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15.3 G PROTEIN-COUPLED RECEPTORS AND THEIR SECOND MESSENGERS 611

FIGURE 15.5 The mechanism of receptor-mediated activation (or in-

hibition) of effectors by means of heterotrimeric G proteins. In step 1, Ligand
the ligand binds to the receptor, altering its conformation and increasing Receptor
its affinity for the G protein to which it binds. In step 2, the G␣ subunit 1
releases its GDP, which is replaced by GTP. In step 3, the G␣ subunit
dissociates from the G␤␥ complex and binds to an effector (in this case γ γ
α α
adenylyl cyclase), activating the effector. The G␤␥ dimer may also bind G protein β β
to an effector (not shown), such as an ion channel or an enzyme. In step GDP GDP
4, activated adenylyl cyclase produces cAMP. In step 5, the GTPase ac-
tivity of G␣ hydrolyzes the bound GTP, deactivating G␣. In step 6, G␣ Effector
reassociates with G␤␥, reforming the trimeric G protein, and the effec-
tor ceases its activity. In step 7, the receptor has been phosphorylated by 2
a GRK and in step 8 the phosphorylated receptor has been bound by an
γ
arrestin molecule, which inhibits the ligand-bound receptor from acti- α
vating additional G proteins. The receptor bound to arrestin is likely to β
be taken up by endocytosis.
GTP GDP

GTP, the G␣ subunit will dissociate from the effector and

reassociate with the ␤␥ subunit to reform the inactive het- 3
erotrimeric G protein (step 6). In a sense, heterotrimeric
γ
G proteins function as molecular timers. They are turned on α
by the interaction with an activated receptor and turn them- β
GTP
selves off by hydrolysis of bound GTP after a certain amount
of time has passed. While they are active, G␣ subunits can
turn on downstream effectors.
Heterotrimeric G proteins come in four flavors, Gs, Gq, 4

Gi, and G12/13. This classification is based on the G␣ sub- γ

units and the effectors to which they couple. The particular re- α
β
sponse elicited by an activated GPCR depends on the type of GTP
G protein with which it interacts, although some GPCRs can ATP cAMP
interact with different G proteins and trigger more than one
physiologic response. Gs family members couple receptors to
adenylyl cyclase. Adenylyl cyclase is activated by GTP-bound 5
Gs subunits. Gq family members contain G␣ subunits that ac-
γ
tivate PLC␤. PLC␤ hydrolyzes phosphatidylinositol bisphos- α
β
phate, producing inositol trisphosphate and diacylglycerol
GDP
(page 616). Activated Gi subunits function by inhibiting +
adenylyl cyclase. G12/13 members are less well characterized than Pi
the other G protein families although their inappropriate acti-
vation has been associated with excessive cell proliferation and 6
malignant transformation. Following its dissociation from the
G␣ subunit, the ␤␥ complex also has a signaling function and γ
α
it can couple to at least four different types of effectors: PLC␤, β
K⫹ ion channels, adenylyl cyclase, and PI 3-kinase. GDP

Termination of the Response We have seen that ligand

binding results in receptor activation. The activated receptors 7
turn on G proteins, and G proteins turn on effectors. To prevent
overstimulation, receptors have to be blocked from continuing GRK P γ
α
to activate G proteins. To regain sensitivity to future stimuli, the ATP P β
GDP
receptor, the G protein, and the effector must all be returned to ADP
their inactive state. Desensitization, the process that blocks ac-
tive receptors from turning on additional G proteins, takes
place in two steps. In the first step, the cytoplasmic domain of 8
the activated GPCR is phosphorylated by a specific type of ki-
nase, called G protein-coupled receptor kinase (GRK ) (Figure P γ
α
15.5, step 7). GRKs form a small family of serine-threonine P β
Arrestin GDP
protein kinases that specifically recognize activated GPCRs.
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612 Chapter 15 CELL SIGNALING AND SIGNAL TRANSDUCTION: COMMUNICATION BETWEEN CELLS

Phosphorylation of the GPCR sets the stage for the returned to the cell surface, the cells remain sensitive to the
second step, which is the binding of proteins, called arrestins ligand.
(Figure 15.5, step 8). Arrestins form a small group of pro- Signaling by the activated G␣ subunit is terminated by a
teins that bind to GPCRs and compete for binding with less complex mechanism: the bound GTP molecule is simply
heterotrimeric G proteins. As a consequence, arrestin binding hydrolyzed to GDP (step 5, Figure 15.5). Thus, the strength
prevents the further activation of additional G proteins. This and duration of the signal are determined in part by the rate of
action is termed desensitization because the cell stops re- GTP hydrolysis by the G␣ subunit. G␣ subunits possess a
sponding to the stimulus, while that stimulus is still acting on weak GTPase activity, which allows them to slowly hydrolyze
the outer surface of the cell. Desensitization is one of the the bound GTP and inactivate themselves. Termination of the
mechanisms that allows a cell to respond to a change in its en- response is accelerated by regulators of G protein signaling
vironment, rather than continuing to “fire” endlessly in the (RGSs). The interaction with an RGS protein increases the
presence of an unchanging environment. The importance of rate of GTP hydrolysis by the G␣ subunit. Once the GTP is
desensitization is illustrated by the observation that mutations hydrolyzed, the G␣-GDP reassociates with the G␤␥ subunits
that interfere with phosphorylation of rhodopsin by a GRK to reform the inactive trimeric complex (step 6) as discussed
lead to the death of the photoreceptor cells in the retina. This above. This returns the system to the resting state.
type of retinal cell death is thought to be one of the causes of The mechanism for transmitting signals across the
blindness resulting from the disease retinitis pigmentosa. plasma membrane by G proteins is of ancient evolutionary
While they are bound to phosphorylated GPCRs, ar- origin and is highly conserved. This is illustrated by an exper-
restin molecules are also capable of binding to clathrin mole- iment in which yeast cells were genetically engineered to ex-
cules that are situated in clathrin-coated pits (page 302). The press a receptor for the mammalian hormone somatostatin.
interaction between bound arrestin and clathrin promotes the When these yeast cells were treated with somatostatin, the
uptake of phosphorylated GPCRs into the cell by endocyto- mammalian receptors at the cell surface interacted with the
sis. Depending on the circumstances, receptors that have been yeast heterotrimeric G proteins at the inner surface of the
removed from the surface by endocytosis may be dephospho- membrane and triggered a response leading to proliferation of
rylated and returned to the plasma membrane. Alternatively, the yeast cells.
internalized receptors are degraded in the lysosomes (see Fig- The effects of certain mutations on the function of G
ure 8.42). If receptors are degraded, the cells lose, at least tem- protein-coupled receptors are discussed in the accompanying
porarily, sensitivity for the ligand in question. If receptors are Human Perspective.

THE HUMAN PERSPECTIVE

Disorders Associated with G Protein-Coupled Receptors
GPCRs represent the largest family of genes encoded by the human gene that encodes rhodopsin, the visual pigment of the rods. Many
genome. Their importance in human biology is reflected by the fact of these mutations lead to premature termination or improper fold-
that over one-third of all prescription drugs act as ligands that bind ing of the rhodopsin protein and its elimination from the cell before
to this huge superfamily of receptors. A number of inherited disor- it reaches the plasma membrane (page 282). Other mutations may
ders have been traced to defects in both GPCRs (Figure 1) and het-
erotrimeric G proteins (Table 1). Retinitis pigmentosa (RP) is an
inherited disease characterized by progressive degeneration of the
NH2
retina and eventual blindness. RP can be caused by mutations in the Extracellular

FIGURE 1 Two-dimensional representation of a “composite” transmem- 2

8
brane receptor showing the approximate sites of a number of mutations 7
responsible for causing human diseases. Most of the mutations (numbers 3
1 4
1, 2, 5, 6, 7, and 8) result in constitutive stimulation of the effector, but 6
others (3 and 4) result in blockage of the receptor’s ability to stimulate
Intracellular 5
the effector. Mutations at sites 1 and 2 are found in the MSH
(melanocyte-stimulating hormone) receptor; 3 in the ACTH (adreno-
corticotrophic hormone) receptor; 4 in the vasopressin receptor; 5 and 6
in the TSH (thyroid-stimulating hormone) receptor; 7 in the LH
(luteinizing hormone) receptor; and 8 in rhodopsin, the light-sensitive
pigment of the retina.
COOH
 15.4 PROTEIN-TYROSINE PHOSPHORYLATION AS A MECHANISM FOR SIGNAL TRANSDUCTION 631

Signaling by the Insulin Receptor kinase domains on the inside of the cell to come into close
physical proximity (Figure 15.22b). Juxtaposition of the ki-
Our bodies spend considerable effort maintaining blood glu-
nase domains leads to trans-autophosphorylation and recep-
cose levels within a narrow range. A decrease in blood glucose
tor activation (Figure 15.22c).
levels can lead to loss of consciousness and coma, as the cen-
Several tyrosine phosphorylation sites have been identi-
tral nervous system depends largely on glucose for its energy
fied in the cytoplasmic region of the insulin receptor. Three of
metabolism. A persistent elevation in blood glucose levels re-
these phosphorylation sites are present in the activation loop.
sults in a loss of glucose, fluids, and electrolytes in the urine
In the unphosphorylated state, the activation loop assumes a
and serious health problems. The levels of glucose in the cir-
conformation in which it occupies the active site. Upon phos-
culation are monitored by the pancreas. When blood glucose
phorylation of the three tyrosine residues, the activation loop
levels fall below a certain level, the alpha cells of the pancreas
assumes a new conformation away from the catalytic cleft.
secrete glucagon. As discussed earlier, glucagon acts through
This new conformation requires a rotation of the small and
GPCRs and stimulates the breakdown of glycogen resulting
large lobes of the kinase domain with respect to each other,
in an increase in blood glucose levels. When glucose levels
thereby bringing residues that are essential for catalysis closer
rise, as occurs after a carbohydrate-rich meal, the beta cells of
together. In addition, the activation loop now leaves the cat-
the pancreas respond by secreting insulin. Insulin functions as
alytic cleft open so that it can bind substrates. Following acti-
an extracellular messenger molecule, informing cells that glu-
vation of the kinase domain, the receptor phosphorylates itself
cose levels are high. Cells that express insulin receptors on
on tyrosine residues that are present adjacent to the mem-
their surface, such as cells in the liver, respond to this message
brane and in the carboxyl-terminal tail (Figure 15.22c).
by increasing glucose uptake, increasing glycogen and triglyc-
eride synthesis, and decreasing gluconeogenesis.
Insulin Receptor Substrates 1 and 2 Most RTKs possess
The Insulin Receptor Is a Protein-Tyrosine Kinase Each autophosphorylation sites that directly recruit SH2 domain-
insulin receptor is composed of an ␣ and a ␤ chain, which are containing signaling proteins (as in Figure 15.17a, c, and d ).
derived from a single precursor protein by proteolytic process- The insulin receptor is an exception to this general rule, be-
ing. The ␣ chain is entirely extracellular and contains the cause it associates instead with a small family of docking
insulin-binding site. The ␤ chain is composed of an extracel- proteins (Figure 15.17b), called insulin-receptor substrates
lular region, a single transmembrane region, and a cytoplasmic (IRSs). The IRSs, in turn, provide the binding sites for SH2
region (Figure 15.22). The ␣ and ␤ chains are linked together domain-containing signaling proteins. Some of the events
by disulfide bonds (Figure 15.22). Two of these ␣␤ het- that occur during insulin signaling are shown in Figure 15.23.
erodimers are held together by disulfide bonds between the Following ligand binding and kinase activation, the insulin
␣ chains. Thus, while most RTKs are thought to be present on receptor autophosphorylates tyrosine 960, which then forms a
the cell surface as monomers, insulin receptors are present as binding site for the phosphotyrosine binding (PTB) domains
stable dimers. Like other RTKs, insulin receptors are inactive of insulin receptor substrates. As indicated in Figure 15.23a,
in the absence of ligand (Figure 15.22a). Recent work sug- IRSs are characterized by the presence of an N-terminal PH
gests that the insulin receptor dimer binds a single insulin domain, a PTB domain, and a long tail containing tyrosine
molecule. This causes repositioning of the ligand-binding phosphorylation sites. The PH domain may interact with
domains on the outside of the cell, which causes the tyrosine phospholipids present at the inside leaflet of the plasma mem-

Insulin

α chain

P P

P P
β chain P P

Tyrosine
kinase
domain
(a) (b) (c)
FIGURE 15.22 The response of the insulin receptor to ligand binding. in the ␤ subunits, which activates the tyrosine kinase activity of the ␤ sub-
(a) The insulin receptor, shown here in schematic form in the inactive units. (c) The activated ␤ subunits phosphorylate tyrosine residues located
state, is a tetramer consisting of two ␣ and two ␤ subunits. (b) Binding of on the cytoplasmic domain of the receptor as well as tyrosine residues on
a single insulin molecule to the ␣ subunits causes a conformational change several insulin receptor substrates (IRSs) that are discussed below.
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632 Chapter 15 CELL SIGNALING AND SIGNAL TRANSDUCTION: COMMUNICATION BETWEEN CELLS

PI3K Grb2 Shp2 PI 3-kinase

N PH PTB YYY YY YY YY Y Y Y YY Y Y Y C PI(4,5)P2 PI(3,4,5)P3

(a)

Plasma membrane
Insulin receptor

PTB domain
3'
5'

4
P PIP3 3 5
Protein synthesis
P
Ras P
P P P
P P
P GTP
P PH domain
P P P Grb2 Sos
P
IRS-1
Glucose uptake P
P
Activated PDK1
P PKB
Other P
signaling
proteins
PI3K Glycogen synthesis
(2 subunits)
(b) (c)

FIGURE 15.23 The role of tyrosine-phosphorylated IRS in activating a molecule for purposes of illustration.) (c) Activation of PI3K leads to the
variety of signaling pathways. (a) Schematic representation of an IRS formation of membrane-bound phosphoinositides, including PIP3. One
polypeptide. The N-terminal portion of the molecule contains a PH do- of the key kinases in numerous signaling pathways is PKB (AKT), which
main that allows it to bind to phosphoinositides of the membrane and a interacts at the plasma membrane with PIP3 by means of a PH domain on
PTB domain that allows it to bind to a specific phosphorylated tyrosine the protein. This interaction changes the conformation of the PKB mole-
residue (#960) on the cytoplasmic domain of an activated insulin receptor. cule, making it a substrate for another PIP3-bound kinase (PDK1), which
Once bound to the insulin receptor, a number of tyrosine residues in the phosphorylates PKB. The second phosphate shown linked to PKB is
IRS may be phosphorylated (indicated as Y). These phosphorylated ty- added by a second kinase, mostly likely mTOR. Once activated, PKB
rosines can serve as binding sites for other proteins, including a lipid dissociates from the plasma membrane and moves into the cytosol and
kinase (PI3K), an adaptor protein (Grb2), and a protein-tyrosine phos- nucleus. PKB is a major component of a number of separate signaling
phatase (Shp2). (b) Phosphorylation of IRSs by the activated insulin pathways that mediate the insulin response. These pathways lead to
receptor is known to activate PI3K and Ras pathways, both of which are translocation of glucose transporters to the plasma membrane, synthesis of
discussed in the chapter. Other pathways that are less well defined are also glycogen, and the synthesis of new proteins in the cell.
activated by IRSs. (The IRS is drawn as an extended, two-dimensional

brane, the PTB domain binds to tyrosine phosphorylation directly as a consequence of binding of its two SH2 domains to
sites on the activated receptor, and the tyrosine phosphoryla- tyrosine phosphorylation sites, phosphorylates phosphoinosi-
tion sites provide docking sites for SH2 domain-containing tides at the 3 position of the inositol ring (Figure 15.23c). The
signaling proteins. At least four members of the IRS family products of this enzyme, which include PI 3,4-bisphosphate
have been identified. Based on the results obtained in knock- (PIP2) and PI 3,4,5-trisphosphate (PIP3), remain in the cytoso-
out experiments in mice, it is thought that IRS-1 and IRS-2 lic leaflet of the plasma membrane where they provide binding
are most relevant to insulin-receptor signaling. sites for PH domain-containing signaling proteins such as the
Autophosphorylation of the activated insulin receptor at serine-threonine kinases PKB and PDK1. As indicated in Fig-
tyrosine 960 provides a binding site for IRS-1 or IRS-2. Only ure 15.23c, PKB (also known as AKT) plays a role in mediat-
after stable association with either IRS-1 or IRS-2 is the acti- ing the response to insulin, as well as to other extracellular
vated insulin receptor able to phosphorylate tyrosine residues signals. Recruitment of PDK1 to the plasma membrane, in
present on these docking proteins (Figure 15.23b). Both IRS-1 close proximity to PKB, provides a setting in which PDK1 can
and IRS-2 contain a large number of potential tyrosine phos- phosphorylate and activate PKB (Figure 15.23c). While phos-
phorylation sites that include binding sites for the SH2 do- phorylation by PDK1 is essential, it is not sufficient for activa-
mains of PI 3-kinase, Grb2, and Shp2 (Figure 15.23a,b). tion of PKB. Activation of PKB also depends on
These proteins associate with the receptor-bound IRS-1 or phosphorylation by a second kinase, most likely mTOR.
IRS-2 and activate downstream signaling pathways.
PI 3-kinase (PI3K) is composed of two subunits, one con- Glucose Transport PKB is directly involved in regulating
taining two SH2 domains and the other containing the cat- glucose transport and glycogen synthesis. The glucose trans-
alytic domain (Figure 15.23b). PI 3-kinase, which is activated porter GLUT4 carries out insulin-dependent glucose trans-
 15.4 PROTEIN-TYROSINE PHOSPHORYLATION AS A MECHANISM FOR SIGNAL TRANSDUCTION 633

Plasma Glucose glycogen synthase activity (Figure 15.23c). Activation of protein

membrane GLUT4
phosphatase 1, an enzyme known to dephosphorylate glycogen
IR
synthase, contributes further to glycogen synthase activation.

Diabetes Mellitus One of the most common human dis-

IRS-1 eases, diabetes mellitus, is caused by defects in insulin signal-
ing. Diabetes occurs in two varieties: type 1, which accounts

Fusion
PI3K for 5–10 percent of the cases, and type 2, which accounts for
the remaining 90–95 percent. Type 1 diabetes is caused by an
GLUT4 inability to produce insulin and is discussed in the Human
PDKI
Perspective of Chapter 17. Type 2 diabetes is a more complex
PKB disease whose incidence is increasing around the world at an
alarming rate. The rising incidence of the disease is most
likely a result of changing lifestyle and eating habits. A high-
calorie diet combined with a sedentary lifestyle is thought to
lead to a chronic increase in insulin secretion. Elevated levels
Cytoplasmic
of insulin overstimulate target cells in the liver and elsewhere
vesicle in the body, which leads to a condition referred to as insulin re-
sistance, in which these target cells stop responding to the
FIGURE 15.24 Regulation of glucose uptake in muscle and fat cells by
presence of the hormone. According to genetic experiments in
insulin. Glucose transporters are stored in the walls of cytoplasmic vesi-
cles that form by budding from the plasma membrane (endocytosis). mice, mutations in the genes for either the insulin receptor or
When the insulin level increases, a signal is transmitted through the IRS-2, which renders cells unable to respond to insulin, cre-
IRS-PI3K-PKB pathway, which triggers the translocation of cytoplas- ates a diabetic phenotype. However, mutations in these genes
mic vesicles to the cell periphery. The vesicles fuse with the plasma are rare in the human population and the exact basis of insulin
membrane (exocytosis), delivering the transporters to the cell surface resistance remains to be established.
where they can mediate glucose uptake. A second pathway leading from
the insulin receptor to GLUT4 translocation is not shown (see Trends Insulin Signaling and Lifespan We saw on page 34 that
Biochem. Sci. 31:215, 2006). (AFTER D. VOET AND J. G. VOET, BIOCHEM- the lifespan of an animal can be significantly increased by re-
ISTRY, 2D ED.; COPYRIGHT © 1995, JOHN WILEY & SONS, INC. REPRINTED BY
ducing its food intake (i.e., calorie restriction). A number of
PERMISSION OF JOHN WILEY & SONS, INC.)
early studies demonstrated that the lifespan of a worm or fruit
fly can also be increased by decreasing the level of insulin (or
insulin-like growth factors) that circulate in their blood-
port from the blood (page 152). In the absence of insulin, stream. Studies of humans support this relationship; humans
GLUT4 is present in membrane vesicles in the cytoplasm of that live exceptionally long lives often exhibit unusually high
insulin responsive cells (Figure 15.24). These vesicles fuse with insulin sensitivity—that is, their tissues respond fully to rela-
the plasma membrane in response to insulin, a process that is tively low circulating insulin levels. Thus, just as increased in-
referred to as GLUT4 translocation. The increase in numbers sulin resistance is associated with poor health, increased
of glucose transporters in the plasma membrane leads to in- insulin sensitivity appears to be associated with good health.
creased glucose uptake (Figure 15.24). GLUT4 translocation It is interesting to note that calorie restriction in laboratory
depends on activation of PI 3-kinase and PKB. This conclu- animals leads to decreased insulin levels and increased insulin
sion is based on experiments showing that inhibitors of PI3K sensitivity, so that these two paths to increased longevity may
block GLUT4 translocation. In addition, overexpression of be acting by the same mechanism.
PI3K or PKB stimulates GLUT4 translocation. It is well
known that many receptors activate PI3K, whereas it is only
Signaling Pathways in Plants
the insulin receptor that stimulates GLUT4 translocation.
This suggests that there is a second pathway downstream of the Plants and animals share certain basic signaling mechanisms,
insulin receptor that is essential for GLUT4 translocation to oc- including the use of Ca2⫹ and phosphoinositide messengers,
cur. Detailed understanding of how the two pathways work to- but other pathways are unique to each major kingdom. For
gether to stimulate GLUT4 translocation is still lacking. example, cyclic nucleotides, which may be the most ubiqui-
Excess glucose that is taken up by muscle and liver cells is tous animal cell messengers, appear to play little, if any, role
stored in the form of glycogen. Glycogen synthesis is carried in plant cell signaling. Receptor tyrosine kinases are also lack-
out by glycogen synthase, an enzyme that is turned off by phos- ing in plant cells. On the other hand, plants contain a type of
phorylation on serine and threonine residues. Glycogen syn- protein kinase that is absent from animal cells.
thase kinase-3 (GSK-3) has been identified as a negative It has long been known that bacterial cells have a protein
regulator of glycogen synthase. GSK-3, in turn, is inactivated kinase that phosphorylates histidine residues and mediates the
following phosphorylation by PKB. Thus, activation of the cell’s response to a variety of environmental signals. Until
PI 3-kinase-PKB pathway in response to insulin leads to a 1993, these enzymes were thought to be restricted to bacterial
decrease in GSK-3 kinase activity, resulting in an increase in cells but were then discovered in both yeast and flowering