions to be coordi-
tion induces synthesis of second messengers. nately regulated by a single type of signaling molecule. In ad-
A second level of amplification is illustrated by the dition, the multiple steps between stimulus and final
cAMP-mediated stimulation of glycogenolysis. As we just response offer possibilities for regulation by other signaling
discussed, cAMP promotes glycogen degradation via a three- pathways, thereby fine-tuning the cellular response. We will
stage cascade, that is, a series of reactions in which the en- encounter other examples of cascades in signaling pathways
zyme catalyzing one step is activated (or inhibited) by the discussed in the next chapter.
product of a previous step (see Figure 13-17a). The amplifi-
cation that occurs in such a cascade depends on the number
Several Mechanisms Regulate Signaling
of steps in it.
Both levels of amplification are depicted in Figure 13-18. from G Protein–Coupled Receptors
For example, blood levels of epinephrine as low as 1010 M Several factors contribute to termination of the response to
can stimulate liver glycogenolysis and release of glucose. An hormones recognized by -adrenergic receptors and other re-
epinephrine stimulus of this magnitude generates an intra- ceptors coupled to Gs. First, the affinity of the receptor for
cellular cAMP concentration of 106 M, an amplification of hormone decreases when the GDP bound to Gs
is replaced
104. Because three more catalytic steps precede the release with a GTP following hormone binding. This increase in the
of glucose, another 104 amplification can occur. In striated Kd of the receptor-hormone complex enhances dissociation of
muscle, the concentrations of the three successive enzymes in the hormone from the receptor. Second, the GTP bound to Gs
the glycogenolytic cascade—protein kinase A, glycogen is quickly hydrolyzed, reversing the activation of adenylyl cy-
phosphorylase kinase, and glycogen phosphorylase—are in a clase and production of cAMP (see Figure 13-11). Third,
1:10:240 ratio, which dramatically illustrates the amplifica- cAMP phosphodiesterase acts to hydrolyze cAMP to 5-AMP,
tion of the effects of epinephrine and cAMP. terminating the cellular response. Thus the continuous pres-
ence of hormone at a high enough concentration is required
for continuous activation of adenylyl cyclase and maintenance
of an elevated cAMP level. Once the hormone concentration
Epinephrine (1010 M) falls sufficiently, the cellular response quickly terminates.
Amplification When a Gs protein–coupled receptor is exposed to hor-
Adenylyl monal stimulation for several hours, several serine and thre-
cyclase onine residues in the cytosolic domain of the receptor
become phosphorylated by protein kinase A (PKA). The
Amplification
phosphorylated receptor can bind its ligand, but ligand bind-
cAMP (106 M) ing leads to reduced activation of adenylyl cyclase; thus the
receptor is desensitized. This is an example of feedback sup-
Protein pression, in which the end product of a pathway (here acti-
kinase A
vated PKA) blocks an early step in the pathway (here,
Amplification receptor activation). Because the activity of PKA is enhanced
Activated by the high cAMP level induced by any hormone that acti-
enzyme vates Gs, prolonged exposure to one such hormone, say, ep-
inephrine, causes desensitization not only of -adrenergic
Amplification receptors but also of Gs protein–coupled receptors that bind
Product different ligands (e.g., glucagon). This cross-regulation is
called heterologous desensitization.
▲ FIGURE 13-18 Amplification of an external signal Additional residues in the cytosolic domain of the -
downstream from a cell-surface receptor. In this example, adrenergic receptor are phosphorylated by a receptor-specific
binding of a single epinephrine molecule to one Gs protein–
enzyme called -adrenergic receptor kinase (BARK), but only
coupled receptor molecule induces synthesis of a large number
when epinephrine or an agonist is bound to the receptor. Be-
of cAMP molecules, the first level of amplification. Four
molecules of cAMP activate two molecules of protein kinase
cause BARK phosphorylates only activated -adrenergic re-
A (PKA), but each activated PKA phosphorylates and activates ceptors, this process is called homologous desensitization.
multiple product molecules. This second level of amplification Prolonged treatment of cells with epinephrine results in ex-
may involve several sequential reactions in which the product of tensive phosphorylation and hence desensitization of the -
one reaction activates the enzyme catalyzing the next reaction. adrenergic receptor by both PKA and BARK.
The more steps in such a cascade, the greater the signal Phosphorylated (desensitized) receptors are constantly
amplification possible. being resensitized owing to dephosphorylation by constitutive
phosphatases. Thus the number of phosphates per receptor additional function of -arrestin in regulating cell-surface re-
molecule reflects how much ligand has been bound in the ceptors initially was suggested by the observation that loss of
recent past (e.g., 1–10 minutes). This means that if a cell is cell surface -adrenergic receptors in response to ligand
constantly being exposed to a certain concentration of a binding is stimulated by overexpression of BARK and -
hormone, that hormone concentration will eventually cease arrestin. Subsequent studies revealed that -arrestin binds
to stimulate the receptor. If the hormone concentration is not only to phosphorylated receptors but also to clathrin and
now increased to a new value, the receptor will activate an associated protein termed AP2, two essential components
downstream signaling pathways but to a lesser extent than of the coated vesicles that are involved in one type of endo-
would occur if the cell were switched from a medium with- cytosis. These interactions promote the formation of coated
out hormone to one with this hormone level. If the hormone pits and endocytosis of the associated receptors, thereby de-
is then completely removed, the receptor becomes com- creasing the number of receptors exposed on the cell surface
pletely dephosphorylated and “reset” to its maximum sen- (Figure 13-19). Eventually the internalized receptors become
sitivity, in which case it can respond to very low levels of dephosphorylated in endosomes, -arrestin dissociates, and
hormone. Thus a feedback loop involving receptor phos- the resensitized receptors recycle to the cell surface, similar to
phorylation and dephosphorylation modulates the activity recycling of the LDL receptor (Chapter 17). Regulation of
of -adrenergic and related Gs protein–coupled receptors, other G protein–coupled receptors also is thought to involve
permitting a cell to adjust receptor sensitivity to the hor- endocytosis of ligand-occupied receptors and their seques-
mone level at which it is being stimulated. tration inside the cell.
Another key participant in regulation of -adrenergic re- As we discuss later, -arrestin also functions as an
ceptors is -arrestin. This cytosolic protein binds to receptors adapter protein in transducing signals from Gs protein–
extensively phosphorylated by BARK and completely in- coupled receptors to the nucleus. The multiple functions of
hibits their interaction with and ability to activate Gs. An -arrestin illustrate the importance of adapter proteins in
both regulating signaling and transducing signals from cell-
surface receptors.
+
NH3
G protein–coupled receptor
Exterior Anchoring Proteins Localize Effects
of cAMP to Specific Subcellular Regions
In many cell types, a rise in the cAMP level may produce a
response that is required in one part of the cell but is un-
wanted, perhaps deleterious, in another part. A family of an-
Cytosol
−
choring proteins localizes PKA isoforms to specific
OOC subcellular locations, thereby restricting cAMP-dependent
P P
AJK-1 AP2 responses to these locations. These proteins, referred to as
Activation of -Arrestin Endocytosis
c-Jun kinase Clathrin A kinase–associated proteins (AKAPs), have a two-domain
MKKY
cascade JNK-1 c-Src
structure with one domain conferring a specific subcellular
location and another that binds to the regulatory subunit of
protein kinase A.
Activation of MAP One anchoring protein (AKAP15) is tethered to the cy-
kinase cascade tosolic face of the plasma membrane near a particular type of
gated Ca2 channel in certain heart muscle cells. In the heart,
▲ FIGURE 13-19 Role of -arrestin in GPCR desensitization activation of -adrenergic receptors by epinephrine (as part
and signal transduction. -Arrestin binds to phosphorylated of the fight-or-flight response) leads to PKA-catalyzed phos-
serine and tyrosine residues in the C-terminal segment of phorylation of these Ca2 channels, causing them to open;
G protein–coupled receptors (GPCRs). Clathrin and AP2, two the resulting influx of Ca2 increases the rate of heart mus-
other proteins bound by -arrestin, promote endocytosis of the
cle contraction. The interaction of AKAP15 with PKA local-
receptor. -Arrestin also functions in transducing signals from
izes PKA next to these channels, thereby reducing the time
activated receptors by binding to and activating several cytosolic
protein kinases. c-Src activates the MAP kinase pathway, leading
that otherwise would be required for diffusion of PKA cat-
to phosphorylation of key transcription factors (Chapter 14). alytic subunits from their sites of generation to their Ca2-
Interaction of -arrestin with three other proteins, including channel substrate.
JNK-1 (a Jun N-terminal kinase), results in phosphorylation and Another A kinase–associated protein (mAKAP) in heart
activation of the c-Jun transcription factor. [Adapted from W. Miller muscle anchors both PKA and cAMP phosphodiesterase
and R. J. Lefkowitz, 2001, Curr. Opin. Cell Biol. 13:139, and K. Pierce (PDE) to the outer nuclear membrane. Because of the close
et al., 2002, Nature Rev. Mol. Cell Biol. 3:639.] proximity of PDE to PKA, a negative feedback loop provides
13.4 • G Protein–Coupled Receptors That Regulate Ion Channels 555
1 2 3
Basal PDE activity = Increased cAMP: PDE phosphorylation
resting state PKA activation and activation; reduction
in cAMP level
cAMP PDE PDE P PDE P
mAKAP C mAKAP C C mAKAP C
R R C PKA R R R R
Cytosol C
Outer
nuclear
membrane
4 Return to resting state
▲ FIGURE 13-20 Localization of protein kinase A (PKA) excess of that which can be degraded by PDE. The resulting
to the nuclear membrane in heart muscle. This A kinase– binding of cAMP to the regulatory (R) subunits of PKA
associated protein mAKAP anchors both PKA and cAMP releases the active catalytic (C) subunits. Step 3 : Subsequent
phosphodiesterase (PDE) to the nuclear membrane, maintaining phosphorylation of PDE by PKA stimulates its catalytic activity,
them in a negative feedback loop that provides close local control thereby driving cAMP levels back to basal and causing
of the cAMP level. Step 1 : The basal level of PDE activity in reformation of the inactive PKA. Subsequent dephosphorylation
the absence of hormone (resting state) keeps cAMP levels of PDE (step 4 ) returns the complex to the resting state.
below those necessary for PKA activation. Step 2 : Activation of [Adapted from K. L. Dodge et al., 2001, EMBO J. 20:1921.]
-adrenergic receptors causes an increase in cAMP level in
close local control of the cAMP level and hence PKA activ- ■ cAMP-dependent activation of protein kinase A (PKA)
ity (Figure 13-20). The localization of PKA near the nuclear mediates the diverse effects of cAMP in different cells. The
membrane also facilitates entry of the catalytic subunits into substrates for PKA and thus the cellular response to
the nucleus, where they phosphorylate and activate certain hormone-induced activation of PKA vary among cell types.
transcription factors (Section 13.6). ■ In liver and muscle cells, activation of PKA induced by
epinephrine and other hormones exerts a dual effect, in-
hibiting glycogen synthesis and stimulating glycogen break-
down via a kinase cascade (see Figure 13-17).
KEY CONCEPTS OF SECTION 13.3
■ Signaling pathways involving second messengers and ki-
G Protein–Coupled Receptors That Activate nase cascades amplify an external signal tremendously (see
or Inhibit Adenylyl Cyclase Figure 13-18).
■ Trimeric G proteins transduce signals from coupled cell- ■ BARK phosphorylates ligand-bound -adrenergic re-
surface receptors to associated effector proteins, which are ceptors, leading to the binding of -arrestin and endocy-
either enzymes that form second messengers or cation tosis of the receptors. The consequent reduction in cell-
channel proteins (see Table 13-1). surface-receptor numbers renders the cell less sensitive to
■ Signals most commonly are transduced by G
, a GTPase additional hormone.
switch protein that alternates between an active (“on”) ■ Localization of PKA to specific regions of the cell by an-
state with bound GTP and inactive (“off”) state with GDP. choring proteins restricts the effects of cAMP to particu-
The and subunits, which remain bound together, oc- lar subcellular locations.
casionally transduce signals.
■ Hormone-occupied receptors act as GEFs for G
pro-
teins, catalyzing dissociation of GDP and enabling GTP to
bind. The resulting change in conformation of switch re-
13.4 G Protein–Coupled Receptors
gions in G
causes it to dissociate from the G subunit That Regulate Ion Channels
and interact with an effector protein (see Figure 13-11).
As we learned in Chapter 7, many neurotransmitter recep-
■ Gs
, which is activated by multiple types of GPCRs, tors are ligand-gated ion channels. These include some types
binds to and activates adenylyl cyclase, enhancing the syn- of glutamate and serotonin receptors, as well as the nicotinic
thesis of 3,5-cyclic AMP (cAMP). acetylcholine receptor found at nerve-muscle synapses. Many
neurotransmitter receptors, however, are G protein–coupled rate of heart muscle contraction by causing a long-lived (sev-
receptors. The effector protein for some of these is a Na or eral seconds) hyperpolarization of the muscle cell membrane.
K channel; neurotransmitter binding to these receptors This can be studied experimentally by direct addition of
causes the associated ion channel to open or close, leading to acetylcholine to heart muscle in culture.
changes in the membrane potential. Other neurotransmitter Activation of the muscarinic acetylcholine receptor,
receptors, as well as odorant receptors in the nose and pho- which is coupled to a Gi protein, leads to opening of associ-
toreceptors in the eye, are G protein–coupled receptors that ated K channels; the subsequent efflux of K ions causes
indirectly modulate the activity of ion channels via the hyperpolarization of the plasma membrane. As depicted in
action of second messengers. In this section, we consider two Figure 13-21, the signal from activated receptors is trans-
G protein–coupled receptors that illustrate the direct and induced to the effector protein by the released G subunit
direct mechanisms for regulating ion channels: the muscarinic rather than by G
·GTP. That G directly activates the K
acetylcholine and Gt-coupled receptors. channel was demonstrated by patch-clamping experiments,
which can measure ion flow through a single ion channel in
a small patch of membrane (see Figure 7-17). When purified
Cardiac Muscarinic Acetylcholine Receptors G protein was added to the cytosolic face of a patch of
Activate a G Protein That Opens K Channels heart muscle plasma membrane, K channels opened imme-
Binding of acetylcholine to nicotinic acetylcholine receptors diately, even in the absence of acetylcholine or other neuro-
in striated muscle cells generates an action potential that trig- transmitters.
gers muscle contraction (see Figure 7-45). In contrast, the
muscarinic acetylcholine receptors in cardiac muscle are in-
Gt-Coupled Receptors Are Activated by Light
hibitory. Binding of acetylcholine to these receptors slows the
The human retina contains two types of photoreceptors, rods
and cones, that are the primary recipients of visual stimula-
tion. Cones are involved in color vision, while rods are
stimulated by weak light like moonlight over a range of
Acetylcholine
K+ channel wavelengths. The photoreceptors synapse on layer upon
Exterior + + layer of interneurons that are innervated by different com-
binations of photoreceptor cells. All these signals are
processed and interpreted by the part of the brain called the
− −
Cytosol Gβγ visual cortex.
Giα
G
Rhodopsin, a G protein–coupled receptor that is acti-
Active muscarinic D vated by light, is localized to the thousand or so flattened
P
acetylcholine receptor membrane disks that make up the outer segment of rod cells
(Figure 13-22). The trimeric G protein coupled to rhodopsin,
GDP
GTP
K+ often called transducin (Gt), is found only in rod cells.
A human rod cell contains about 4 107 molecules of
+ + + + + rhodopsin, which consists of the seven-spanning protein
opsin to which is covalently bound the light-absorbing pig-
− − − − −
ment 11-cis-retinal. Upon absorption of a photon, the retinal
Giα
Gβγ moiety of rhodopsin is very rapidly converted to the all-trans
G isomer, causing a conformational change in the opsin portion
T
P that activates it (Figure 13-23). This is equivalent to the con-
formational change that occurs upon ligand binding by other
▲ FIGURE 13-21 Operational model of muscarinic G protein–coupled receptors. The resulting form in which
acetylcholine receptor in the heart muscle plasma membrane. opsin is covalently bound to all-trans-retinal is called meta-
These receptors are linked via a trimeric G protein to K channels. rhodopsin II, or activated opsin. Analogous to other G
Binding of acetylcholine triggers activation of the Gi
subunit and
protein–coupled receptors, this light-activated form of
its dissociation from the G subunit in the usual way (see Figure
rhodopsin interacts with and activates an associated G pro-
13-11). In this case, the released G subunit (rather than Gi
·GTP)
binds to and opens the associated effector, a K channel. The
tein (i.e., Gt). Activated opsin is unstable and spontaneously
increase in K permeability hyperpolarizes the membrane, which dissociates into its component parts, releasing opsin and all-
reduces the frequency of heart muscle contraction. Though not trans-retinal, thereby terminating visual signaling. In the
shown here, activation is terminated when the GTP bound to dark, free all-trans-retinal is converted back to 11-cis-retinal,
Gi
is hydrolyzed to GDP and Gi
·GDP recombines with G . which can then rebind to opsin, re-forming rhodopsin.
[See K. Ho et al., 1993, Nature 362:31, and Y. Kubo et al., 1993, Nature In the dark, the membrane potential of a rod cell is about
362:127.] 30 mV, considerably less than the resting potential (60 to
(a) (b)
Outer
segment
Disks
containing
rhodopsin
Microtubules
Mitochondria
Rough
Golgi endoplasmic
reticulum
Inner Cilium
segment
Nucleus Basal
body
Synaptic
body
Human rod cell 0.5 m
▲ FIGURE 13-22 Human rod cell. (a) Schematic diagram of an of the rod cell indicated by the bracket in (a). This region includes
entire rod cell. At the synaptic body, the rod cell forms a synapse the junction of the inner and outer segments. [Part (b) from R. G.
with one or more bipolar interneurons. Rhodopsin, a light-sensitive Kessel and R. H. Kardon, 1979, Tissues and Organs: A Text-Atlas of
G protein–coupled receptor, is located in the flattened membrane Scanning Electron Microscopy, W. H. Freeman and Company, p. 91.]
disks of the outer segment. (b) Electron micrograph of the region
90 mV) typical of neurons and other electrically active The more photons absorbed by rhodopsin, the more
cells. As a consequence of this depolarization, rod cells in the channels are closed, the fewer Na ions cross the membrane
dark are constantly secreting neurotransmitters, and the from the outside, the more negative the membrane potential
bipolar interneurons with which they synapse are continually becomes, and the less neurotransmitter is released. This
being stimulated. The depolarized state of the plasma mem- change is transmitted to the brain where it is perceived as
brane of resting rod cells is due to the presence of a large light. Remarkably, a single photon absorbed by a resting rod
number of open nonselective ion channels that admit Na cell produces a measurable response, a decrease in the mem-
and Ca2, as well as K. Absorption of light by rhodopsin brane potential of about 1 mV, which in amphibians lasts a
leads to closing of these channels, causing the membrane second or two. Humans are able to detect a flash of as few as
potential to become more negative. five photons.
11-cis -Retinal moiety Activation of Rhodopsin Induces Closing

of cGMP-Gated Cation Channels
H3C CH3 CH3 cis
11 The key transducing molecule linking activated opsin to the
12 closing of cation channels in the rod-cell plasma membrane
Lysine side chain is the second messenger cyclic GMP (cGMP). Rod outer seg-
CH3 H3C ments contain an unusually high concentration (≈0.07 mM)
+ of cGMP, which is continuously formed from GTP in a re-
C N (CH2)4 Opsin
action catalyzed by guanylyl cyclase that appears to be un-
H H affected by light. However, light absorption by rhodopsin
Rhodopsin induces activation of a cGMP phosphodiesterase, which hy-
drolyzes cGMP to 5-GMP. As a result, the cGMP concen-
Light-induced tration decreases upon illumination. The high level of cGMP
isomerization
(<10−2 s)
H+
all-trans -Retinal moiety FIGURE 13-23 The light-triggered step in vision. The
light-absorbing pigment 11-cis-retinal is covalently bound to the
trans amino group of a lysine residue in opsin, the protein portion of
H3C CH3 CH3 CH3 rhodopsin. Absorption of light causes rapid photoisomerization of
11 the cis-retinal to the all-trans isomer, forming the unstable
C N (CH2)4 Opsin*
12
intermediate meta-rhodopsin II, or activated opsin, which
H activates Gt proteins. Within seconds all-trans-retinal dissociates
CH3 from opsin and is converted by an enzyme back to the cis
Meta-rhodopsin II isomer, which then rebinds to another opsin molecule.
(activated opsin) [See J. Nathans, 1992, Biochemistry 31:4923.]
Light
Cytosol
Disk membrane
Disk lumen
1 2 3 Inactive 4 Active
O O* PDE PDE Rod
Gβγ plasma
β β membrane
γ γ γ γ
Gtα G G α G α G
D T T T
P P P P
G G 5
T D
P P cGMP GMP
6
Low
Na+
▲ FIGURE 13-24 Operational model for rhodopsin-induced closing of cytosolic
Ca2+
cGMP ( )
cation channels in rod cells. In dark-adapted rod cells, a high level of cGMP keeps
Closed cGMP-gated
nucleotide-gated nonselective cation channels open. Light absorption generates ion channel
activated opsin, O* (step 1 ), which binds inactive GDP-bound Gt protein and mediates
replacement of GDP with GTP (step 2 ). The free Gt
·GTP generated then activates High Dark-adapted
cGMP phosphodiesterase (PDE) by binding to its inhibitory subunits (step 3 ) and cytosolic state
cGMP
dissociating them from the catalytic
and subunits (step 4 ). Relieved of their inhibition, ( )
Na+
the
and subunits convert cGMP to GMP (step 5 ). The resulting decrease in
Ca2+
cytosolic cGMP leads to dissociation of cGMP from the nucleotide-gated channels in the
Open cGMP-gated
plasma membrane and closing of the channels (step 6 ). The membrane then becomes ion channel
transiently hyperpolarized. [Adapted from V. Arshavsky and E. Pugh, 1998, Neuron 20:11.]
present in the dark acts to keep cGMP-gated cation channels N

open; the light-induced decrease in cGMP leads to channel Rhodopsin
closing, membrane hyperpolarization, and reduced neuro-
transmitter release. Exterior
As depicted in Figure 13-24, cGMP phosphodiesterase is
the effector protein for Gt. The free Gt
·GTP complex that
is generated after light absorption by rhodopsin binds to the
two inhibitory subunits of cGMP phosphodiesterase, re-
leasing the active catalytic
and subunits, which then con-
vert cGMP to GMP. This is another example of how
signal-induced removal of an inhibitor can quickly activate
an enzyme, a common mechanism in signaling pathways. A Cytosol
single molecule of activated opsin in the disk membrane can C Sites of lipid
attachment
activate 500 Gt
molecules, each of which in turn activates C
N
cGMP phosphodiesterase; this is the primary stage of signal GTPase
C
amplification in the visual system. Even though activation domain
of cGMP phosphodiesterase leads to a decrease in a second Gγ
messenger, cGMP, this activation occurs by the same general GDP
mechanism described earlier except that absorption of light Gtα
by rhodopsin rather than ligand binding is the activating sig-
nal (see Figure 13-11). Helical
domain
Conversion of active Gt
·GTP back to inactive Gt
·GDP
Gβ
is accelerated by a GTPase-activating protein (GAP) specific
for Gt
·GTP. In mammals Gt
normally remains in the ac- N
tive GTP-bound state for only a fraction of a second. Thus
cGMP phosphodiesterase rapidly becomes inactivated, and N
the cGMP level gradually rises to its original level when the
▲ FIGURE 13-25 Structural models of rhodopsin and its
light stimulus is removed. This allows rapid responses of the
associated Gt protein. The structures of rhodopsin and the Gt
eye toward moving or changing objects. and G subunits were obtained by x-ray crystallography. The
Recent x-ray crystallographic studies reveal how the sub- C-terminal segment of rhodopsin is not shown in this model. The
units of Gt protein interact with each other and with light- orientation of Gt
with respect to rhodopsin and the membrane
activated rhodopsin and provide clues about how binding is hypothetical; it is based on the charge and hydrophobicity of
of GTP leads to dissociation of G
from G (Figure 13-25). the protein surfaces and the known rhodopsin-binding sites on
Two surfaces of Gt
interact with G: an N-terminal region Gt
. As in other trimeric G proteins, the Gt
and G subunits
near the membrane surface and the two adjacent switch I contain covalently attached lipids that are thought to be inserted
and switch II regions, which are found in all G
proteins. Al- into the membrane. In the GDP-bound form shown here (GDP,
though G and G also contact each other, G does not con- red), the
subunit (gray) and the subunit (light blue) interact
tact Gt
. with each other, as do the and (purple) subunits, but the
Studies with adrenergic receptors discussed earlier indi- small subunit, which contains just two
helices, does not
cate that ligand binding to a G protein–coupled receptor contact the
subunit. Several segments of the
subunit are
thought to interact with an activated receptor, causing a
causes the transmembrane helices in the receptor to slide rel-
conformational change that promotes release of GDP and binding
ative to one another, resulting in conformational changes in
of GTP. Binding of GTP, in turn, induces large conformational
the cytosolic loops that create a binding site for the coupled
changes in the switch regions of Gt
, leading to dissociation of
trimeric G protein. The crystallographic structures in Figure Gt
from G . The structure of a Gs
subunit in the GTP-bound
13-25 suggest that the nucleotide-binding domain of Gt
, to- form, which interacts with an effector protein, is shown in Figure
gether with the lipid anchors at the C-terminus of G and the 13-14b. [Adapted from H. Hamm, 2001, Proc. Nat’l. Acad. Sci. USA
N-terminus of Gt
, form a surface that binds to light- 98:4819, and D. G. Lambright et al., 1996, Nature 379:311.]
activated rhodopsin (O* in Figure 13-24), promoting the re-
lease of GDP from Gt
and the subsequent binding of GTP.
The subsequent conformational changes in Gt
, particularly Direct support for the role of cGMP in rod-cell activity
those within switches I and II, disrupt the molecular inter- has been obtained in patch-clamping studies using isolated
actions between Gt
and G , leading to their dissociation. patches of rod outer-segment plasma membrane, which con-
The structural studies with rhodopsin and Gt are consistent tains abundant cGMP-gated cation channels. When cGMP is
with data concerning other G protein–coupled receptors and added to the cytosolic surface of these patches, there is a
are thought to be generally applicable to all receptors of this rapid increase in the number of open ion channels. The effect
type. occurs in the absence of protein kinases or phosphatases, and
cGMP acts directly on the channels to keep them open, in- vation in bright light. Because the extent of opsin phos-
dicating that these are nucleotide-gated channels. Like the phorylation is proportional to the amount of time each
voltage-gated K channels discussed in Chapter 7, the opsin molecule spends in the light-activated form, it is a
cGMP-gated channel protein contains four subunits, each measure of the background (ambient) level of light. Under
of which is able to bind a cGMP molecule (see Figure high-light conditions, phosphorylated opsin is abundant
7-36a). Three or four cGMP molecules must bind per and activation of Gt is reduced; thus, a greater increase in
channel in order to open it; this allosteric interaction light level will be necessary to generate a visual signal.
makes channel opening very sensitive to small changes in When the level of ambient light is reduced, the opsins be-
cGMP levels. come dephosphorylated and the ability to activate Gt in-
creases; in this case, fewer additional photons will be
necessary to generate a visual signal.
Rod Cells Adapt to Varying
At high ambient light (such as noontime outdoors), the
Levels of Ambient Light level of opsin phosphorylation is such that the protein -
Cone cells are insensitive to low levels of illumination, and arrestin binds to the C-terminal segment of opsin. The bound
the activity of rod cells is inhibited at high light levels. Thus -arrestin prevents interaction of Gt with O*, totally block-
when we move from daylight into a dimly lighted room, we ing formation of the active Gt
·GTP complex and causing a
are initially blinded. As the rod cells slowly become sensi- shutdown of all rod-cell activity. The mechanism by which
tive to the dim light, we gradually are able to see and distin- rod-cell activity is controlled by rhodopsin kinase and ar-
guish objects. This process of visual adaptation permits a rod restin is similar to adaptation (or desensitization) of other
cell to perceive contrast over a 100,000-fold range of ambi- G protein–coupled receptors to high ligand levels.
ent light levels; as a result, differences in light levels, rather A second mechanism of visual adaptation appears unique
than the absolute amount of absorbed light, are used to form to rod cells. In dark-adapted cells virtually all the Gt
and
visual images. G subunits are in the outer segments. But exposure for
One process contributing to visual adaptation involves 10 minutes to moderate daytime intensities of light causes
phosphorylation of activated opsin (O*) by rhodopsin ki- over 80 percent of the Gt
and G subunits to move out of
nase (Figure 13-26). This rod-cell enzyme is analogous to the outer segments into other cellular compartments (Figure
-adrenergic receptor kinase (BARK) discussed previously. 13-27). The mechanism by which these proteins move is not
Each opsin molecule has three principal serine phosphory- yet known, but as a result of this adaptation Gt proteins are
lation sites; the more sites that are phosphorylated, the less physically unable to bind activated opsin. As occurs in other
able O* is to activate Gt and thus induce closing of cGMP- signaling pathways, multiple mechanisms are thus used to in-
gated cation channels. Indeed, rod cells from mice with mu- activate signaling during visual adaptation, presumably to
tant rhodopsins bearing zero or only one of these serine allow strict control of activation of the signaling pathway
residues show a much slower than normal rate of deacti- over broad ranges of illumination.
Rhodopsin Activated
Overview Animation: Extracellular Signaling
(dark adapted) opsin

High Very high
Low light ATP ADP light O* light O*
O* O*
MEDIA CONNECTIONS
Rhodopsin Arrestin
kinase
P P P P P P P
Cytosol
Arrestin
Activation of Slightly reduced Greatly reduced No

Gtα Gtα activation Gtα activation Gtα activation
▲ FIGURE 13-26 Role of opsin phosphorylation in Gt

is inversely proportional to the number of phosphorylated
adaptation of rod cells to changes in ambient light residues. Thus the higher the ambient light level, the greater the
levels. Light-activated opsin (O*), but not dark-adapted extent of opsin phosphorylation and the larger the increase
rhodopsin, is a substrate for rhodopsin kinase. The extent in light level needed to activate the same number of Gt
of opsin phosphorylation is directly proportional to the (transducin) molecules. At very high light levels, arrestin binds to
amount of time each opsin molecule spends in the light- the completely phosphorylated opsin, forming a complex that
activated form and thus to the average ambient light level cannot activate transducin at all. [See L. Lagnado and D. Baylor, 1992,
over the previous few minutes. The ability of O* to activate Neuron 8:995, and A. Mendez et al., 2000, Neuron 28:153.]
13.5 • G Protein–Coupled Receptors That Activate Phospholipase C 561
■ The effector protein activated by Gt

·GTP is cGMP
phosphodiesterase. Reduction in the cGMP level by this
enzyme leads to closing of cGMP-gated Na/Ca2 chan-
nels, hyperpolarization of the membrane, and decreased re-
lease of neurotransmitter (see Figure 13-24).
■ As with other G
proteins, binding of GTP to
Gt
causes conformational changes in the protein that
disrupt its molecular interactions with G and
enable Gt
·GTP to bind to its downstream effector (see
Figure 13-25).
■ Phosphorylation of light-activated opsin by rhodopsin
kinase and subsequent binding of arrestin to phosphory-
lated opsin inhibit its ability to activate transducin (see
Figure 13-26). This general mechanism of adaptation, or
desensitization, is utilized by other GPCRs at high ligand
levels.
13.5 G Protein–Coupled Receptors

That Activate Phospholipase C
▲ EXPERIMENTAL FIGURE 13-27 Movement of Gt from
outer segments of rod cells contributes to visual adaptation. In this section, we discuss GPCR-triggered signal-transduction
As shown by immunofluorescence staining of retinas of dark- pathways involving several other second messengers and
adapted rats, both the
and subunits of transducin (G
t and the mechanisms by which they regulate various cellular ac-
Gt) are localized to the outer segments (OS) of rod cells, where tivities. A number of these second messengers are derived
they can be activated by rhodopsin photoreceptors in the from phosphatidylinositol (PI). The inositol group in this
membrane disks (see Figure 13-22). After several minutes of phospholipid, which extends into the cytosol adjacent to
bright light most of the transducin
and subunits have moved the membrane, can be reversibly phosphorylated at several
to the inner segment (IS) of the rod cells, where they cannot positions by the combined actions of various kinases
interact with active opsin; this contributes to desensitization of and phosphatases. These reactions yield several different
rod cells at high light intensities. [From M. Sokolov et al., 2002, membrane-bound phosphoinositides, two of which are de-
Neuron 33:95. Courtesy of Vadim Arshavsky, Harvard Medical School.] picted in Figure 13-28.
The levels of many phosphoinositides in cells are dy-
namically regulated by extracellular signals, especially
those that bind to receptor tyrosine kinases or cytokine re-
ceptors, which we cover in the next chapter. The phospho-
inositide PIP2 (PI 4,5-bisphosphate) binds many cytosolic
KEY CONCEPTS OF SECTION 13.4 proteins to the plasma membrane. Some of these proteins
G Protein–Coupled Receptors That Regulate are required for forming and remodeling the actin cy-
Ion Channels toskeleton (Chapter 19); others are required for binding of
proteins important for endocytosis and vesicle fusions
■ The cardiac muscarinic acetylcholine receptor is a GPCR (Chapter 17).
whose effector protein is a K channel. Receptor activa- PIP2 is also cleaved by the plasma-membrane – associated
tion causes release of the G subunit, which opens K enzyme phospholipase C (PLC) to generate two important
channels (see Figure 13-21). The resulting hyperpolariza- second messengers: 1,2-diacylglycerol (DAG), a lipophilic
tion of the cell membrane slows the rate of heart muscle molecule that remains associated with the membrane, and in-
contraction. ositol 1,4,5-trisphosphate (IP3), which diffuses in the cytosol
■ Rhodopsin, the photosensitive GPCR in rod cells, (see Figure 13-28). We refer to downstream events involv-
comprises the opsin protein linked to 11-cis-retinal. The ing these two second messengers collectively as the IP3/DAG
light-induced isomerization of the 11-cis-retinal moiety pathway. Hormone binding to receptors coupled to either a
produces activated opsin, which then activates the cou- Go or a Gq protein (see Table 13-1) induces activation of the
pled trimeric G protein transducin (Gt) by catalyzing ex- isoform of phospholipase C (PLC) by the general mech-
change of free GTP for bound GDP on the Gt
subunit. anism outlined in Figure 13-11.
Cytosolic leaflet
1,2-Diacylglycerol
(DAG)
C O C O C O C O C O C O C O C O
O O O O O O O O
ATP ADP ATP ADP
CH2 CH CH2 CH2 CH CH2 CH2 CH CH2 CH2 CH CH2
O PI-4 kinase O PIP-5 kinase O OH
−O P −O −
O P
O OH P O OH O P Phospholipase C
6 5 5
O O O
Inositol
OH OH OH
1 4 4 4 P
OH HO OH HO OH HO
OH P P 5
2 3 P
OH
4
Phosphatidylinositol PI 4-phosphate PI 4,5-bisphosphate 1 OH HO
(PI) (PIP) (PIP2) P
Inositol 1,4,5-
trisphosphate
(IP3)
▲ FIGURE 13-28 Synthesis of DAG and IP3 from PIP and PIP2. Cleavage of PIP2 by phospholipase C (PLC) yields
membrane-bound phosphatidylinositol (PI). Each membrane- the two important second messengers DAG and IP3. [See A. Toker
bound PI kinase places a phosphate (yellow circles) on a specific and L. C. Cantley, 1997, Nature 387:673, and C. L. Carpenter and L. C.
hydroxyl group on the inositol ring, producing the phosphoinositides Cantley, 1996, Curr. Opin. Cell Biol. 8:153.]
Inositol 1,4,5-Trisphosphate (IP3) Triggers Release brane and ER membrane actively pump Ca2 from the cytosol
of Ca2 from the Endoplasmic Reticulum to the cell exterior and ER lumen, respectively. Furthermore,
within a second of its generation, one specific phosphate
Most intracellular Ca2 ions are sequestered in the mitochon- on IP3 is hydrolyzed, yielding inositol 1,4-bisphosphate, which
dria and in the lumen of the endoplasmic reticulum (ER) and does not stimulate Ca2 release from the ER.
other vesicles. Cells employ various mechanisms for regulating Without some means for replenishing depleted stores of
the concentration of Ca2 ions in the cytosol, which usually is intracellular Ca2, a cell would soon be unable to increase
kept below 0.2 M. For instance, Ca2 ATPases pump cyto- the cytosolic Ca2 level in response to hormone-induced
solic Ca2 ions across the plasma membrane to the cell exterior IP3. Patch-clamping studies have revealed that a plasma-
or into the lumens of intracellular Ca2-storing compartments membrane Ca2 channel, called the TRP channel or the
(see Figure 7-7). As we discuss below, a small rise in cytosolic store-operated channel, opens in response to depletion of ER
Ca2 induces a variety of cellular responses, and thus the cy- Ca2 stores (see Figure 13-29). In a way that is not under-
tosolic concentration of Ca2 is carefully controlled. stood, depletion of Ca2 in the ER lumen leads to a confor-
Binding of many hormones to their cell-surface receptors mational change in the IP3-gated Ca2 channel that allows
on liver, fat, and other cells induces an elevation in cytosolic it to bind to the TRP Ca2 channel in the plasma membrane,
Ca2 even when Ca2 ions are absent from the surrounding causing the latter to open. Indeed, expression in cells of a
extracellular fluid. In this situation, Ca2 is released into the specific fragment of the ER membrane IP3-gated Ca2 chan-
cytosol from the ER lumen through operation of the IP3- nel prevents opening of the TRP channel upon depletion of
gated Ca2 channel in the ER membrane. This large protein ER Ca2 stores, implicating an interaction between the two
is composed of four identical subunits, each containing an Ca2 channels in opening the TRP channel.
IP3-binding site in the N-terminal cytosolic domain. IP3 bind- Opening of IP3-gated Ca2 channels is potentiated by cy-
ing induces opening of the channel, allowing Ca2 ions to tosolic Ca2 ions, which increase the affinity of these chan-
exit from the ER into the cytosol (Figure 13-29). When var- nel receptors for IP3, resulting in greater release of stored
ious phosphorylated inositols found in cells are added to Ca2. Higher concentrations of cytosolic Ca2, however, in-
preparations of ER vesicles, only IP3 causes release of Ca2 hibit IP3-induced release of Ca2 from intracellular stores
ions from the vesicles. This simple experiment demonstrates by decreasing the affinity of the receptor for IP3. This com-
the specificity of the IP3 effect. plex regulation of IP3-gated Ca2 channels in ER membranes
The IP3-mediated rise in the cytosolic Ca2 level is only by cytosolic Ca2 can lead to rapid oscillations in the cy-
transient because Ca2 ATPases located in the plasma mem- tosolic Ca2 level when the IP3 pathway in cells is stimu-
13.5 • G Protein–Coupled Receptors That Activate Phospholipase C 563
Store-operated
Ca2+
FIGURE 13-29 IP3/DAG pathway
TRP Ca2+ and the elevation of cytosolic Ca2.
channel
This pathway can be triggered by ligand
Signaling Pathways
Focus Animation: Second Messengers in
MEDIA CONNECTIONS
Phospholipase C
binding to certain G protein–coupled
Exterior receptors and several other receptor
Plasma 1 DAG 5 types, leading to activation of
membrane
7 phospholipase C. Cleavage of PIP2 by
Cytosol PKC phospholipase C yields IP3 and DAG
PIP2 IP3 (step 1 ). After diffusing through the
4 cytosol, IP3 interacts with and opens
6 Ca2 channels in the membrane of the
PKC Phosphorylation endoplasmic reticulum (step 2 ), causing
of substrates
release of stored Ca2 ions into the
Protein cytosol (step 3 ). One of various cellular
kinase C
responses induced by a rise in cytosolic
Ca2 is recruitment of protein kinase C
IP3 (PKC) to the plasma membrane (step 4 ),
3
IP3-gated where it is activated by DAG (step 5 ).
2 Ca2+ channel The activated kinase can phosphorylate
various cellular enzymes and receptors,
thereby altering their activity (step 6 ). As
endoplasmic reticulum Ca2 stores are
depleted, the IP3-gated Ca2 channels
bind to and open store-operated TRP
Ca2 channels in the plasma membrane,
Ca2+ allowing influx of extracellular Ca2
Endoplasmic reticulum
(step 7 ). [Adapted from J. W. Putney, 1999,
Proc. Nat’l. Acad. Sci. USA 96:14669.]
lated. For example, stimulation of hormone-secreting cells in regulate glycogen metabolism by phosphorylating and thus
the pituitary by luteinizing hormone–releasing hormone inhibiting glycogen synthase. Protein kinase C also phospho-
(LHRH) causes rapid, repeated spikes in the cytosolic Ca2 rylates various transcription factors; depending on the cell
level; each spike is associated with a burst in secretion of type; these induce synthesis of mRNAs that trigger cell
luteinizing hormone (LH). The purpose of the fluctuations of proliferation.
Ca2, rather than a sustained rise in cytosolic Ca2, is not
understood. One possibility is that a sustained rise in Ca2
may be toxic to cells. Ca2/Calmodulin Complex Mediates Many
Cellular Responses to External Signals
Ligand binding to several types of receptors, in addition to
Diacylglycerol (DAG) Activates Protein Kinase C,
G protein–coupled receptors, can activate a phospholipase C
Which Regulates Many Other Proteins isoform, leading to an IP3-mediated increase in the cytosolic
After its formation by hydrolysis of PIP2 or other phospho- level of free Ca2. Such localized increases in cytosolic Ca2
inositides, DAG remains associated with the plasma mem- in specific cell types are critical to its function as a second mes-
brane. The principal function of DAG is to activate a family of senger. For example, acetylcholine stimulation of G protein–
protein kinases collectively termed protein kinase C (PKC). In coupled receptors in secretory cells of the pancreas and
the absence of hormone stimulation, protein kinase C is pres- parotid gland induces an IP3-mediated rise in Ca2 that trig-
ent as a soluble cytosolic protein that is catalytically inactive. gers the fusion of secretory vesicles with the plasma mem-
A rise in the cytosolic Ca2 level causes protein kinase C to brane and release of their contents into the extracellular
bind to the cytosolic leaflet of the plasma membrane, where space. In blood platelets, the rise in Ca2 induced by throm-
the membrane-associated DAG can activate it. Thus activation bin stimulation triggers a conformational change in these cell
of protein kinase C depends on an increase of both Ca2 ions fragments leading to their aggregation, an important step in
and DAG, suggesting an interaction between the two branches plugging holes in blood vessels. Secretion of insulin from pan-
of the IP3/DAG pathway (see Figure 13-29). creatic cells also is triggered by Ca2, although the increase
The activation of protein kinase C in different cells re- in Ca2 occurs by a different mechanism (see Figure 15-7).
sults in a varied array of cellular responses, indicating that A small cytosolic protein called calmodulin, which is
it plays a key role in many aspects of cellular growth and me- ubiquitous in eukaryotic cells, functions as a multipurpose
tabolism. In liver cells, for instance, protein kinase C helps switch protein that mediates many cellular effects of Ca2
ions. Binding of Ca2 to four sites on calmodulin yields a novel signaling molecule and provides another example of
complex that interacts with and modulates the activity of cGMP functioning as a second messenger.
many enzymes and other proteins (see Figure 3-28). Because
Ca2 binds to calmodulin in a cooperative fashion, a small
change in the level of cytosolic Ca2 leads to a large change Signal-Induced Relaxation of Vascular
in the level of active calmodulin. One well-studied enzyme Smooth Muscle Is Mediated by cGMP-
activated by the Ca2/calmodulin complex is myosin light- Activated Protein Kinase G
chain kinase, which regulates the activity of myosin in mus-
cle cells (Chapter 19). Another is cAMP phosphodiesterase, Nitroglycerin has been used for over a century as
the enzyme that degrades cAMP to 5-AMP and terminates a treatment for the intense chest pain of angina. It
its effects. This reaction thus links Ca2 and cAMP, one of was known to slowly decompose in the body to ni-
many examples in which two second messengers interact to tric oxide (NO), which causes relaxation of the smooth mus-
fine-tune certain aspects of cell regulation. cle cells surrounding the blood vessels that “feed” the heart
In certain cells, the rise in cytosolic Ca2 following re- muscle itself, thereby increasing the diameter of the blood
ceptor signaling via PLC-generated IP3 leads to activation of vessels and increasing the flow of oxygen-bearing blood to
specific transcription factors. In some cases, Ca2/calmod- the heart muscle. One of the most intriguing discoveries in
ulin activates protein kinases that, in turn, phosphorylate modern medicine is that NO, a toxic gas found in car ex-
transcription factors, thereby modifying their activity and haust, is in fact a natural signaling molecule. ❚
regulating gene expression. In other cases, Ca2/calmodulin
activates a phosphatase that removes phosphate groups from Definitive evidence for the role of NO in inducing relax-
a transcription factor. An important example of this mecha- ation of smooth muscle came from a set of experiments in
nism involves T cells of the immune system in which Ca2 which acetylcholine was added to experimental preparations
ions enhance the activity of an essential transcription factor, of the smooth muscle cells that surround blood vessels. Di-
NFAT (nuclear factor of activated T cells). In unstimulated rect application of acetylcholine to these cells caused them to
cells, phosphorylated NFAT is located in the cytosol. Fol- contract, the expected effect of acetylcholine on these muscle
lowing receptor stimulation and elevation of cytosolic Ca2, cells. But addition of acetylcholine to the lumen of small iso-
the Ca2/calmodulin complex binds to and activates cal- lated blood vessels caused the underlying smooth muscles to
cineurin, a protein-serine phosphatase. Activated calcineurin relax, not contract. Subsequent studies showed that in re-
then dephosphorylates key phosphate residues on cytosolic sponse to acetylcholine the endothelial cells that line the
NFAT, exposing a nuclear localization sequence that allows lumen of blood vessels were releasing some substance that
NFAT to move into the nucleus and stimulate expression of in turn triggered muscle cell relaxation. That substance
genes essential for activation of T cells. turned out to be NO.
The Ca2/calmodulin complex also plays a key role in We now know that endothelial cells contain a Go
controlling the diameter of blood vessels and thus their abil- protein–coupled receptor that binds acetylcholine and acti-
ity to deliver oxygen to tissues. This pathway involves a vates phospholipase C, leading to an increase in the level of
FIGURE 13-30 Regulation of Lumen of Acetylcholine

contractility of arterial smooth muscle blood vessel
by nitric oxide (NO) and cGMP. Nitric
Acetylcholine Phospholipase
oxide is synthesized in endothelial cells GPCR C
in response to acetylcholine and the IP3
subsequent elevation in cytosolic Ca2. Endothelial
NO diffuses locally through tissues and cells
Ca2+/Calmodulin
activates an intracellular NO receptor
with guanylyl cyclase activity in nearby
smooth muscle cells. The resulting rise NO synthase
in cGMP leads to activation of protein

kinase G (PKG), relaxation of the muscle, Arginine + O2 Citrulline + NO
and thus vasodilation. The cell-surface
receptor for atrial natriuretic factor (ANF) NO NO
also has intrinsic guanylyl cyclase activity GTP
NO
(not shown); stimulation of this receptor receptor Protein
cGMP kinase G
on smooth muscle cells also leads to
PPi
increased cGMP and subsequent muscle
relaxation. PPi pyrophosphate. [See
C. S. Lowenstein et al., 1994, Ann. Intern. Smooth muscle cells RELAXATION
Med. 120:227.] OF MUSCLE CELL
13.6 • Activation of Gene Transcription by G Protein–Coupled Receptors 565
cytosolic Ca2. After Ca2 binds to calmodulin, the resulting ■ Stimulation of the acetylcholine GPCR on endothelial
complex stimulates the activity of NO synthase, an enzyme cells induces an increase in cytosolic Ca2 and subsequent
that catalyzes formation of NO from O2 and the amino acid synthesis of NO. After diffusing into surrounding smooth
arginine. Because NO has a short half-life (2–30 seconds), it muscle cells, NO activates intracellular guanylate cyclase
can diffuse only locally in tissues from its site of synthesis. to synthesize cGMP (see Figure 13-30).
In particular NO diffuses from the endothelial cell into ■ Synthesis of cGMP in vascular smooth muscle cells leads
neighboring smooth muscle cells, where it triggers muscle re- to activation of protein kinase G, which triggers a path-
laxation (Figure 13-30). way leading to muscle relaxation and vasodilation.
The effect of NO on smooth muscle is mediated by the
second messenger cGMP, which can be formed by an intra- ■ cGMP is also produced in vascular smooth muscle cells
cellular NO receptor expressed by smooth muscle cells. Bind- by stimulation of cell-surface receptors that have intrinsic
ing of NO to the heme group in this receptor leads to a guanylate cyclase activity. These include receptors for atrial
conformational change that increases its intrinsic guanylyl natriuretic factor (ANF).
cyclase activity, leading to a rise in the cGMP level. Most of
the effects of cGMP are mediated by a cGMP-dependent pro-
tein kinase, also known as protein kinase G (PKG). In vas- 13.6 Activation of Gene Transcription
cular smooth muscle, protein kinase G activates a signaling
pathway that results in inhibition of the actin-myosin com- by G Protein–Coupled Receptors
plex, relaxation of the cell, and dilation of the blood vessel. As mentioned early in this chapter, intracellular signal-
In this case, cGMP acts indirectly via protein kinase G, transduction pathways can have short-term and long-term
whereas in rod cells cGMP acts directly by binding to and effects on the cell. Short-term effects (seconds to minutes)
thus opening cation channels in the plasma membrane. result from modulation of the activity of preexisting enzymes
Relaxation of vascular smooth muscle also is triggered by or other proteins, leading to changes in cell metabolism or
binding of atrial natriuretic factor (ANF) and some other function. Most of the pathways activated by G protein–
peptide hormones to their receptors on smooth muscle cells. coupled receptors fall into this category. However, GPCR sig-
The cytosolic domain of these cell-surface receptors, like the naling pathways also can have long-term effects (hours to
intracellular NO receptor, possesses intrinsic guanylyl cyclase days) owing to activation or repression of gene transcription,
activity. When an increased blood volume stretches cardiac leading in some cases to cell proliferation or to differentia-
muscle cells in the heart atrium, they release ANF. Circulat- tion into a different type of cell. Earlier we discussed how a
ing ANF binds to ANF receptors in smooth muscle cells sur- signal-induced rise in cytosolic Ca2 can lead to activation of
rounding blood vessels, inducing activation of guanylyl transcription factors. Here we consider other mechanisms
cyclase activity and formation of cGMP. Subsequent activa- by which some G protein–coupled receptors regulate gene
tion of protein kinase G causes dilation of the vessel by the expression.
mechanism described above. This vasodilation reduces blood
pressure and counters the stimulus that provoked the initial
release of ANF. Membrane-Localized Tubby Transcription Factor
Is Released by Activation of Phospholipase C
KEY CONCEPTS OF SECTION 13.5 The tubby gene, which is expressed primarily in
certain areas of the brain involved in control of
G Protein–Coupled Receptors That Activate eating behavior, first attracted attention because of
Phospholipase C its involvement in obesity. Mice bearing mutations in the
■ Simulation of some GPCRs and other cell-surface re- tubby gene develop adult-onset obesity, and certain aspects
ceptors leads to activation of phospholipase C, which gen- of their metabolism resemble that of obese humans.
erates two second messengers: diffusible IP3 and mem- Sequencing of the cloned tubby gene suggested that its en-
brane-bound DAG (see Figure 13-28). coded protein contains both a DNA-binding domain and a
transcription-activation domain (Chapter 11). However, the
■ IP3 triggers opening of IP3-gated Ca2 channels in the
Tubby protein was found to be localized near the plasma
endoplasmic reticulum and elevation of cytosolic free
membrane, making it an unlikely candidate as a transcription
Ca2. In response to elevated cytosolic Ca2, protein ki-
factor. Subsequent studies revealed that Tubby binds tightly
nase C is recruited to the plasma membrane, where it is
to PIP2, anchoring the protein to the plasma membrane (Fig-
activated by DAG (see Figure 13-29).
ure 13-31). Hormone binding to Go- or Gq-coupled receptors,
■ The Ca2/calmodulin complex regulates the activity of which activate phospholipase C, leads to hydrolysis of PIP2
many different proteins, including cAMP phosphodi- and release of Tubby into the cytosol. Tubby then enters the
esterase, nitric oxide synthase, and protein kinases or phos- nucleus and activates transcription of a still unknown gene or
phatases that control the activity of various transcription genes. Identification of these genes should provide clues about
factors. how their encoded proteins relate to obesity. ❚
Phospholipase C
FIGURE 13-31 Activation of the Tubby transcription
factor following ligand binding to receptors coupled to Go or
DAG Exterior
Gq. In resting cells, Tubby is bound tightly to PIP2 in the plasma
PIP2 membrane. Receptor stimulation (not shown) leads to activation
IP3 of phospholipase C, hydrolysis of PIP2, and release of Tubby into
1
Cytosol the cytosol ( 1 ). Directed by two functional nuclear localization
sequences (NLS) in its N-terminal domain, Tubby translocates into
Transcriptional the nucleus ( 2 ) and activates transcription of target genes ( 3 ). It
activation
domain is not known whether IP3 remains bound to Tubby. [Adapted from
Tubby
S. Santagata et al., 2001, Science 292:2041.]
DNA binding
domain
Nucleus
Transcription
FIGURE 13-32 Activation of gene expression Gs protein–coupled Adenylyl

following ligand binding to Gs protein–coupled receptor cyclase
receptors. Receptor stimulation ( 1 ) leads to activation of
Exterior
PKA ( 2 ). Catalytic subunits of PKA translocate to the
nucleus ( 3 ) and there phosphorylate and activate the 1
transcription factor CREB ( 4 ). Phosphorylated CREB
associates with the co-activator CBP/P300 ( 5 ) to
Cytosol
stimulate various target genes controlled by the CRE PKA
regulatory element. See the text for details. [See K. A. Lee
C R R C 2
and N. Masson, 1993, Biochim. Biophys. Acta 1174:221, and
D. Parker et al., 1996, Mol. Cell Biol. 16(2):694.]
cAMP
Overview Animation: Extracellular Signaling
R R
C
C
3
MEDIA CONNECTIONS
ATP
CREB C
ADP
4 CBP/P300
Nucleus
P
5
Basal transcription
P P machinery
CRE
Transcription
Perspectives for the Future 567
CREB Links cAMP Signals to Transcription in part from activation of the MAP kinase cascade. As just de-
In mammalian cells, an elevation in the cytosolic cAMP level scribed, the GPCR-arrestin complex can trigger this cascade.
stimulates the expression of many genes. For instance, in- Another, perhaps more important, way that activation
creased cAMP in certain endocrine cells induces production of -adrenergic receptors promotes cardiac hypertrophy in-
of somatostatin, a peptide that inhibits release of various volves another type of receptor. The Gs protein activated
hormones; in liver cells, cAMP induces synthesis of several by -adrenergic receptors can somehow lead to activation
enzymes involved in converting three-carbon compounds to of a specific extracellular metal-containing protease that, in
glucose. turn, cleaves the transmembrane precursor of epidermal
All genes regulated by cAMP contain a cis-acting DNA growth factor (EGF). The soluble EGF released into the ex-
sequence, the cAMP-response element (CRE), that binds the tracellular space binds to and activates EGF receptors on the
phosphorylated form of a transcription factor called CRE- same cell in an autocrine fashion. As we learn in the next
binding (CREB) protein, which is found only in the nucleus. chapter, the EGF receptor belongs to the receptor tyrosine ki-
As discussed previously, binding of neurotransmitters and nase (RTK) class of receptors, which commonly trigger the
hormones to Gs protein–coupled receptors activates adenylyl MAP kinase cascade leading to cell proliferation. Similar
cyclase, leading to an increase in cAMP and subsequent re- cross-talk between two types of receptors occurs in many
lease of the active catalytic subunit of PKA. Some of the cat- other signaling systems. Just as no cell lives in isolation, no
alytic subunits then translocate to the nucleus and receptor and no signal-transduction pathway function by
phosphorylate serine-133 on CREB protein. themselves. ❚
Phosphorylated CREB protein binds to CRE-containing
target genes and also interacts with a co-activator termed
KEY CONCEPTS OF SECTION 13.6
CBP/300, which links CREB to the basal transcriptional ma-
chinery, thereby permitting CREB to stimulate transcription Activation of Gene Transcription by G Protein–Coupled
(Figure 13-32). Earlier studies suggested that phosphoryla- Receptors
tion induced a conformational change in CREB protein, but ■ Activation of phospholipase C by receptors coupled to
more recent work indicates that CBP/P300 binds specifically Go or Gq proteins releases the Tubby transcription factor,
to phosphoserine-133 in activated CREB. As discussed in which is bound to PIP2 embedded in the plasma membrane
Chapter 11, other signal-regulated transcription factors rely of resting cells (see Figure 13-31).
on CBP/P300 to exert their activating effect. Thus this co-
activator plays an important role in integrating signals from ■ Signal-induced activation of protein kinase A (PKA) of-
multiple signaling pathways that regulate gene transcription. ten leads to phosphorylation of CREB protein, which to-
gether with the CBP/300 co-activator stimulates transcrip-
tion of many target genes (see Figure 13-32).
GPCR-Bound Arrestin Activates Several Kinase
■ The GPCR-arrestin complex activates several cytosolic
Cascades That Control Gene Expression kinases, initiating cascades that lead to transcriptional ac-
We saw earlier that binding of -arrestin to phosphorylated tivation of many genes controlling cell growth.
serines in the cytosolic domain of G protein–coupled recep-
tors both blocks activation of G
and mediates endocytosis
of the GPCR-arrestin complex. Perhaps surprisingly, the
GPCR-arrestin complex also acts as a scaffold for binding PERSPECTIVES FOR THE FUTURE
and activating several cytosolic kinases (see Figure 13-19).
These include c-Src, which activates the MAP kinase path- Very soon we will know the identity of all the pieces in many
way and other pathways leading to transcription of genes signal-transduction pathways, but putting the puzzle to-
needed for cell division. A complex of three arrestin-bound gether to predict cellular responses remains elusive. For
proteins, including a Jun N-terminal kinase (JNK-1), initiates instance, we can enumerate the G proteins, kinases, phos-
a kinase cascade that ultimately activates the c-Jun tran- phatases, arrestins, and other proteins that participate in sig-
scription factor. Activated c-Jun promotes expression of cer- naling from -adrenergic receptors in liver cells, but we are
tain growth-promoting enzymes and other proteins that help still far from being able to predict, quantitatively, how liver
cells respond to some stresses. cells react over time to a given dose of adrenaline. In part this
is because complex feedback (and in some cases feed-for-
Binding of epinephrine to the -adrenergic receptors ward) loops regulate the activity of multiple enzymes and
in heart muscle stimulates glycogenolysis and en- other components in the pathway. Although biochemical and
hances the rate of muscle contraction. Prolonged cell biological experiments tell us how these interactions
treatment with epinephrine, however, induces proliferation of occur, we cannot describe quantitatively the rates or extent
these cardiac muscle cells. In extreme cases, such cardiac hy- of these reactions in living cells.
pertrophy causes failure of the heart muscle, a major cause of The emerging field of biological systems analysis attempts
heart disease. This epinephrine-induced cell proliferation results to develop an integrated view of a cell’s response to external

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13.

3 • G Protein–Coupled Receptors That Activate or Inhibit Adenylyl Cyclase 545

13.3 G Protein–Coupled ▲ FIGURE 13-10 Schematic diagram of the general

The signal-transducing G proteins contain three subunits Hormone

The G Subunit of G Proteins Cycles Gβγ Gα

mation, causing it to bind to the G

Some bacterial toxins contain a subunit that pene- NH3+

activate the transducing G

cult to crystallize, scientists prepared two protein fragments

Stimulatory Epinephrine Inhibitory PGE1

HOCH2 HOCH2 HOCH2

HOCH2 HOCH2 HOCH2

HOCH2 HOCH2 HOCH2

(a) Increased cAMP

GPK GPK P GS GS P Inhibition of

PKA Protein kinase A

mAKAP C mAKAP C C mAKAP C

4 Return to resting state

11-cis -Retinal moiety Activation of Rhodopsin Induces Closing

present in the dark acts to keep cGMP-gated cation channels N

(dark adapted) opsin

Activation of Slightly reduced Greatly reduced No

▲ FIGURE 13-26 Role of opsin phosphorylation in Gt

■ The effector protein activated by Gt

13.5 G Protein–Coupled Receptors

FIGURE 13-30 Regulation of Lumen of Acetylcholine

in cGMP leads to activation of protein

FIGURE 13-32 Activation of gene expression Gs protein–coupled Adenylyl

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