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Biosignaling
1. Hormone Experiments in Cell-Free Systems In the 1950s, Earl W. Sutherland, Jr., and his
colleagues carried out pioneering experiments to elucidate the mechanism of action of epinephrine
and glucagon. Given what you have learned in this chapter about hormone action, interpret each
of the experiments described below. Identify substance X and indicate the significance of the
results.
(a) Addition of epinephrine to a homogenate of normal liver resulted in an increase in the activity of
glycogen phosphorylase. However, if the homogenate was first centrifuged at a high speed and
epinephrine or glucagon was added to the clear supernatant fraction that contains phosphory-
lase, no increase in the phosphorylase activity occurred.
(b) When the particulate fraction from the centrifugation in (a) was treated with epinephrine,
substance X was produced. The substance was isolated and purified. Unlike epinephrine,
substance X activated glycogen phosphorylase when added to the clear supernatant fraction of
the centrifuged homogenate.
(c) Substance X was heat-stable; that is, heat treatment did not affect its capacity to activate phos-
phorylase. (Hint: Would this be the case if substance X were a protein?) Substance X was nearly
identical to a compound obtained when pure ATP was treated with barium hydroxide. (Fig. 8–6
will be helpful.)
2. Effect of Dibutyryl cAMP versus cAMP on Intact Cells The physiological effects of epinephrine
should in principle be mimicked by addition of cAMP to the target cells. In practice, addition of
cAMP to intact target cells elicits only a minimal physiological response. Why? When the structurally
related derivative dibutyryl cAMP (shown below) is added to intact cells, the expected physiological
response is readily apparent. Explain the basis for the difference in cellular response to these two
substances. Dibutyryl cAMP is widely used in studies of cAMP function.
chapter
12
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S-132 Chapter 12 Biosignaling
3. Effect of Cholera Toxin on Adenylyl Cyclase The gram-negative bacterium Vibrio cholerae
produces a protein, cholera toxin (M
r
90,000), that is responsible for the characteristic symptoms of
cholera: extensive loss of body water and Na
⫹
through continuous, debilitating diarrhea. If body fluids
and Na
⫹
are not replaced, severe dehydration results; untreated, the disease is often fatal. When the
cholera toxin gains access to the human intestinal tract it binds tightly to specific sites in the plasma
membrane of the epithelial cells lining the small intestine, causing adenylyl cyclase to undergo pro-
longed activation (hours or days).
(a) What is the effect of cholera toxin on [cAMP] in the intestinal cells?
(b) Based on the information above, suggest how cAMP normally functions in intestinal epithelial
cells.
(c) Suggest a possible treatment for cholera.
4. Mutations in PKA Explain how mutations in the R or C subunit of cAMP-dependent protein kinase
(PKA) might lead to (a) a constantly active PKA or (b) a constantly inactive PKA.
5. Therapeutic Effects of Albuterol The respiratory symptoms of asthma result from constriction of
the bronchi and bronchioles of the lungs caused by contraction of the smooth muscle of their walls.
This constriction can be reversed by raising the [cAMP] in the smooth muscle. Explain the therapeutic
effects of albuterol, a b-adrenergic agonist taken (by inhalation) for asthma. Would you expect this
drug to have any side effects? How might one design a better drug that did not have these effects?
Dibutyryl cAMP
(N
6
,O
2
⬘-Dibutyryl adenosine 3⬘,5⬘-cyclic monophosphate)
(CH
2
)
2
CH
3
CH
2
C
(CH
2
)
2
CH
3
C
O
OO O
O
H
H
H
H
N
NH
N
NN
O
P
O
O
⫺
6. Termination of Hormonal Signals Signals carried by hormones must eventually be terminated.
Describe several different mechanisms for signal termination.
7. Using FRET to Explore Protein-Protein Interactions in Vivo Figure 12–8 shows the interaction
between -arrestin and the -adrenergic receptor. How would you use FRET (see Box 12–3) to
demonstrate this interaction in living cells? Which proteins would you fuse? Which wavelengths would
you use to illuminate the cells, and which would you monitor? What would you expect to observe if
the interaction occurred? If it did not occur? How might you explain the failure of this approach to
demonstrate this interaction?
8. EGTA Injection EGTA (ethylene glycol-bis(b-aminoethyl ether)-N,N,N⬘,N⬘-tetraacetic acid) is a
chelating agent with high affinity and specificity for Ca
2⫹
. By microinjecting a cell with an appropriate
Ca
2⫹
-EGTA solution, an experimenter can prevent cytosolic [Ca
2⫹
] from rising above 10
⫺7
M
. How
would EGTA microinjection affect a cell’s response to vasopressin (see Table 12–4)? To glucagon?
9. Amplification of Hormonal Signals Describe all the sources of amplification in the insulin receptor
system.
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10. Mutations in ras How does a mutation in ras that leads to formation of a Ras protein with no GTPase
activity affect a cell’s response to insulin?
11. Differences among G Proteins Compare the G proteins G
s
, which acts in transducing the signal
from b-adrenergic receptors, and Ras. What properties do they share? How do they differ? What is the
functional difference between G
s
and G
i
?
12. Mechanisms for Regulating Protein Kinases Identify eight general types of protein kinases found
in eukaryotic cells, and explain what factor is directly responsible for activating each type.
13. Nonhydrolyzable GTP Analogs Many enzymes can hydrolyze GTP between the band gphosphates.
The GTP analog b,g-imidoguanosine 5⬘-triphosphate Gpp(NH)p, shown below, cannot be hydrolyzed
between the band gphosphates. Predict the effect of microinjection of Gpp(NH)p into a myocyte on
the cell’s response to b-adrenergic stimulation.
Gpp(NH)p
(b,g-imidoguanosine 5⬘-triphosphate)
CH
2
OO
OHOH
H
H
H
H
N
O
N
HN
H
2
N
N
O
⫺
P
O
O
O
⫺
P
O
⫺
O
P
O
⫺
O
H
N
14. Use of Toxin Binding to Purify a Channel Protein ␣-Bungarotoxin is a powerful neurotoxin
found in the venom of a poisonous snake (Bungarus multicinctus). It binds with high specificity to
the nicotinic acetylcholine receptor (AChR) protein and prevents the ion channel from opening. This
interaction was used to purify AChR from the electric organ of torpedo fish.
(a) Outline a strategy for using ␣-bungarotoxin covalently bound to chromatography beads to purify
the AChR protein. (Hint: See Fig. 3–17c.)
(b) Outline a strategy for the use of [
125
I]␣-bungarotoxin to purify the AChR protein.
Answer
15. Resting Membrane Potential A variety of unusual invertebrates, including giant clams, mussels,
and polychaete worms, live on the fringes of deep-sea hydrothermal vents, where the temperature
is 60 ⬚C.
(a) The adductor muscle of a giant clam has a resting membrane potential of ⫺95 mV. Given the
intracellular and extracellular ionic compositions shown below, would you have predicted this
membrane potential? Why or why not?
Concentration (m
M
)
Ion Intracellular Extracellular
Na
⫹
50 440
K
⫹
400 20
Cl
⫺
21 560
Ca
2⫹
0.4 10
(b) Assume that the adductor muscle membrane is permeable to only one of the ions listed above.
Which ion could determine the V
m
?
Answer
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16. Membrane Potentials in Frog Eggs Fertilization of a frog oocyte by a sperm cell triggers ionic
changes similar to those observed in neurons (during movement of the action potential) and initiates
the events that result in cell division and development of the embryo. Oocytes can be stimulated to
divide without fertilization by suspending them in 80 m
M
KCl (normal pond water contains 9 m
M
KCl).
(a) Calculate how much the change in extracellular [KCl] changes the resting membrane potential of
the oocyte. (Hint: Assume the oocyte contains 120 m
M
K
⫹
and is permeable only to K
⫹
.)
Assume a temperature of 20 ⬚C.
(b) When the experiment is repeated in Ca
2⫹
-free water, elevated [KCl] has no effect. What does this
suggest about the mechanism of the KCl effect?
Answer
17. Excitation Triggered by Hyperpolarization In most neurons, membrane depolarization leads to
the opening of voltage-dependent ion channels, generation of an action potential, and ultimately an in-
flux of Ca
2⫹
, which causes release of neurotransmitter at the axon terminus. Devise a cellular strategy
by which hyperpolarization in rod cells could produce excitation of the visual pathway and passage
of visual signals to the brain. (Hint: The neuronal signaling pathway in higher organisms consists of a
series of neurons that relay information to the brain (see Fig. 12–36). The signal released by one
neuron can be either excitatory or inhibitory to the following, postsynaptic neuron.)
18. Genetic “Channelopathies” There are many genetic diseases that result from defects in ion channels.
For each of the following, explain how the molecular defect might lead to the symptoms described.
(a) A loss-of-function mutation in the gene encoding the ␣subunit of the cGMP-gated cation channel
of retinal cone cells leads to a complete inability to distinguish colors.
(b) Loss-of-function alleles of the gene encoding the ␣subunit of the ATP-gated K
⫹
channel shown
in Figure 23–28 lead to a condition known as congenital hyperinsulinism—persistently high lev-
els of insulin in the blood.
(c) Mutations affecting the subunit of the ATP-gated K
⫹
channel that prevent ATP binding lead to
neonatal diabetes—persistently low levels of insulin in the blood in newborn babies.
Answer
19. Visual Desensitization Oguchi disease is an inherited form of night blindness. Affected individuals
are slow to recover vision after a flash of bright light against a dark background, such as the headlights
of a car on the freeway. Suggest what the molecular defect(s) might be in Oguchi disease. Explain in
molecular terms how this defect would account for night blindness.
20. Effect of a Permeant cGMP Analog on Rod Cells An analog of cGMP, 8-Br-cGMP, will permeate
cellular membranes, is only slowly degraded by a rod cell’s PDE activity, and is as effective as cGMP in
opening the gated channel in the cell’s outer segment. If you suspended rod cells in a buffer containing
a relatively high [8-Br-cGMP], then illuminated the cells while measuring their membrane potential,
what would you observe?
21. Hot and Cool Taste Sensations The sensations of heat and cold are transduced by a group of
temperature-gated cation channels. For example, TRPV1, TRPV3, and TRPM8 are usually closed, but
open under the following conditions: TRPV1 at ⱖ43 ⬚C; TRPV3 at ⱖ33 ⬚C; and TRPM8 at ⬍25 ⬚C.
These channels are expressed in sensory neurons known to be responsible for temperature sensation.
(a) Propose a reasonable model to explain how exposing a sensory neuron containing TRPV1 to high
temperature leads to a sensation of heat.
(b) Capsaicin, one of the active ingredients in “hot” peppers, is an agonist of TRPV1. Capsaicin
shows 50% activation of the TRPV1 response at a concentration (i.e., it has an EC
50
) of 32 n
M
.
Explain why even a very few drops of hot pepper sauce can taste very “hot” without actually
burning you.
(c) Menthol, one of the active ingredients in mint, is an agonist of TRPM8 (EC
50
⫽30
M
) and
TRPV3 (EC
50
⫽20
M
). What sensation would you expect from contact with low levels of
menthol? With high levels?
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Answer
22. Oncogenes, Tumor-Suppressor Genes, and Tumors For each of the following situations, provide a
plausible explanation for how it could lead to unrestricted cell division.
(a) Colon cancer cells often contain mutations in the gene encoding the prostaglandin E
2
receptor.
PGE
2
is a growth factor required for the division of cells in the gastrointestinal tract.
(b) Kaposi sarcoma, a common tumor in people with untreated AIDS, is caused by a virus carrying a
gene for a protein similar to the chemokine receptors CXCR1 and CXCR2. Chemokines are cell-
specific growth factors.
(c) Adenovirus, a tumor virus, carries a gene for the protein E1A, which binds to the retinoblastoma
protein, pRb. (Hint: See Fig. 12–49.)
(d) An important feature of many oncogenes and tumor suppressor genes is their cell-type speci-
ficity. For example, mutations in the PGE
2
receptor are not typically found in lung tumors.
Explain this observation. (Note that PGE
2
acts through a GPCR in the plasma membrane.)
Answer
23. Mutations in Tumor Suppressor Genes and Oncogenes Explain why mutations in tumor
suppressor genes are recessive (both copies of the gene must be defective for the regulation of cell
division to be defective), whereas mutations in oncogenes are dominant.
24. Retinoblastoma in Children Explain why some children with retinoblastoma develop multiple tu-
mors of the retina in both eyes, whereas others have a single tumor in only one eye.
25. Specificity of a Signal for a Single Cell Type Discuss the validity of the following proposition. A
signaling molecule (hormone, growth factor, or neurotransmitter) elicits identical responses in differ-
ent types of target cells if they contain identical receptors.
Data Analysis Problem
26. Exploring Taste Sensation in Mice Figure 12–42 shows the signal-transduction pathway for sweet
taste in mammals. Pleasing tastes are an evolutionary adaptation to encourage animals to consume
nutritious foods. Zhao and coauthors (2003) examined the two major pleasurable taste sensations:
sweet and umami. Umami is a “distinct savory taste” triggered by amino acids, especially aspartate and
glutamate, and probably encourages animals to consume protein-rich foods. Monosodium glutamate
(MSG) is a flavor enhancer that exploits this sensitivity.
At the time the article was published, specific taste receptor proteins (labeled SR in Fig. 12–42)
for sweet and umami had been tentatively characterized. Three such proteins were known—T1R1,
T1R2, and T1R3—which function as heterodimeric receptor complexes: T1R1-T1R3 was tentatively
identified as the umami receptor, and T1R2-T1R3 as the sweet receptor. It was not clear how taste
sensation was encoded and sent to the brain, and two possible models had been suggested. In the cell-
based model, individual taste-sensing cells express only one kind of receptor; that is, there are “sweet
cells,” “bitter cells,” “umami cells,” and so on, and each type of cell sends its information to the brain
via a different nerve. The brain “knows” which taste is detected by the identity of the nerve fiber that
transmits the message. In the receptor-based model, individual taste-sensing cells have several kinds
of receptors and send different messages along the same nerve fiber to the brain, the message depend-
ing on which receptor is activated. Also unclear at the time was whether there was any interaction be-
tween the different taste sensations, or whether parts of one taste-sensing system were required for
other taste sensations.
(a) Previous work had shown that different taste receptor proteins are expressed in nonoverlapping
sets of taste receptor cells. Which model does this support? Explain your reasoning.
Zhao and colleagues constructed a set of “knockout mice”—mice homozygous for loss-of-function
alleles for one of the three receptor proteins, T1R1, T1R2, or T1R3—and double-knockout mice with
nonfunctioning T1R2 and T1R3. The researchers measured the taste perception of these mice by mea-
suring their “lick rate” of solutions containing different taste molecules. Mice will lick the spout of a
feeding bottle with a pleasant-tasting solution more often than one with an unpleasant-tasting solution.
The researchers measured relative lick rates: how often the mice licked a sample solution compared
with water. A relative lick rate of 1 indicated no preference; ⬍1, an aversion; and ⬎1, a preference.
(b) All four types of knockout strains had the same responses to salt and bitter tastes as did wild-
type mice. Which of the above issues did this experiment address? What do you conclude from
these results?
S-140 Chapter 12 Biosignaling
The researchers then studied umami taste reception by measuring the relative lick rates of the dif-
ferent mouse strains with different quantities of MSG in the feeding solution. Note that the solutions also
contained inosine monophosphate (IMP), a strong potentiator of umami taste reception (and a common
ingredient in ramen soups, along with MSG), and ameloride, which suppresses the pleasant salty taste
imparted by the sodium of MSG. The results are shown in the graph.
MSG + IMP + ameloride (mM)
Wild type and
T1R2 knockout
T1R1 knockout
T1R3 knockout
1 10 100
10
1
Relative lick rate
(c) Are these data consistent with the umami taste receptor consisting of a heterodimer of T1R1 and
T1R3? Why or why not?
(d) Which model(s) of taste encoding does this result support? Explain your reasoning.
Zhao and coworkers then performed a series of similar experiments using sucrose as a sweet taste.
These results are shown below.
Sucrose (mM)
Wild type and
T1R1 knockout
T1R3 knockout
T1R2-T1R3
double knockout T1R2 knockout
1 100 1000
20
1
Relative lick rate
(e) Are these data consistent with the sweet taste receptor consisting of a heterodimer of T1R2 and
T1R3? Why or why not?
(f) There were some unexpected responses at very high sucrose concentrations. How do these com-
plicate the idea of a heterodimeric system as presented above?
In addition to sugars, humans also taste other compounds (e.g., the peptides monellin and aspar-
tame) as sweet; mice do not taste these as sweet. Zhao and coworkers inserted into T1R2 knockout
mice a copy of the human T1R2 gene under the control of the mouse T1R2 promoter. These modified
mice now tasted monellin and saccharin as sweet. The researchers then went further, adding to T1R1
knockout mice the RASSL protein—a G protein–linked receptor for the synthetic opiate spiradoline; the
RASSL gene was under the control of a promoter that could be induced by feeding the mice tetracycline.
These mice did not prefer spiradoline in the absence of tetracycline; in the presence of tetracycline, they
showed a strong preference for nanomolar concentrations of spiradoline.
(g) How do these results strengthen Zhao and coauthors’ conclusions about the mechanism of taste
sensation?
Chapter 12 Biosignaling S-141
Answer
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