Untitled
CHAPTER
Signal Transduction Pathways:
An Introduction to Information
Metabolism
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In Chapter 13 you learned how biological membranes serve as semipermeable boundaries that isolate the cell from its surroundings and separate intracellular compartments from one another. That chapter also described how selective, con trolled breaching of the membrane barrier generates changing ion gradients across the bilayer thereby producing electrical signals. In this chapter you will learn how molecules external to the cell bind to integral membrane protein receptors to initiate specific responses within the cell. The text describes how these binding and transmission mechanisms lead to an amplification of the initial signal and to specific effects that adapt the cell to its environment through effects on intracellular enzymes and regulatory proteins. It also describes how disorders in these pathways of information flow can lead to diseases.
After a brief overview of signal transduction, the text describes the structure of the seven-helix transmembrane b-adrenergic receptor and indicates how it transmits to the intracellular side of the plasma membrane a signal arising from binding the hormone epinephrine on the extracellular surface of the cell. The common features of the G proteins are presented next. The description of the information-transmission pathway from hormone stimulus to G proteins to adenylate cyclase is completed by a discussion of how cAMP activates specific protein kinases to modulate the activities of the phosphorylated target proteins. A small number of hormone molecules outside the cell results in an amplified response because each activated enzyme in the triggered cascade forms numerous products. There are many distinct seven-helix transmembrane hormone receptors.
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The text next describes an analogous hormone-stimulated system—the phosphoionosi tide cascade. In this system, the hormone activates, by means of G proteins, a specific phospholipase (phospholipase C) that cleaves a plasma membrane phospholipid, phosphatidyl inositol 4,5-bisphosphate (PIP2), to form two second messengers. The inositol phosphate derivative, inositol 1,4,5-trisphosphate (IP3), which is short-lived, triggers the opening of ion channels so that the Ca;2 concentration in the cytosol is increased. The remnant of the PIP2 molecule, diacylglycerol (DAG), is also a second messenger that activates protein kinase C.
The increased Ca;2 levels and the activated protein kinase C affect a variety of biochemical reactions. The authors then describe the structure of Ca;2-binding proteins, focusing on calmodulin, and explain how the binding of the ion is highly specific and leads to a large conformational change in the protein—qualities desirable in molecules serving as Ca2; sensors and signal transducers.
The text then introduces another class of receptors, the transmembrane receptor tyro sine kinases that are often activated by a ligand-induced dimerization. The activated dimers phosphorylate some of their own tyrosine residues to provide docking sites for effector proteins on the cytosolic side of the membrane. Once bound, these effector enzymes are themselves phosphorylated and thereby activated by the tyrosine receptor kinase. A description of the susceptibility of signal transduction pathways to malfunctions that produce cancer follows, and the roles of oncogenes and their normal cellular counterparts (proto-oncogenes) in cell growth and differentiation are presented next. A discussion of the evolutionary relationships of the signal transduction pathways closes the chapter. In addition to Chapter 13, you should review covalent modification of proteins in Chapter 10, phospholipids in Chapter 12, and ion gradients in Chapter 14.
LEARNING OBJECTIVES
When you have mastered this chapter, you should be able to complete the following objectives.
Seven-Transmembrane-Helix Receptors Change Conformation in Response
to Ligand Binding and Activate G Proteins (Text Section 15.1) 1. Define information metabolism.
2. List the components of signal transduction cascades.
3. Draw a generalized molecular circuit based on a signal transduction cascade. Outline the roles of membrane receptors, ligands, primary messengers, second messengers, and protein
phosphorylation in the process.
4. Explain how a small number of hormone molecules outside the cell can effect a change involving many molecules within the cell, and consider that the cascade must be curtailed after being initiated.
5. Describe the topology of the structure of the b-adrenergic receptor, which binds epinephrine.
6. Locate guanyl nucleotide-binding proteins (G proteins) in the cell and describe their struc tures, catalytic characteristics, and molecular mechanisms of activation and inactivation.
Describe the roles of G proteins in coupling a hormone-receptor complex to adenylate cyclase and in amplifying the stimulus.
7. Describe the role of GTP and GTPase in G protein activity.
8. Appreciate that families of G proteins enable diverse hormones to effect a variety of phys iologic functions.
SIGNAL TRANSDUCTION PATHWAYS: AN INTRODUCTION TO INFORMATION METABOLISM
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9. Recognize the structure of adenosine 3„,5„-monophosphate (cyclic AMP, or cAMP), and write the reaction that forms it. Appreciate that cAMP is hydrolyzed by a cAMP phosphodiesterase and converts it to 5„-AMP.
10. Describe the mechanism by which cAMP modulates the activity of protein kinase A (PKA) to cAMP.
11. List the steps in a G protein-cAMP cascade that contribute to the amplification of the hor monal stimulus, and explain how the amplified response is achieved.
The Hydrolysis of Phosphatidyl Inositol Bisphosphate by Phospholipase C
Generates Two Messengers (Text Section 15.2) 12. Draw the structure of phosphatidyl inositol 4,5-bisphosphate (PIP2).
13. Write the reaction catalyzed by phospholipase C to produce the second messengers inos-itol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). Note that there are several forms of mammalian phospholipase C.
14. Outline the phosphoinositide cascade, and note the diversity of the elicited physiologic re sponses.
15. Describe the effects of IP3 on the IP3-gated channel. Describe the effects of Ca2; released from endoplasmic reticulum of smooth muscle, and list some biochemical processes affected by an increased intracellular Ca2; concentration.
16. Describe the biochemical fates of the second messengers produced from PIP2. Note that the phosphoinositide cascade often produces arachidonate—a precursor of the prostaglandin family of hormones.
Calcium Ion Is a Ubiquitous Cytosolic Messenger (Text Section 15.3) 17. Outline the features that suit Ca2; in its role as a eukaryotic signaling ion.
18. Describe the structure of calmodulin and its biochemical function. Relate calmodulin to the calmodulin-dependent protein kinase (CaM Kinase) and the Ca2+-ATP ion pump. Note the value of calcium ionophores, calcium buffers, and fluorescent indicators in studying the functions of Ca2; in cells.
19. Describe the EF hand structural motif of calcium-binding proteins, explain how it binds Ca2; and describe how ion binding affects its structure.
Some Receptors Dimerize in Response to Ligand Binding and Signal
by Cross-Phosphorylation (Text Section 15.4) 20. Describe how the quaternary structure (monomer-dimer equilibrium) of the human growthhormone receptor changes upon binding human growth hormone.
21. Outline the consequences of externally induced dimerization on intracellular protein ki-nase activity. Relate the roles of the JAK and STAT proteins in signal transduction.
22. Describe the general structures of the receptor tyrosine kinases and outline the process that converts them from inactive proteins to active enzymes. List some of the hormones that activate tyrosine kinases.
23. Define autophosphorylation and crosstalk as it relates to signal transduction.
24. Note the role of autophosphorylation of the kinase in the signal transduction process.
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25. Name some members of the small G protein family, and distinguish their structures from those of the heterotrimeric G proteins.
26. Appreciate the essential roles of the receptor tyrosine kinases and small G proteins in controlling cell growth and differentiation.
Defects in Signaling Pathways Can Lead to Cancer and Other Diseases
(Text Section 15.5) 27. Define cancer in terms of cell growth.
28. Describe the effect of Rous sarcoma virus gene product (v-src) on cell growth and note the relationship of v-src to its cellular counterpart, c-src. Describe the relationship between proto-oncogene and oncogene.
29. Explain the role of SH2 and SH3 domains in tyrosine kinase function. Appreciate that proto-oncogenes, which provide normal, essential functions in cell growth and proliferation, can give rise to cancer upon mutation to oncogenes.
30. Outline the biochemical mechanism of v-ras protein–induced cancer and note the role of the normal (noncarcinogenic) c-ras protein in cellular growth. Appreciate that a diminished GTPase activity, in this case, leads to cancer.
31. Explain how an inhibitor of a specific protein kinase might be an effective anticancer drug.
32. Explain the molecular mechanisms causing cholera and pertussis.
Recurring Features of Signal-Transduction Pathways Reveal Evolutionary
Relationships (Text Section 15.6) 33. List some of the superfamilies of proteins involved in signal transduction pathways.
34. Discern the consequences on the structure of a protein that cycles between a form bind ing a nucleoside triphosphate or a nucleoside diphosphate, and appreciate how such a system functions as a molecular switch.
35. Provide examples of the conservation of signaling pathways between organisms.
SELF-TEST
Seven-Transmembrane-Helix Receptors Change Conformation in Response
to Ligand Binding and Activate G Proteins 1. Provide a brief definition of information metabolism and contrast it with the traditional definition of metabolism.
2. Signal transduction cascades are produced by molecular assemblies of which of the following components?
(a) enzymes (b) regulatory proteins (c) receptors (d) transmembrane channels (e) nuclear pores (a) GTP is associated with the a subunit of a guanyl nucleotide–binding protein (G protein).
(b) GTP reduces the magnitude of the hormone response because it is converted to cGMP—a compound that antagonizes the effects of cAMP.
(c) GTP maintains the steady-state level of cAMP by rephosphorylating AMP to ATP in a nucleotide kinase-catalyzed reaction.
(d) GTP activates G protein so that its Ga-GTP subunit interacts with adenylate cy clase.
(e) GTP couples the stimulus from a hormone-receptor complex or an activated re ceptor to a system that produces an allosteric effector.
(f) The effect of GTP on hormone response is antagonized by the GTPase activity of the Ga subunit of the G protein.
(g) A single GTP binding event with a stimulatory G protein leads to the formation of one cAMP molecule.
8. Which of the following are correct statements about G proteins and their functioning in cAMP-mediated hormonal systems?
(a) G proteins bind hormones.
(b) G proteins are integral membrane proteins.
(c) G proteins are heterotrimers.
(d) G proteins bind adenylate cyclase.
(e) In their GDP form and in the absence of hormone, G proteins bind to hormone re ceptors and are converted to their GTP forms.
(f) When G protein in the GDP form binds to a hormone-receptor complex, GTP exchanges with GDP.
(g) The a subunit of G proteins is a GTPase.
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CHAPTER 15
9. If cells with b-adrenergic receptors are exposed for extended times to epinephrine, a hormone that causes activation of adenylate cyclase, the G protein fails to carry out efficiently the GDP-GTP exchange reaction and adenylate cyclase is no longer activated. What is this phenomenon called, what is its biological function, and how does it occur?
10. Both cAMP and AMP contain one adenine base, one ribose, and one phosphorus atom.
How are they different?
11. Which of the following statements about cAMP and its functioning in hormone action are correct?
(a) Most effects of cAMP in eukaryotic cells are exerted through the activation of pro tein kinase A (PKA).
(b) Cyclic AMP binds the catalytic subunits of PKA and activates the enzyme alloster ically.
(c) Cyclic AMP binds the regulatory subunits of PKA and activates the enzyme by releasing the catalytic subunits.
(d) Cyclic AMP is bound by the activated hormone receptor and PKA simultaneously to convey the hormonal signal in order to activate the kinase.
12. During cAMP-mediated hormone activation, what are the three steps at which amplifi cation occurs?
13. Which of the following statements about adenosine 3„,5„-monophosphate (cyclic AMP, or cAMP) are correct?
(a) ATP is converted to cAMP by the enzyme adenylate cyclase in one step.
(b) Cyclic AMP contains a phosphorous atom in a phosphodiester bond.
(c) ATP reacts with adenosine to form cAMP and ADP in a reaction catalyzed by adeny late kinase.
(d) Cyclic AMP is converted to 5„-AMP by a phosphodiesterase-catalyzed reaction with H2O.
(e) Cyclic AMP is susceptible to hydrolysis to Pi and the ribonucleoside adenosine by phosphomonoesterases.
14. Which of the following statements about cAMP and the second-messenger mechanism of hormone function are correct?
(a) The hormonal stimulus leads to increased amounts of adenylate cyclase.
(b) The formation of a hormone-receptor complex leads to the activation of adenylate cyclase.
(c) Cyclic AMP acts as an allosteric modulator to affect the activities of specific protein kinases.
(d) Cyclic AMP interacts with a hormone-receptor complex to dissociate the hormone.
(e) The hormone-receptor complex enters the cell and affects the activities of target enzymes.
15. Why do you think the cells of one kind of tissue respond to a given hormone, whereas cells of another tissue may not do so?
16. Suppose a patient is suffering from a disorder in which adenylate cyclase is impaired and, as a result, cAMP levels are not readily increased by hormones. Explain why the infusion of cAMP probably will not remedy the problem.
SIGNAL TRANSDUCTION PATHWAYS: AN INTRODUCTION TO INFORMATION METABOLISM
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The Hydrolysis of Phosphatidyl Inositol Bisphosphate by Phospholipase C
Generates Two Messengers 17. Which of the following statements about the phosphoinositide cascade are correct?
(a) The phosphoinositide cascade depends on the hydrolysis of a phospholipid com ponent of the plasma membrane.
(b) A polypeptide hormone interacts with a GM1 ganglioside on the cell surface to trig ger the phosphoinositide cascade.
(c) In some cases, a G protein system acts to transduce the stimulus from the receptor to the phosphoinositidase.
(d) At least four kinds of phospholipase C play a crucial role in the phosphoinositide cascade.
(e) The phosphoinositide cascade directly produces a unique second messenger molecule.
18. Which of the following are the second messengers that are produced by the phospho inositide cascade?
(a) Phosphatidyl inositol 4,5-bisphosphate (PIP2) (b) Inositol 1,4,5-trisphosphate (IP3) (c) Inositol 4-phosphate (d) Inositol 1,3,4,5-tetrakisphosphate (e) Inositol 1,3,4-trisphosphate (f) Diacylglycerol (DAG) 19. Which of the following statements about inositol 1,4,5-trisphosphate (IP3) are correct?
(a) IP3 leads to the uptake of Ca2; by the endoplasmic reticulum and the sarcoplasmic reticulum.
(b) IP3 may be rapidly inactivated by either a phosphatase or a kinase.
(c) IP3 opens calcium ion channels in the membranes of the endoplasmic reticulum and the sarcoplasmic reticulum.
(d) IP3 reacts with CTP to form CDP-inositol phosphate, a precursor of PIP2.
(e) IP3 acts by altering the intracellular-to-extracellular Na;-to-K; ratio, thereby alter ing the transmembrane potential.
20. Which of the following statements about the actions or targets of the second messengers of the phosphoinositide cascade are correct?
(a) Diacylglycerol (DAG) activates protein kinase C (PKC).
(b) Most of the effects of IP3 and DAG are antagonistic.
(c) DAG increases the affinity of PKC for Ca2;.
(d) PKC requires Ca2; for its activity.
21. How is a pseudosubstrate involved in the regulation of PKC?
Calcium Ion Is a Ubiquitous Cytosolic Messenger 22. Which of the following statements about Ca2; and its roles in the regulation of cellular metabolism are correct?
(a) The solubility product of calcium phosphate is small; therefore, low Ca2; levels must be maintained in the cell to avoid its precipitation.
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CHAPTER 15 (b) Intracellular Ca2; is maintained at concentrations that are several orders of magni tude less than the extracellular concentration by ATP-dependent Ca2; pumps.
(c) The transient opening of ion channels in the plasma membrane or endoplasmic reticulum can rapidly raise cytosolic Ca2; levels.
(d) The binding of Ca2; by a protein can induce a large conformational change because the ion simultaneously coordinates to several anionic groups within the protein.
(e) Ca2; is bound by a family of regulatory proteins that have a characteristic EF hand, helix-loop-helix structure.
(f) When calmodulin binds Ca2; at its low-affinity site, it undergoes a conformational change that allows the complex to interact with target proteins.
23. Explain how a Ca2; ionophore could mimic the effects of a hormone.
24. If it were incubated with cells in vitro, why would EGTA prevent either a Ca2;-triggering hormone or a Ca2; ionophore from acting?
25. Which of the following answers complete the sentence correctly? Calmodulin
(a) is a member of the EF hand family of calcium-binding proteins.
(b) activates target molecules by recognizing negatively charged b sheets.
(c) serves as a calcium sensor in most eukaryotic cells.
(d) is activated when intracellular Ca2; concentrations rise above 0.5 mM.
(e) activates CAM kinase II, which then phosphorylates many different proteins.
(f)
undergoes a large conformational change upon binding Ca2; ions.
Some Receptors Dimerize in Response to Ligand Binding and Signal
by Cross-Phosphorylation 26. Which of the following answers complete the sentence correctly? Receptor tyrosine kinases (a) are seven-transmembrane-helix receptors.
(b) are integral membrane enzymes.
(c) activate their targets via the G protein cascade.
(d) are often activated by ligand-induced dimerization.
(e) can phosphorylate themselves on their cytoplasmic domains when activated.
(f) that have been activated by hormone binding are recognized by target proteins having SH2 (src protein homology region 2) sequences.
(g) are so named because they contain extraordinarily high amounts of tyrosine.
27. Which of the following statements about the tyrosine kinases or hormones that affect them are correct?
(a) Epidermal growth factor (EGF) stimulates epidermal and epithelial cells to divide.
(b) EGF is a protein kinase that phosphorylates tyrosine residues.
(c) EGF and insulin share the common mechanism of dimerization for signal trans duction across the plasma membrane.
(d) Receptors for EGF and insulin are integral membrane proteins.
(e) Some oncogenes encode tyrosine kinases.
(f) Specialized adaptor proteins (such as JAK2 and STAT5) link the phosphorylation of the EGF receptor to the stimulation of cell growth.
SIGNAL TRANSDUCTION PATHWAYS: AN INTRODUCTION TO INFORMATION METABOLISM
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28. Match each compound in the left column with its characteristic from the right column.
(a) Cyclic AMP (1) is cleaved by phospholipase C.
(b) GTP (2) binds the regulatory subunits of specific (c) G proteins protein kinases.
(d) Adenylate cyclase (3) converts cAMP to AMP.
(e) PIP2 (4) exchanges with GDP on Ga subunits.
(f)
Ca2; (5) is a downstream hormone product of
(g) IP3 PIP2 catabolism.
(h) DAG (6) binds epinephrine.
(i) A specific phosphodiesterase (7) is a second messenger arising from PIP2.
(j) Insulin receptor (8) is a small G protein GTPase.
(k) Ras (9) transduces hormone stimulus from an (l)
b-Adrenergic receptor activated 7TM membrane receptor to (m) Arachidonic acid adenylate cyclase.
(10) has inducible tyrosine kinase activity.
(11) is activated by Ga-GTP.
(12) has its intracellular concentration increased by IP3.
(13) activates protein kinase C.
Defects in Signaling Pathways Can Lead to Cancer and Other Diseases 29. Which of the following answers complete the sentence correctly? A mammalian protein, src, (a) has a viral counterpart, v-src, that is oncogenic.
(b) is a proto-oncogene.
(c) is a component of a signaling pathway for cell growth and differentiation.
(d) can be converted to an oncogene by the alteration of some of its C-terminal amino acids.
(e) is a protein tyrosine kinase.
30. Which of the following statements about hormones in mammals are correct?
(a) Hormones are enzymes.
(b) Hormones are synthesized in specific tissues.
(c) Hormones are secreted into the blood.
(d) Hormones alter one or more activities in the cells to which they are targeted.
(e) Hormones display specificity toward the tissues with which they interact.
(f) Hormones are involved in biochemical amplification systems.
31. Cholera toxin (choleragen) (a) inactivates a G protein by locking it in the off state (inactivates the GTPase).
(b) A subunit enters the cell and ADP-ribosylates the GaS subunit of a G protein.
(c) B subunit interacts with a GM1 ganglioside on the target-cell surface.
(d) causes the activation of protein kinase A, which opens a membrane channel and inhibits a Na;-H; exchanger.
(e) causes the retention of Cl: in the cell.
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Recurring Features of Signal-Transduction Pathways Reveal
Evolutionary Relationships 32. What is the key feature of the superfamily of proteins that include the G protein Ga sub units, Ras family, and proteins that cycle between ATP-bound and ADP-bound forms?
ANSWERS TO SELF-TEST
1. Information metabolism is the collection of biochemical reactions that allows cells to respond to their changing environments. It includes signal reception, processing, amplification, and connection to processes such as gene expression, membrane permeability, and enzyme activity. Ordinary metabolism is defined as the integrated and regulated sum of the reactions within a cell that allows it to extract energy and reducing power from its environment and to synthesize the building blocks necessary to form its constituents. (See p. 373 of the text.)
2. a, b, c, d
3. (a) 4, (b) 3, (c) 1, (d) 2 4. A signal, in the form of a molecule or a photon, interacts with a part of a 7TM on the outside surface of the cell. This interaction causes a conformational change in the protein that is transmitted to the inside of the cell.
5. b, d, e. G proteins are heterotrimers and alternate between states in which GTP or GDP is bound.
6. GDP is bound to G proteins when they are inactive, and GTP when they are activated.
The 7TM receptor, when activated by binding its cognate signaling molecule outside the cell, catalyzes the exchange on a G protein of GDP by GTP inside the cell to activate the G protein. An intrinsic GTPase of the G protein converts GTP to GDP to cause inactivation.
7. a, d, e, f. Answer (g) is incorrect because the GTP form of the G protein activates adeny late cyclase and it forms many cAMP molecules; that is, an amplification occurs.
8. c, d, f, g. Answers (a) and (b) are incorrect because G proteins are peripheral membrane proteins inside cells. They do not bind the hormone but rather the activated hormonereceptor complex, and they carry the signal to adenylate cyclase. Answer (e) is incorrect because the hormone receptor must have the hormone bound to it or it must have been activated by hormone binding before the G protein will bind.
9. The phenomenon is called desensitization or adaptation. It allows the system to adapt to a given level of hormone so that it can respond to changes in hormone concentrations rather than to absolute amounts. Desensitization is effected by phosphorylation by badrenergic receptor kinase at multiple seine and threonine sites on the carboxyl-terminal region of the b-adrenergic receptor when it has epinephrine bound to it. These covalent modifications of the hormone-receptor complex allow b-arrestin to bind it and further inhibit, but not completely prevent, the GDP-GTP exchange. These events thereby decrease the activation of adenylate cyclase. However, the desensitized receptor can still respond to an increase in epinephrine concentrations. Ultimately, a phosphatase reverses the effects of the modification and resensitizes the receptor.
10. AMP has a single phosphomonoester attached to the 5„-hydroxyl of the adenosine moi ety. cAMP has its single phosphate group attached to both the 5„ and 3„ hydroxyls of its adenosine to form a phosphodiester bond.
11. a, c 12. A single hormone molecule combines with a single receptor to form several stimulatory Ga-GTP molecules. Each of these stimulatory molecules activates an adenylate cyclase molecule to form many cAMP molecules. The cAMP molecules activate protein kinases, mainly PKA molecules, each of which can phosphorylate many target enzymes.
13, a, b, d. Answer (e) is incorrect because a phosphomonoesterase cannot cleave a phosphodiester-linked phosphate.
14. b, c. Answer (a) is incorrect because the hormone leads to an increase in the activity of adenylate cyclase, not an increase in the amount of the enzyme. Answer (e) is incorrect because the hormone need not enter the cell to carry out its action.
15. The simplest explanation for the tissue specificity of hormones is the presence or absence of receptors for particular hormones on the extracellular surfaces of the tissues. Whether or not a given cell type has a given hormone receptor depends upon which genes have been expressed within it.
16. Aside from the likelihood that serum phosphodiesterases might destroy it, cAMP is a polar molecule that does not readily traverse the plasma membrane. Even if a more hydrophobic derivative, such as dibutyryl-cAMP, were used to overcome the permeability problem, there would be no tissue specificity, and all cells would have increased cAMP levels, leading to a massive, nonspecific response.
17. a, c, d. Answer (e) is incorrect because two messengers are formed.
18. b, f. Answer (a) is incorrect because PIP2 is the precursor of the second messengers. The other incorrect choices are all downstream products of IP3 metabolism.
19. b, c. Answer (a) is incorrect because IP3 causes the release, not the uptake, of Ca2;.
Answer (b) is correct because not only does a phosphatase act on IP3 but a specific kinase phosphorylates it to form the inactive tetrakisphosphate derivative. Answer (d) is incorrect because free inositol reacts with CDP-diacylglycerol to form phosphatidyl inositol, which is then phosphorylated to form PIP2.
20. a, c, d. Answer (b) is incorrect because most of the effects of IP3 and Ca2; are synergis tic, not antagonistic.
21. The N-terminal domain of PKC contains an amino acid sequence similar to that of its substrates, which interact with the active site formed by a C-terminal domain. This pseudosubstrate sequence lacks the critical target serine residue, and, consequently, although it binds to the active site, it cannot be phosphorylated. It thus occludes the active site until DAG binds and displaces it, thereby freeing the active site to react with the true protein substrates.
22. a, b, c, d, e, f
23. The ionophore allows Ca2; to enter cells by rendering the membrane permeable to the ion. Since the extracellular Ca2; concentration is higher than the intracellular concentration, the ion enters the cell and the cytosolic level increases. Because some hormones act to raise intracellular Ca2; levels in order to carry out their physiological roles, the ionophore could lead to the same response.
24. EGTA is a specific Ca2; chelator. It would bind tightly to the ion and markedly lower Ca2; concentration in the extracellular medium. Consequently, when a hormone or a Ca2; ionophore acted to allow Ca2; influx, none could occur because the concentration gradient of Ca2; would be insufficient.
25. a, c, d, e, f. Answer (b) is incorrect because activated calmodulin recognizes comple mentary positively charged amphipathic a helices on target proteins. Complementary hydrophobic interactions also contribute to the recognition.
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CHAPTER 15 26. b, d, e, f. Answer (g) is incorrect because the name arises from the amino acid that they phosphorylate in their target proteins.
27. a, d, e, f. Answer (b) is incorrect because the hormone itself does not have tyrosine ki nase activity; only the activated receptor is an active tyrosine kinase. Answer (c) is incorrect because the insulin receptor exists as a dimer and merely requires binding insulin to activate its intrinsic tyrosine kinase activity.
28. (a) 2, (b) 4, (c) 9, (d) 11, (e) 1, (f) 12, (g) 7, (h) 13, (i) 3, (j) 10, (k) 8, (l) 6, (m) 5 29. a, b, c, d, e 30. b, c, d, e, f 31. b, c, d. Choleragen stabilizes the GTP form of the G protein to keep it in the activated state, resulting in a loss of Cl: and H2O from the cell.
32. All the members of this superfamily undergo conformational changes upon going from the NTP-bound to NDP-bound forms. The conformational changes allow them to behave as molecular switches; in one form they interact with other proteins differently than when in the alternate form.
PROBLEMS
1. Based on the material so far covered in the text and your general understanding of reg ulation and signal transduction, list properties that a substance should have for it to be classified as a hormone.
2. Bee venom is particularly rich in phospholipase A2, an enzyme that hydrolytically re moves the fatty acyl residue at position 2 of phospholipids. The action of phospholipase A2 on phosphatidyl choline is shown in Figure 15-1. One of the mediators of the inflammatory response following a bee sting (swelling, redness, pain, heat, and loss of function) is lysophosphatidyl choline, the remainder of the phospholipid following the hydrolysis of the fatty acyl residue at position 2. Lysophosphatidyl choline stimulates mast cells to release histamine, which triggers the inflammatory response.
FIGURE 15.1 Action of phospholipase A2 on phosphatidyl choline.
O
K
O
CH JOJCJR
2
1
K
J R CJOJCJH
O
CH
2
3
J
K
J
A CH JOJPJOJCH JCH JN+JCH
2
2
2
2
3
J
J
O CH3 (a) Explain the major point of similarity between the system described here and one (phospholipase C hydrolysis of PIP2) described in Section 15.2 in the text.
(b) Suppose that the hydrolysis product of phosphatidyl choline that is important as a mediator of the inflammatory response were unknown. Suggest an experiment that might help establish the identity of the active agent.
3. Suppose that epinephrine stimulates the conversion of compound A to compound B in liver cells by means of a regulatory cascade involving G protein (text, pp. 400–401), cAMP, protein kinase A, and enzymes E1 and E2 as shown in Figure 15-2. Assume that each catalytically active enzyme subunit in the regulatory cascade has a turnover number of 1000 s:1. Assume further that 10 G-GTP are formed for each molecule of epinephrine bound to receptor. Calculate the theoretical number of molecules of A that would be converted to molecules of B per second as a result of the interaction of one molecule of epinephrine with its receptor on a liver cell membrane.
FIGURE 15.2 Hypothetical regulatory cascade for problem 3.
D
≈ Ga$ GDPDGa$ GTP
D
GTP GTP ≈
ATPDcAMP+PPi
R C +2cAMPDC D +(R-cAMP)
2
2
2
2
≈
(E )
D(ED ) 1 inact
1 act
≈
(E )
D(E ) 2 inact
D 2 act
≈
ADB
4. In the early days of research on insulin action, it was not known whether insulin might enter cells and directly mediate intracellular effects or whether it might act through a second messenger. In a classic experiment, Pedro Cuatrecasas attached insulin covalently to sepharose beads many times the size of fat cells and showed that the addition of the insulin-sepharose complexes to isolated fat cells gave the same stimulation of glucose oxidation as did addition of insulin alone.
(a) What conclusion might follow from this experiment? Explain.
(b) What assumptions have you made about the effects of adding sepharose without at tached insulin to fat cells and about the attachment of the insulin to the sepharose bead?
5. Suppose that you are trying to isolate a receptor for a polypeptide hormone from liver cells. Suggest an effective means of purification involving specialized-column chromatography and a highly purified polypeptide hormone. Also explain how you would get the receptor off the column.
6. In kinetic studies on the interaction of human growth hormone with its receptor, each functional receptor dimer was found to bind one hormone molecule, and the monomer receptors needed to dimerize in order to transduce the signal from the hormone. Explain how this occurs.
7. To be effective, intracellular signal substances must be readily inactivated when their ef fects are no longer needed. Give a method of inactivation for each of the following classes of intracellular messengers:
(a) G proteins (b) cyclic nucleotides (c) phosphoproteins (d) calcium ion (e) inositol 1,4,5-trisphosphate (IP3) (f)
diacylglycerol
8. A tissue is known to increase cyclic AMP production upon stimulation by a certain hor mone. Addition of an analog of GTP in which the terminal phosphate group is replaced by a sulfate to a homogenate of the tissue results in sustained production of cyclic AMP.
Propose an explanation for this observation.
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9. Just as steady-state kinetic studies of enzymes can yield valuable information about re action mechanisms, so can equilibrium binding studies on the interaction of hormonal ligands with receptors. Typically, in such studies, radioactively labeled hormones are incubated with a receptor preparation long enough for equilibrium to be achieved. The amounts of free and bound hormone may be readily measured because insoluble hormone-receptor complexes may be separated rapidly from free hormone by filtration. A typical linear plotting form for such data is the Scatchard plot (Figure 15-3), in which the bound/free ratio is plotted against the amount bound. The slope of such a line is equal to :1/K, where K is the equilibrium dissociation constant for the ligand-receptor complex; the intercept on the B/F axis is Bmax/F; and the intercept on the B axis is Bmax, a value that can be used to estimate the number of receptors in the system. (A Scatchard plot is the equilibrium binding analog of the Eadie-Hofstee plot for steady-state enzyme kinetic data, presented in problem 6 on page 224 of the text. B is the analog of V of the Eadie-Hofstee plot, Bmax is the analog of Vmax, and F is the analog of S.) When a system consists of a ligand and a highly purified receptor, the expected linear plot is usually obtained. On the other hand, when insulin is incubated with a tissue homogenate known to contain insulin receptors, the resulting plot is a curve that is concave upward (Figure 15-4). Propose two reasons that might account for the more complex observations in the latter system. (Hint: Think about the fact that tissue homogenates contain many molecular components. Also, think about some mechanisms used to explain allosterism.)
FIGURE 15.3 Scatchard plot for a ligand and highly purified receptor.
B
D
max
F
-1
D
Slope=
K
B
F
D
Bmax
B FIGURE 15.4 Scatchard plot for insulin and liver cell homogenate.
B
F
B
10. What properties of Ca2; render it so useful as a messenger in cells? What protein is often used in cells to “sense” Ca2;? How does the cell overcome the problem of the low solubility product of Ca2; with Pi, phosphorylated compounds, and carboxyl groups?
SIGNAL TRANSDUCTION PATHWAYS: AN INTRODUCTION TO INFORMATION METABOLISM
15
11. What signal-transduction functions do SH2 domains in proteins serve?
12. After the insulin-insulin receptor complex autophosphorylates itself, a series of down stream events carries the signal to molecules directly involved in promoting, among other things, the entry of glucose into muscle and adipose cells. Insulin thus promotes a lowering of the blood glucose (hypoglycemia). When a strain of mice had both copies of the gene (Akt2) for a particular serine-threonine kinase (a protein kinase B isoform) ablated, the “knockout” mice could no longer lower their blood glucose by taking it into muscle cells upon administration of insulin. (Isoenzymes are sometimes called isoforms. [See section 16.35 of the text to see how lactate dehydrogenase exemplifies isoenzymes.])
What conclusions can you draw about the role of the Akt2 isoform of protein kinase B in glucose homeostasis? Can you think of alternative explanations for the observation with the knockout mice?
ANSWERS TO PROBLEMS
1. The major criteria for classifying a substance as a hormone are: (1) In order to carry messages from one tissue to another, it should be produced by one type of cell and have effects on another type of cell.
(2) Its effects should involve the chemical amplification of the original signal.
(3) It should be produced in response to a stimulus, and its production should cease upon cessation of the stimulus.
(4) It should be selectively destroyed following cessation of the stimulus.
(5) The addition of the purified substance to tissues should mimic physiologic re sponses produced in vivo.
(6) Specific inhibitors of the physiologic response should also abolish the response elicited by the addition of the purified substance to tissues.
(7) Specific receptors for the hormone should exist and should be more abundant in tissues that are more sensitive to the hormone.
2. (a) The bee venom system resembles the phosphoinositide cascade discussed on pages 403–405 of the text. In that system, a membrane phospholipid is also converted into an active mediator of the response of several hormones.
(b) One could inject each of the hydrolysis products—lysophosphatidyl choline and the fatty acid—into tissues separately to see which might elicit the inflammatory response.
3. The theoretical number of molecules of A converted to B per second would be 1013. One molecule of epinephrine would result in the production of 10 G-GTP. Each activated a subunit would stimulate adenylate cyclase to produce 1000 cAMP molecules for a total of 10,000 molecules of cAMP. Each of these cAMP molecules would activate one catalytic subunit of protein kinase. (Remember that a molecule of protein kinase exists as an R2C2 complex. Two cAMP molecules combine with two R subunits to give two catalytically active C subunits.) Each of the 10,000 active C subunits would result in the production of 1000 molecules of active E1, for a total of 107 molecules of active E1. Each molecule of active E1 would in turn activate 1000 molecules of E2 for a total of 1010 molecules of active E2. Since each molecule of active E2 would convert 1000 molecules of A to B per second, the total would be 1000¥1010=1013 per second. (Note: This is a greatly oversimplified example, but it illustrates the profound chemical amplification that can occur in systems under hormonal control.)
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4. (a) A reasonable conclusion is that insulin need not enter the cell to have an effect. The results are consistent with the notion that insulin affects cells by combining with a membrane receptor site outside the cell thereby causing some second messenger to be formed within the cell that mediates the effects. Note that the experiment does not prove that insulin fails to enter cells.
(b) You likely assumed that the addition of sepharose alone gave no stimulation of glu cose oxidation. You also have to assume that the covalent attachment of the insulin to the sepharose is stable so that free insulin is not formed during the course of the experiment.
5. The most useful technique would be affinity chromatography. Covalently attach the pu rified hormone to sepharose or some other form of an insoluble bead (see problem 4), and use the hormone-bead complex to fill a column. Homogenize liver cells, and add the homogenate to the column. Receptors should stick on the column because their hormone-binding sites are complementary in shape to the covalently bound hormone. You could elute the receptors from the column as hormone-receptor complexes by adding free hormone to compete with the hormone that is bound to the column.
6. A given molecule of growth hormone contains two domains, each of which binds a re ceptor monomer. Thus, a single molecule of growth hormone could be bound by two receptors, bringing them together to form the activated hormone-receptor dimer complex.
7. (a) G proteins are active while GTP is bound, but become inactive as GTP is hydrolyzed to GDP and phosphate.
(b) Cyclic nucleotides are converted by phosphodiesterases to 5„-mononucleotides.
(c) Phosphates are cleaved from phosphoproteins by protein phosphatases.
(d) Calcium ions are pumped from the cell interior into the extracellular fluid or in tracellular storage organelles, for example, the endoplasmic reticulum .
(e) Inositol 1,4,5-trisphosphate can be degraded to inositol and inorganic phosphate by the sequential action of phosphatases or it can be phosphorylated by a kinase to form inositol 1,3,4,5-tetrakisphosphate.
(f) Diacylglycerol may be converted to phosphatidate, or hydrolyzed to glycerol and fatty acids.
8. The observation could be explained if the sulfate-containing analog of GTP bound to G protein, stimulating cyclic AMP production, but could not be hydrolyzed to GDP and sulfate by the GTPase activity of G. Thus the production of cyclic AMP would persist.
9. A homogenate of a tissue containing insulin receptors contains other molecules that may bind to insulin less specifically than do insulin receptors. Thus we could have a whole population of insulin-binding components, each with a different affinity for insulin. The result would be a Scatchard plot that is concave upward. The second reason for such behavior has its analog in allosteric enzyme behavior. Remember that binding of substrate to one subunit of an enzyme may increase or decrease the binding affinity of other subunits for substrate. (You may wish to review the discussion of the sequential and concerted models for allosterism on pp. 268–269 of the text.) In the case of the interaction of insulin with its receptor, we could postulate that binding of insulin to some receptors on a cell may inhibit binding of insulin to other receptor molecules, an instance of so-called negative cooperativity. This would also result in a Scatchard plot that is concave upward.
10. Energy requiring molecular pumps maintain a steep concentration gradient of Ca2; across the plasma membrane between the outside and inside of the cell and across the membrane between intracellular organelles and the cytoplasm. When the membrane, for instance, is rendered permeable to Ca2; as a result of the opening of a Ca2; channel, a flux of ions passes through the membrane raising the cytoplasmic Ca2; concentration. Such a sudden increase in Ca2; can act as a signal to Ca2;-sensing proteins within the cell.
Calmodulin binds Ca2; and interacts with several proteins and enzymes as a consequence of the binding. Because Ca2; can interact simultaneously with several anionic amino acid side chains, the carbonyls of the peptide backbone, or the carbonyls of Gln and Asn, it can cause large conformational changes in the protein to which it binds. Conformational changes in response to binding a ligand are the hallmarks of a molecular switch. Thus, the ability to rapidly change its concentration and to effect large conformational changes renders Ca2; an effective intracellular messenger. The cell avoids precipitating the Ca2; salts of its intracellular components by maintaining the Ca2; concentration below the solubility product for various compounds. Endergonic pumps and exchangers maintain the low intracellular Ca2; concentrations.
11. SH2 domains bind to peptides or sections of proteins that contain phosphotyrosine residues in particular sequence contexts. The formation of phosphorylated tyrosine residues in a receptor often results from hormone activation of the receptor. The phosphorylated tyrosine-containing peptides in the receptor can be recognized and bound by other proteins that have SH2 domains. The SH2 domain allows different proteins to respond to and be affected by the phosphorylated tyrosines that arise in proteins as a result of a signal transduction event.
12. The simplest interpretation of the observation is that this particular protein kinase B iso form is directly involved in mediating the ability of insulin to lower blood glucose concentrations by facilitating its entry into muscle cells. The kinase presumably acts by phosphorylating a target molecule that, in turn, facilitates the movement of a glucose transporter (GULT4) to the surface of the cell. An alternative explanation could be that the lack of the Akt2 kinase during the growth of the knockout mouse led to the failure to synthesize a molecule that was, itself, the active component in the insulin-signaling pathway. The mutation-induced lack of the protein in some other tissue could also have caused the effect if that tissue normally supplied a compound needed in the muscle cells for the insulin response. For instance, adipose tissue is known to affect glucose uptake by muscle cells. The gene deletion could have affected the ability of adipose tissue to make that compound. Further experiments would be required to verify the simplest conclusion. (This problem is based on Cho, H., Mu, J., Kim, J. K., Thorvaldsen, J. L., et al.
(2001). Insulin resistance and a diabetes mellitus-like syndrome in mice lacking the protein kinase Akt2 (PKBb). Science 292: 1728–1731.)
EXPANDED SOLUTIONS TO TEXT PROBLEMS 1. epinephrine to cAMP: There are two significant amplification stages. The binding of one epinephrine to a receptor stimulates the formation of many molecules of Gas. In turn, each molecule of Gas (when bound to adenylate cyclase) stimulates the formation of many molecules of cAMP.
human growth hormone to STAT5: There is one amplification event in this part of the pathway. Each hormone-/receptor-binding event activates a kinase (JAK2), which then can phosphorylate many molecules of STAT5.
EGF to Ras: All of the reactions up to Ras are stoichiometric (no amplification). An EGF/receptor complex autoactivates its own tyrosine kinase. The receptor’s phosphotyrosine then recruits Grb-2, which recruits Sos, which binds and activates Ras.
(Downstream from Ras, amplification does occur through a chain of protein phosphorylations [Figure 15.32].)
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2. The common feature between glutamate and phospho-Ser (or phospho-Thr) is the pres ence of a negative charge (at physiological pH). The negative charge on the glutatmate side chain therefore may sometimes fulfill the role of the negative charge on the phosphate.
3. No. Phospho-Ser and phospho-Thr are significantly smaller than phospho-Tyr. The phos phate of these smaller side chains probably will not reach sufficiently far into the deep binding pocket to make a favorable electrostatic interaction with a counter (+) charge.
4. In the dark the catalytic subunits (a and b) of cGMP phosphodiesterase (PDE) are in hibited by a pair of g subunits. Ta-GTP, the active form of transducin, activates PDE by prying away its g subunits.
The R2C2 complex of protein kinase A (PKA) is inactive. The binding of cAMP to the R (regulatory) chains releases catalytically active C chains.
The regulatory domain of protein kinase C (PKC) inhibits the catalytic domain by occupying the substrate binding site. The binding of diacylglycerol in the presence of Ca2; disrupts this interaction and enables a protein substrate to enter.
5. In the pseudosubstrate sequence A-R-K-G-A*-L-R-Q-K (Section 15.2.2), the central ala nine (A*) is critical in preventing activity. A* replaces the serine or threonine of a substrate sequence and is not phosphorylated. The other underlined residues are identical or similar to the consensus substrate sequence: X-R-XX (S,T)-Hyd-R-X, where Hyd refers to “hydrophobic” (e.g., Leu in the pseudosequence). The mutations therefore should be expected not to be at the underlined positions, but rather at any three of the four positions corresponding to X, that is either the K, G, Q, or initial A in the pseudosubstrate sequence.
6. Some growth factors act by binding two receptor molecules and causing the receptor to dimerize. Antibodies, with two identical binding sites, could similarly cause receptor dimerization and initiate the signaling process.
7. The mutated a-subunit would be defective for signaling because it would be turned “on” at all times, even in the absence of an activated receptor. The inability to turn “off” the signaling pathway would be a serious flaw.
8. The mutated hormone would bind to only one of its receptors. Receptor dimerization and signaling would be blocked. The mutated hormone would nevertheless be useful for studies of the interactions at the binding interface that remains active. For example, one would expect that it should be easier to co-crystallize the receptor with the mutant hormone (in a single binding motif) than with the native hormone (making two different binding interfaces).
9. Calcium is slowed because its intracellular concentration is low and it binds tightly to larger molecules, including proteins. The effective molecular weight of the diffusing complex therefore is large.
10. Epinephrine initiates a pathway that raises the level of cAMP within the muscle cell. The higher level of cAMP ultimately will mobilize glucose (make more glucose available).
Inhibitors of cAMP phosphodiesterase also will raise the level of cAMP within the cell.
Therefore the phosphodiesterase inhibitors will act similarly to epinephrine to increase
the mobilization of glucose.
11. It is reasonable to propose that the nerve growth factor will cause dimerization, au tophosphorylation, and activation of its receptor protein tyrosine kinase upon binding.
The active tyrosine kinase then should phosphorylate and activate a gamma (non-beta) isoform of phospholipase C. Active PLC then would release both diacylglycerol and inositol 1,4,5-trisphosphate from phosphatidyl inositol 4,5-bisphosphate (PIP2). Therefore, the concentration of the second messenger inositol 1,4,5-trisphosphate, as well as of diacylglycerol, would be expected to increase.
SIGNAL TRANSDUCTION PATHWAYS: AN INTRODUCTION TO INFORMATION METABOLISM
19
12. There are several similarities. Both adenylate cyclase and DNA polymerases use ATP as a substrate. In addition, both enzymes release pyrophosphate while forming a new phosphodiester bond. The key difference is that the adenylate cyclase forms a new intramolecular bond, whereas DNA polymerases join molecules by forming new intermolecular bonds.
13. (a) From the graphs, approximately 10:7 M of X, 3 * 10:6 M of Y, or 10:3 M of Z.
(b) Hormone X achieves maximal binding in the lowest concentration range and there fore has the highest binding affinity.
(c) For each hormone, the trend for the activation of adenylate cyclase is similar to the trend for hormone/receptor binding. Therefore, it is likely that the hormone/receptor complex plays a direct role in the mechanism of activation of adenylate cyclase.
(d) A requirement for GTP in addition to hormone would suggest that a Gs protein may be required. The trend for Gas activity should then be measured as a function of the concentration of hormones X, Y, and Z. This could be done by monitoring GTP/GDP exchange activity. Gs protein, the receptor, and unlabeled GDP should be preincubated in the absence of GTP and hormone. Then labeled GTP could be added together with varying amounts of a hormone X, Y, or Z. One would then test for the association of labeled GTP with protein when the proteins are subjected to precipitation, electrophoresis, or chromatography.
14. (a) The ligand X may “stick” to a few sites other than the specific receptor. These sites should not be counted.
(b) The experiment allows the background nonspecific binding to be determined. The large excess of nonradioactive ligand will bind to all of the authentic receptor sites.
The remaining (residual) background binding of the labeled ligand will be revealed as nonspecific binding (line labeled “nonspecific binding”).
(c) The plateau indicates that the ligand binding sites can be saturated. The sites can be saturated because in fact there exist only a discrete number of receptor molecules per cell. (Alternatively, if the cell uptake of ligand were to continue to increase without reaching a plateau, the result would indicate the absence of a specific receptor, and a different uptake mechanism would be operating.)
15. Paying attention to the units, we set up an equation to divide the binding activity by the specific activity and by the number of cells, and finally multiply by Avogadro’s number to convert moles to molecules: 104 cpm
1
protein
mg
6*1023
molecules
protein
mg 1010 cells 103 mmole 600 molecules .
1012 cpm
cell
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Signal Transduction Pathways:
An Introduction to Information
Metabolism
1
5
In Chapter 13 you learned how biological membranes serve as semipermeable boundaries that isolate the cell from its surroundings and separate intracellular compartments from one another. That chapter also described how selective, con trolled breaching of the membrane barrier generates changing ion gradients across the bilayer thereby producing electrical signals. In this chapter you will learn how molecules external to the cell bind to integral membrane protein receptors to initiate specific responses within the cell. The text describes how these binding and transmission mechanisms lead to an amplification of the initial signal and to specific effects that adapt the cell to its environment through effects on intracellular enzymes and regulatory proteins. It also describes how disorders in these pathways of information flow can lead to diseases.
After a brief overview of signal transduction, the text describes the structure of the seven-helix transmembrane b-adrenergic receptor and indicates how it transmits to the intracellular side of the plasma membrane a signal arising from binding the hormone epinephrine on the extracellular surface of the cell. The common features of the G proteins are presented next. The description of the information-transmission pathway from hormone stimulus to G proteins to adenylate cyclase is completed by a discussion of how cAMP activates specific protein kinases to modulate the activities of the phosphorylated target proteins. A small number of hormone molecules outside the cell results in an amplified response because each activated enzyme in the triggered cascade forms numerous products. There are many distinct seven-helix transmembrane hormone receptors.
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The text next describes an analogous hormone-stimulated system—the phosphoionosi tide cascade. In this system, the hormone activates, by means of G proteins, a specific phospholipase (phospholipase C) that cleaves a plasma membrane phospholipid, phosphatidyl inositol 4,5-bisphosphate (PIP2), to form two second messengers. The inositol phosphate derivative, inositol 1,4,5-trisphosphate (IP3), which is short-lived, triggers the opening of ion channels so that the Ca;2 concentration in the cytosol is increased. The remnant of the PIP2 molecule, diacylglycerol (DAG), is also a second messenger that activates protein kinase C.
The increased Ca;2 levels and the activated protein kinase C affect a variety of biochemical reactions. The authors then describe the structure of Ca;2-binding proteins, focusing on calmodulin, and explain how the binding of the ion is highly specific and leads to a large conformational change in the protein—qualities desirable in molecules serving as Ca2; sensors and signal transducers.
The text then introduces another class of receptors, the transmembrane receptor tyro sine kinases that are often activated by a ligand-induced dimerization. The activated dimers phosphorylate some of their own tyrosine residues to provide docking sites for effector proteins on the cytosolic side of the membrane. Once bound, these effector enzymes are themselves phosphorylated and thereby activated by the tyrosine receptor kinase. A description of the susceptibility of signal transduction pathways to malfunctions that produce cancer follows, and the roles of oncogenes and their normal cellular counterparts (proto-oncogenes) in cell growth and differentiation are presented next. A discussion of the evolutionary relationships of the signal transduction pathways closes the chapter. In addition to Chapter 13, you should review covalent modification of proteins in Chapter 10, phospholipids in Chapter 12, and ion gradients in Chapter 14.
LEARNING OBJECTIVES
When you have mastered this chapter, you should be able to complete the following objectives.
Seven-Transmembrane-Helix Receptors Change Conformation in Response
to Ligand Binding and Activate G Proteins (Text Section 15.1) 1. Define information metabolism.
2. List the components of signal transduction cascades.
3. Draw a generalized molecular circuit based on a signal transduction cascade. Outline the roles of membrane receptors, ligands, primary messengers, second messengers, and protein
phosphorylation in the process.
4. Explain how a small number of hormone molecules outside the cell can effect a change involving many molecules within the cell, and consider that the cascade must be curtailed after being initiated.
5. Describe the topology of the structure of the b-adrenergic receptor, which binds epinephrine.
6. Locate guanyl nucleotide-binding proteins (G proteins) in the cell and describe their struc tures, catalytic characteristics, and molecular mechanisms of activation and inactivation.
Describe the roles of G proteins in coupling a hormone-receptor complex to adenylate cyclase and in amplifying the stimulus.
7. Describe the role of GTP and GTPase in G protein activity.
8. Appreciate that families of G proteins enable diverse hormones to effect a variety of phys iologic functions.
SIGNAL TRANSDUCTION PATHWAYS: AN INTRODUCTION TO INFORMATION METABOLISM
3
9. Recognize the structure of adenosine 3„,5„-monophosphate (cyclic AMP, or cAMP), and write the reaction that forms it. Appreciate that cAMP is hydrolyzed by a cAMP phosphodiesterase and converts it to 5„-AMP.
10. Describe the mechanism by which cAMP modulates the activity of protein kinase A (PKA) to cAMP.
11. List the steps in a G protein-cAMP cascade that contribute to the amplification of the hor monal stimulus, and explain how the amplified response is achieved.
The Hydrolysis of Phosphatidyl Inositol Bisphosphate by Phospholipase C
Generates Two Messengers (Text Section 15.2) 12. Draw the structure of phosphatidyl inositol 4,5-bisphosphate (PIP2).
13. Write the reaction catalyzed by phospholipase C to produce the second messengers inos-itol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). Note that there are several forms of mammalian phospholipase C.
14. Outline the phosphoinositide cascade, and note the diversity of the elicited physiologic re sponses.
15. Describe the effects of IP3 on the IP3-gated channel. Describe the effects of Ca2; released from endoplasmic reticulum of smooth muscle, and list some biochemical processes affected by an increased intracellular Ca2; concentration.
16. Describe the biochemical fates of the second messengers produced from PIP2. Note that the phosphoinositide cascade often produces arachidonate—a precursor of the prostaglandin family of hormones.
Calcium Ion Is a Ubiquitous Cytosolic Messenger (Text Section 15.3) 17. Outline the features that suit Ca2; in its role as a eukaryotic signaling ion.
18. Describe the structure of calmodulin and its biochemical function. Relate calmodulin to the calmodulin-dependent protein kinase (CaM Kinase) and the Ca2+-ATP ion pump. Note the value of calcium ionophores, calcium buffers, and fluorescent indicators in studying the functions of Ca2; in cells.
19. Describe the EF hand structural motif of calcium-binding proteins, explain how it binds Ca2; and describe how ion binding affects its structure.
Some Receptors Dimerize in Response to Ligand Binding and Signal
by Cross-Phosphorylation (Text Section 15.4) 20. Describe how the quaternary structure (monomer-dimer equilibrium) of the human growthhormone receptor changes upon binding human growth hormone.
21. Outline the consequences of externally induced dimerization on intracellular protein ki-nase activity. Relate the roles of the JAK and STAT proteins in signal transduction.
22. Describe the general structures of the receptor tyrosine kinases and outline the process that converts them from inactive proteins to active enzymes. List some of the hormones that activate tyrosine kinases.
23. Define autophosphorylation and crosstalk as it relates to signal transduction.
24. Note the role of autophosphorylation of the kinase in the signal transduction process.
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CHAPTER 15
25. Name some members of the small G protein family, and distinguish their structures from those of the heterotrimeric G proteins.
26. Appreciate the essential roles of the receptor tyrosine kinases and small G proteins in controlling cell growth and differentiation.
Defects in Signaling Pathways Can Lead to Cancer and Other Diseases
(Text Section 15.5) 27. Define cancer in terms of cell growth.
28. Describe the effect of Rous sarcoma virus gene product (v-src) on cell growth and note the relationship of v-src to its cellular counterpart, c-src. Describe the relationship between proto-oncogene and oncogene.
29. Explain the role of SH2 and SH3 domains in tyrosine kinase function. Appreciate that proto-oncogenes, which provide normal, essential functions in cell growth and proliferation, can give rise to cancer upon mutation to oncogenes.
30. Outline the biochemical mechanism of v-ras protein–induced cancer and note the role of the normal (noncarcinogenic) c-ras protein in cellular growth. Appreciate that a diminished GTPase activity, in this case, leads to cancer.
31. Explain how an inhibitor of a specific protein kinase might be an effective anticancer drug.
32. Explain the molecular mechanisms causing cholera and pertussis.
Recurring Features of Signal-Transduction Pathways Reveal Evolutionary
Relationships (Text Section 15.6) 33. List some of the superfamilies of proteins involved in signal transduction pathways.
34. Discern the consequences on the structure of a protein that cycles between a form bind ing a nucleoside triphosphate or a nucleoside diphosphate, and appreciate how such a system functions as a molecular switch.
35. Provide examples of the conservation of signaling pathways between organisms.
SELF-TEST
Seven-Transmembrane-Helix Receptors Change Conformation in Response
to Ligand Binding and Activate G Proteins 1. Provide a brief definition of information metabolism and contrast it with the traditional definition of metabolism.
2. Signal transduction cascades are produced by molecular assemblies of which of the following components?
(a) enzymes (b) regulatory proteins (c) receptors (d) transmembrane channels (e) nuclear pores (a) GTP is associated with the a subunit of a guanyl nucleotide–binding protein (G protein).
(b) GTP reduces the magnitude of the hormone response because it is converted to cGMP—a compound that antagonizes the effects of cAMP.
(c) GTP maintains the steady-state level of cAMP by rephosphorylating AMP to ATP in a nucleotide kinase-catalyzed reaction.
(d) GTP activates G protein so that its Ga-GTP subunit interacts with adenylate cy clase.
(e) GTP couples the stimulus from a hormone-receptor complex or an activated re ceptor to a system that produces an allosteric effector.
(f) The effect of GTP on hormone response is antagonized by the GTPase activity of the Ga subunit of the G protein.
(g) A single GTP binding event with a stimulatory G protein leads to the formation of one cAMP molecule.
8. Which of the following are correct statements about G proteins and their functioning in cAMP-mediated hormonal systems?
(a) G proteins bind hormones.
(b) G proteins are integral membrane proteins.
(c) G proteins are heterotrimers.
(d) G proteins bind adenylate cyclase.
(e) In their GDP form and in the absence of hormone, G proteins bind to hormone re ceptors and are converted to their GTP forms.
(f) When G protein in the GDP form binds to a hormone-receptor complex, GTP exchanges with GDP.
(g) The a subunit of G proteins is a GTPase.
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CHAPTER 15
9. If cells with b-adrenergic receptors are exposed for extended times to epinephrine, a hormone that causes activation of adenylate cyclase, the G protein fails to carry out efficiently the GDP-GTP exchange reaction and adenylate cyclase is no longer activated. What is this phenomenon called, what is its biological function, and how does it occur?
10. Both cAMP and AMP contain one adenine base, one ribose, and one phosphorus atom.
How are they different?
11. Which of the following statements about cAMP and its functioning in hormone action are correct?
(a) Most effects of cAMP in eukaryotic cells are exerted through the activation of pro tein kinase A (PKA).
(b) Cyclic AMP binds the catalytic subunits of PKA and activates the enzyme alloster ically.
(c) Cyclic AMP binds the regulatory subunits of PKA and activates the enzyme by releasing the catalytic subunits.
(d) Cyclic AMP is bound by the activated hormone receptor and PKA simultaneously to convey the hormonal signal in order to activate the kinase.
12. During cAMP-mediated hormone activation, what are the three steps at which amplifi cation occurs?
13. Which of the following statements about adenosine 3„,5„-monophosphate (cyclic AMP, or cAMP) are correct?
(a) ATP is converted to cAMP by the enzyme adenylate cyclase in one step.
(b) Cyclic AMP contains a phosphorous atom in a phosphodiester bond.
(c) ATP reacts with adenosine to form cAMP and ADP in a reaction catalyzed by adeny late kinase.
(d) Cyclic AMP is converted to 5„-AMP by a phosphodiesterase-catalyzed reaction with H2O.
(e) Cyclic AMP is susceptible to hydrolysis to Pi and the ribonucleoside adenosine by phosphomonoesterases.
14. Which of the following statements about cAMP and the second-messenger mechanism of hormone function are correct?
(a) The hormonal stimulus leads to increased amounts of adenylate cyclase.
(b) The formation of a hormone-receptor complex leads to the activation of adenylate cyclase.
(c) Cyclic AMP acts as an allosteric modulator to affect the activities of specific protein kinases.
(d) Cyclic AMP interacts with a hormone-receptor complex to dissociate the hormone.
(e) The hormone-receptor complex enters the cell and affects the activities of target enzymes.
15. Why do you think the cells of one kind of tissue respond to a given hormone, whereas cells of another tissue may not do so?
16. Suppose a patient is suffering from a disorder in which adenylate cyclase is impaired and, as a result, cAMP levels are not readily increased by hormones. Explain why the infusion of cAMP probably will not remedy the problem.
SIGNAL TRANSDUCTION PATHWAYS: AN INTRODUCTION TO INFORMATION METABOLISM
7
The Hydrolysis of Phosphatidyl Inositol Bisphosphate by Phospholipase C
Generates Two Messengers 17. Which of the following statements about the phosphoinositide cascade are correct?
(a) The phosphoinositide cascade depends on the hydrolysis of a phospholipid com ponent of the plasma membrane.
(b) A polypeptide hormone interacts with a GM1 ganglioside on the cell surface to trig ger the phosphoinositide cascade.
(c) In some cases, a G protein system acts to transduce the stimulus from the receptor to the phosphoinositidase.
(d) At least four kinds of phospholipase C play a crucial role in the phosphoinositide cascade.
(e) The phosphoinositide cascade directly produces a unique second messenger molecule.
18. Which of the following are the second messengers that are produced by the phospho inositide cascade?
(a) Phosphatidyl inositol 4,5-bisphosphate (PIP2) (b) Inositol 1,4,5-trisphosphate (IP3) (c) Inositol 4-phosphate (d) Inositol 1,3,4,5-tetrakisphosphate (e) Inositol 1,3,4-trisphosphate (f) Diacylglycerol (DAG) 19. Which of the following statements about inositol 1,4,5-trisphosphate (IP3) are correct?
(a) IP3 leads to the uptake of Ca2; by the endoplasmic reticulum and the sarcoplasmic reticulum.
(b) IP3 may be rapidly inactivated by either a phosphatase or a kinase.
(c) IP3 opens calcium ion channels in the membranes of the endoplasmic reticulum and the sarcoplasmic reticulum.
(d) IP3 reacts with CTP to form CDP-inositol phosphate, a precursor of PIP2.
(e) IP3 acts by altering the intracellular-to-extracellular Na;-to-K; ratio, thereby alter ing the transmembrane potential.
20. Which of the following statements about the actions or targets of the second messengers of the phosphoinositide cascade are correct?
(a) Diacylglycerol (DAG) activates protein kinase C (PKC).
(b) Most of the effects of IP3 and DAG are antagonistic.
(c) DAG increases the affinity of PKC for Ca2;.
(d) PKC requires Ca2; for its activity.
21. How is a pseudosubstrate involved in the regulation of PKC?
Calcium Ion Is a Ubiquitous Cytosolic Messenger 22. Which of the following statements about Ca2; and its roles in the regulation of cellular metabolism are correct?
(a) The solubility product of calcium phosphate is small; therefore, low Ca2; levels must be maintained in the cell to avoid its precipitation.
8
CHAPTER 15 (b) Intracellular Ca2; is maintained at concentrations that are several orders of magni tude less than the extracellular concentration by ATP-dependent Ca2; pumps.
(c) The transient opening of ion channels in the plasma membrane or endoplasmic reticulum can rapidly raise cytosolic Ca2; levels.
(d) The binding of Ca2; by a protein can induce a large conformational change because the ion simultaneously coordinates to several anionic groups within the protein.
(e) Ca2; is bound by a family of regulatory proteins that have a characteristic EF hand, helix-loop-helix structure.
(f) When calmodulin binds Ca2; at its low-affinity site, it undergoes a conformational change that allows the complex to interact with target proteins.
23. Explain how a Ca2; ionophore could mimic the effects of a hormone.
24. If it were incubated with cells in vitro, why would EGTA prevent either a Ca2;-triggering hormone or a Ca2; ionophore from acting?
25. Which of the following answers complete the sentence correctly? Calmodulin
(a) is a member of the EF hand family of calcium-binding proteins.
(b) activates target molecules by recognizing negatively charged b sheets.
(c) serves as a calcium sensor in most eukaryotic cells.
(d) is activated when intracellular Ca2; concentrations rise above 0.5 mM.
(e) activates CAM kinase II, which then phosphorylates many different proteins.
(f)
undergoes a large conformational change upon binding Ca2; ions.
Some Receptors Dimerize in Response to Ligand Binding and Signal
by Cross-Phosphorylation 26. Which of the following answers complete the sentence correctly? Receptor tyrosine kinases (a) are seven-transmembrane-helix receptors.
(b) are integral membrane enzymes.
(c) activate their targets via the G protein cascade.
(d) are often activated by ligand-induced dimerization.
(e) can phosphorylate themselves on their cytoplasmic domains when activated.
(f) that have been activated by hormone binding are recognized by target proteins having SH2 (src protein homology region 2) sequences.
(g) are so named because they contain extraordinarily high amounts of tyrosine.
27. Which of the following statements about the tyrosine kinases or hormones that affect them are correct?
(a) Epidermal growth factor (EGF) stimulates epidermal and epithelial cells to divide.
(b) EGF is a protein kinase that phosphorylates tyrosine residues.
(c) EGF and insulin share the common mechanism of dimerization for signal trans duction across the plasma membrane.
(d) Receptors for EGF and insulin are integral membrane proteins.
(e) Some oncogenes encode tyrosine kinases.
(f) Specialized adaptor proteins (such as JAK2 and STAT5) link the phosphorylation of the EGF receptor to the stimulation of cell growth.
SIGNAL TRANSDUCTION PATHWAYS: AN INTRODUCTION TO INFORMATION METABOLISM
9
28. Match each compound in the left column with its characteristic from the right column.
(a) Cyclic AMP (1) is cleaved by phospholipase C.
(b) GTP (2) binds the regulatory subunits of specific (c) G proteins protein kinases.
(d) Adenylate cyclase (3) converts cAMP to AMP.
(e) PIP2 (4) exchanges with GDP on Ga subunits.
(f)
Ca2; (5) is a downstream hormone product of
(g) IP3 PIP2 catabolism.
(h) DAG (6) binds epinephrine.
(i) A specific phosphodiesterase (7) is a second messenger arising from PIP2.
(j) Insulin receptor (8) is a small G protein GTPase.
(k) Ras (9) transduces hormone stimulus from an (l)
b-Adrenergic receptor activated 7TM membrane receptor to (m) Arachidonic acid adenylate cyclase.
(10) has inducible tyrosine kinase activity.
(11) is activated by Ga-GTP.
(12) has its intracellular concentration increased by IP3.
(13) activates protein kinase C.
Defects in Signaling Pathways Can Lead to Cancer and Other Diseases 29. Which of the following answers complete the sentence correctly? A mammalian protein, src, (a) has a viral counterpart, v-src, that is oncogenic.
(b) is a proto-oncogene.
(c) is a component of a signaling pathway for cell growth and differentiation.
(d) can be converted to an oncogene by the alteration of some of its C-terminal amino acids.
(e) is a protein tyrosine kinase.
30. Which of the following statements about hormones in mammals are correct?
(a) Hormones are enzymes.
(b) Hormones are synthesized in specific tissues.
(c) Hormones are secreted into the blood.
(d) Hormones alter one or more activities in the cells to which they are targeted.
(e) Hormones display specificity toward the tissues with which they interact.
(f) Hormones are involved in biochemical amplification systems.
31. Cholera toxin (choleragen) (a) inactivates a G protein by locking it in the off state (inactivates the GTPase).
(b) A subunit enters the cell and ADP-ribosylates the GaS subunit of a G protein.
(c) B subunit interacts with a GM1 ganglioside on the target-cell surface.
(d) causes the activation of protein kinase A, which opens a membrane channel and inhibits a Na;-H; exchanger.
(e) causes the retention of Cl: in the cell.
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CHAPTER 15
Recurring Features of Signal-Transduction Pathways Reveal
Evolutionary Relationships 32. What is the key feature of the superfamily of proteins that include the G protein Ga sub units, Ras family, and proteins that cycle between ATP-bound and ADP-bound forms?
ANSWERS TO SELF-TEST
1. Information metabolism is the collection of biochemical reactions that allows cells to respond to their changing environments. It includes signal reception, processing, amplification, and connection to processes such as gene expression, membrane permeability, and enzyme activity. Ordinary metabolism is defined as the integrated and regulated sum of the reactions within a cell that allows it to extract energy and reducing power from its environment and to synthesize the building blocks necessary to form its constituents. (See p. 373 of the text.)
2. a, b, c, d
3. (a) 4, (b) 3, (c) 1, (d) 2 4. A signal, in the form of a molecule or a photon, interacts with a part of a 7TM on the outside surface of the cell. This interaction causes a conformational change in the protein that is transmitted to the inside of the cell.
5. b, d, e. G proteins are heterotrimers and alternate between states in which GTP or GDP is bound.
6. GDP is bound to G proteins when they are inactive, and GTP when they are activated.
The 7TM receptor, when activated by binding its cognate signaling molecule outside the cell, catalyzes the exchange on a G protein of GDP by GTP inside the cell to activate the G protein. An intrinsic GTPase of the G protein converts GTP to GDP to cause inactivation.
7. a, d, e, f. Answer (g) is incorrect because the GTP form of the G protein activates adeny late cyclase and it forms many cAMP molecules; that is, an amplification occurs.
8. c, d, f, g. Answers (a) and (b) are incorrect because G proteins are peripheral membrane proteins inside cells. They do not bind the hormone but rather the activated hormonereceptor complex, and they carry the signal to adenylate cyclase. Answer (e) is incorrect because the hormone receptor must have the hormone bound to it or it must have been activated by hormone binding before the G protein will bind.
9. The phenomenon is called desensitization or adaptation. It allows the system to adapt to a given level of hormone so that it can respond to changes in hormone concentrations rather than to absolute amounts. Desensitization is effected by phosphorylation by badrenergic receptor kinase at multiple seine and threonine sites on the carboxyl-terminal region of the b-adrenergic receptor when it has epinephrine bound to it. These covalent modifications of the hormone-receptor complex allow b-arrestin to bind it and further inhibit, but not completely prevent, the GDP-GTP exchange. These events thereby decrease the activation of adenylate cyclase. However, the desensitized receptor can still respond to an increase in epinephrine concentrations. Ultimately, a phosphatase reverses the effects of the modification and resensitizes the receptor.
10. AMP has a single phosphomonoester attached to the 5„-hydroxyl of the adenosine moi ety. cAMP has its single phosphate group attached to both the 5„ and 3„ hydroxyls of its adenosine to form a phosphodiester bond.
11. a, c 12. A single hormone molecule combines with a single receptor to form several stimulatory Ga-GTP molecules. Each of these stimulatory molecules activates an adenylate cyclase molecule to form many cAMP molecules. The cAMP molecules activate protein kinases, mainly PKA molecules, each of which can phosphorylate many target enzymes.
13, a, b, d. Answer (e) is incorrect because a phosphomonoesterase cannot cleave a phosphodiester-linked phosphate.
14. b, c. Answer (a) is incorrect because the hormone leads to an increase in the activity of adenylate cyclase, not an increase in the amount of the enzyme. Answer (e) is incorrect because the hormone need not enter the cell to carry out its action.
15. The simplest explanation for the tissue specificity of hormones is the presence or absence of receptors for particular hormones on the extracellular surfaces of the tissues. Whether or not a given cell type has a given hormone receptor depends upon which genes have been expressed within it.
16. Aside from the likelihood that serum phosphodiesterases might destroy it, cAMP is a polar molecule that does not readily traverse the plasma membrane. Even if a more hydrophobic derivative, such as dibutyryl-cAMP, were used to overcome the permeability problem, there would be no tissue specificity, and all cells would have increased cAMP levels, leading to a massive, nonspecific response.
17. a, c, d. Answer (e) is incorrect because two messengers are formed.
18. b, f. Answer (a) is incorrect because PIP2 is the precursor of the second messengers. The other incorrect choices are all downstream products of IP3 metabolism.
19. b, c. Answer (a) is incorrect because IP3 causes the release, not the uptake, of Ca2;.
Answer (b) is correct because not only does a phosphatase act on IP3 but a specific kinase phosphorylates it to form the inactive tetrakisphosphate derivative. Answer (d) is incorrect because free inositol reacts with CDP-diacylglycerol to form phosphatidyl inositol, which is then phosphorylated to form PIP2.
20. a, c, d. Answer (b) is incorrect because most of the effects of IP3 and Ca2; are synergis tic, not antagonistic.
21. The N-terminal domain of PKC contains an amino acid sequence similar to that of its substrates, which interact with the active site formed by a C-terminal domain. This pseudosubstrate sequence lacks the critical target serine residue, and, consequently, although it binds to the active site, it cannot be phosphorylated. It thus occludes the active site until DAG binds and displaces it, thereby freeing the active site to react with the true protein substrates.
22. a, b, c, d, e, f
23. The ionophore allows Ca2; to enter cells by rendering the membrane permeable to the ion. Since the extracellular Ca2; concentration is higher than the intracellular concentration, the ion enters the cell and the cytosolic level increases. Because some hormones act to raise intracellular Ca2; levels in order to carry out their physiological roles, the ionophore could lead to the same response.
24. EGTA is a specific Ca2; chelator. It would bind tightly to the ion and markedly lower Ca2; concentration in the extracellular medium. Consequently, when a hormone or a Ca2; ionophore acted to allow Ca2; influx, none could occur because the concentration gradient of Ca2; would be insufficient.
25. a, c, d, e, f. Answer (b) is incorrect because activated calmodulin recognizes comple mentary positively charged amphipathic a helices on target proteins. Complementary hydrophobic interactions also contribute to the recognition.
12
CHAPTER 15 26. b, d, e, f. Answer (g) is incorrect because the name arises from the amino acid that they phosphorylate in their target proteins.
27. a, d, e, f. Answer (b) is incorrect because the hormone itself does not have tyrosine ki nase activity; only the activated receptor is an active tyrosine kinase. Answer (c) is incorrect because the insulin receptor exists as a dimer and merely requires binding insulin to activate its intrinsic tyrosine kinase activity.
28. (a) 2, (b) 4, (c) 9, (d) 11, (e) 1, (f) 12, (g) 7, (h) 13, (i) 3, (j) 10, (k) 8, (l) 6, (m) 5 29. a, b, c, d, e 30. b, c, d, e, f 31. b, c, d. Choleragen stabilizes the GTP form of the G protein to keep it in the activated state, resulting in a loss of Cl: and H2O from the cell.
32. All the members of this superfamily undergo conformational changes upon going from the NTP-bound to NDP-bound forms. The conformational changes allow them to behave as molecular switches; in one form they interact with other proteins differently than when in the alternate form.
PROBLEMS
1. Based on the material so far covered in the text and your general understanding of reg ulation and signal transduction, list properties that a substance should have for it to be classified as a hormone.
2. Bee venom is particularly rich in phospholipase A2, an enzyme that hydrolytically re moves the fatty acyl residue at position 2 of phospholipids. The action of phospholipase A2 on phosphatidyl choline is shown in Figure 15-1. One of the mediators of the inflammatory response following a bee sting (swelling, redness, pain, heat, and loss of function) is lysophosphatidyl choline, the remainder of the phospholipid following the hydrolysis of the fatty acyl residue at position 2. Lysophosphatidyl choline stimulates mast cells to release histamine, which triggers the inflammatory response.
FIGURE 15.1 Action of phospholipase A2 on phosphatidyl choline.
O
K
O
CH JOJCJR
2
1
K
J R CJOJCJH
O
CH
2
3
J
K
J
A CH JOJPJOJCH JCH JN+JCH
2
2
2
2
3
J
J
O CH3 (a) Explain the major point of similarity between the system described here and one (phospholipase C hydrolysis of PIP2) described in Section 15.2 in the text.
(b) Suppose that the hydrolysis product of phosphatidyl choline that is important as a mediator of the inflammatory response were unknown. Suggest an experiment that might help establish the identity of the active agent.
3. Suppose that epinephrine stimulates the conversion of compound A to compound B in liver cells by means of a regulatory cascade involving G protein (text, pp. 400–401), cAMP, protein kinase A, and enzymes E1 and E2 as shown in Figure 15-2. Assume that each catalytically active enzyme subunit in the regulatory cascade has a turnover number of 1000 s:1. Assume further that 10 G-GTP are formed for each molecule of epinephrine bound to receptor. Calculate the theoretical number of molecules of A that would be converted to molecules of B per second as a result of the interaction of one molecule of epinephrine with its receptor on a liver cell membrane.
FIGURE 15.2 Hypothetical regulatory cascade for problem 3.
D
≈ Ga$ GDPDGa$ GTP
D
GTP GTP ≈
ATPDcAMP+PPi
R C +2cAMPDC D +(R-cAMP)
2
2
2
2
≈
(E )
D(ED ) 1 inact
1 act
≈
(E )
D(E ) 2 inact
D 2 act
≈
ADB
4. In the early days of research on insulin action, it was not known whether insulin might enter cells and directly mediate intracellular effects or whether it might act through a second messenger. In a classic experiment, Pedro Cuatrecasas attached insulin covalently to sepharose beads many times the size of fat cells and showed that the addition of the insulin-sepharose complexes to isolated fat cells gave the same stimulation of glucose oxidation as did addition of insulin alone.
(a) What conclusion might follow from this experiment? Explain.
(b) What assumptions have you made about the effects of adding sepharose without at tached insulin to fat cells and about the attachment of the insulin to the sepharose bead?
5. Suppose that you are trying to isolate a receptor for a polypeptide hormone from liver cells. Suggest an effective means of purification involving specialized-column chromatography and a highly purified polypeptide hormone. Also explain how you would get the receptor off the column.
6. In kinetic studies on the interaction of human growth hormone with its receptor, each functional receptor dimer was found to bind one hormone molecule, and the monomer receptors needed to dimerize in order to transduce the signal from the hormone. Explain how this occurs.
7. To be effective, intracellular signal substances must be readily inactivated when their ef fects are no longer needed. Give a method of inactivation for each of the following classes of intracellular messengers:
(a) G proteins (b) cyclic nucleotides (c) phosphoproteins (d) calcium ion (e) inositol 1,4,5-trisphosphate (IP3) (f)
diacylglycerol
8. A tissue is known to increase cyclic AMP production upon stimulation by a certain hor mone. Addition of an analog of GTP in which the terminal phosphate group is replaced by a sulfate to a homogenate of the tissue results in sustained production of cyclic AMP.
Propose an explanation for this observation.
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CHAPTER 15
9. Just as steady-state kinetic studies of enzymes can yield valuable information about re action mechanisms, so can equilibrium binding studies on the interaction of hormonal ligands with receptors. Typically, in such studies, radioactively labeled hormones are incubated with a receptor preparation long enough for equilibrium to be achieved. The amounts of free and bound hormone may be readily measured because insoluble hormone-receptor complexes may be separated rapidly from free hormone by filtration. A typical linear plotting form for such data is the Scatchard plot (Figure 15-3), in which the bound/free ratio is plotted against the amount bound. The slope of such a line is equal to :1/K, where K is the equilibrium dissociation constant for the ligand-receptor complex; the intercept on the B/F axis is Bmax/F; and the intercept on the B axis is Bmax, a value that can be used to estimate the number of receptors in the system. (A Scatchard plot is the equilibrium binding analog of the Eadie-Hofstee plot for steady-state enzyme kinetic data, presented in problem 6 on page 224 of the text. B is the analog of V of the Eadie-Hofstee plot, Bmax is the analog of Vmax, and F is the analog of S.) When a system consists of a ligand and a highly purified receptor, the expected linear plot is usually obtained. On the other hand, when insulin is incubated with a tissue homogenate known to contain insulin receptors, the resulting plot is a curve that is concave upward (Figure 15-4). Propose two reasons that might account for the more complex observations in the latter system. (Hint: Think about the fact that tissue homogenates contain many molecular components. Also, think about some mechanisms used to explain allosterism.)
FIGURE 15.3 Scatchard plot for a ligand and highly purified receptor.
B
D
max
F
-1
D
Slope=
K
B
F
D
Bmax
B FIGURE 15.4 Scatchard plot for insulin and liver cell homogenate.
B
F
B
10. What properties of Ca2; render it so useful as a messenger in cells? What protein is often used in cells to “sense” Ca2;? How does the cell overcome the problem of the low solubility product of Ca2; with Pi, phosphorylated compounds, and carboxyl groups?
SIGNAL TRANSDUCTION PATHWAYS: AN INTRODUCTION TO INFORMATION METABOLISM
15
11. What signal-transduction functions do SH2 domains in proteins serve?
12. After the insulin-insulin receptor complex autophosphorylates itself, a series of down stream events carries the signal to molecules directly involved in promoting, among other things, the entry of glucose into muscle and adipose cells. Insulin thus promotes a lowering of the blood glucose (hypoglycemia). When a strain of mice had both copies of the gene (Akt2) for a particular serine-threonine kinase (a protein kinase B isoform) ablated, the “knockout” mice could no longer lower their blood glucose by taking it into muscle cells upon administration of insulin. (Isoenzymes are sometimes called isoforms. [See section 16.35 of the text to see how lactate dehydrogenase exemplifies isoenzymes.])
What conclusions can you draw about the role of the Akt2 isoform of protein kinase B in glucose homeostasis? Can you think of alternative explanations for the observation with the knockout mice?
ANSWERS TO PROBLEMS
1. The major criteria for classifying a substance as a hormone are: (1) In order to carry messages from one tissue to another, it should be produced by one type of cell and have effects on another type of cell.
(2) Its effects should involve the chemical amplification of the original signal.
(3) It should be produced in response to a stimulus, and its production should cease upon cessation of the stimulus.
(4) It should be selectively destroyed following cessation of the stimulus.
(5) The addition of the purified substance to tissues should mimic physiologic re sponses produced in vivo.
(6) Specific inhibitors of the physiologic response should also abolish the response elicited by the addition of the purified substance to tissues.
(7) Specific receptors for the hormone should exist and should be more abundant in tissues that are more sensitive to the hormone.
2. (a) The bee venom system resembles the phosphoinositide cascade discussed on pages 403–405 of the text. In that system, a membrane phospholipid is also converted into an active mediator of the response of several hormones.
(b) One could inject each of the hydrolysis products—lysophosphatidyl choline and the fatty acid—into tissues separately to see which might elicit the inflammatory response.
3. The theoretical number of molecules of A converted to B per second would be 1013. One molecule of epinephrine would result in the production of 10 G-GTP. Each activated a subunit would stimulate adenylate cyclase to produce 1000 cAMP molecules for a total of 10,000 molecules of cAMP. Each of these cAMP molecules would activate one catalytic subunit of protein kinase. (Remember that a molecule of protein kinase exists as an R2C2 complex. Two cAMP molecules combine with two R subunits to give two catalytically active C subunits.) Each of the 10,000 active C subunits would result in the production of 1000 molecules of active E1, for a total of 107 molecules of active E1. Each molecule of active E1 would in turn activate 1000 molecules of E2 for a total of 1010 molecules of active E2. Since each molecule of active E2 would convert 1000 molecules of A to B per second, the total would be 1000¥1010=1013 per second. (Note: This is a greatly oversimplified example, but it illustrates the profound chemical amplification that can occur in systems under hormonal control.)
16
CHAPTER 15
4. (a) A reasonable conclusion is that insulin need not enter the cell to have an effect. The results are consistent with the notion that insulin affects cells by combining with a membrane receptor site outside the cell thereby causing some second messenger to be formed within the cell that mediates the effects. Note that the experiment does not prove that insulin fails to enter cells.
(b) You likely assumed that the addition of sepharose alone gave no stimulation of glu cose oxidation. You also have to assume that the covalent attachment of the insulin to the sepharose is stable so that free insulin is not formed during the course of the experiment.
5. The most useful technique would be affinity chromatography. Covalently attach the pu rified hormone to sepharose or some other form of an insoluble bead (see problem 4), and use the hormone-bead complex to fill a column. Homogenize liver cells, and add the homogenate to the column. Receptors should stick on the column because their hormone-binding sites are complementary in shape to the covalently bound hormone. You could elute the receptors from the column as hormone-receptor complexes by adding free hormone to compete with the hormone that is bound to the column.
6. A given molecule of growth hormone contains two domains, each of which binds a re ceptor monomer. Thus, a single molecule of growth hormone could be bound by two receptors, bringing them together to form the activated hormone-receptor dimer complex.
7. (a) G proteins are active while GTP is bound, but become inactive as GTP is hydrolyzed to GDP and phosphate.
(b) Cyclic nucleotides are converted by phosphodiesterases to 5„-mononucleotides.
(c) Phosphates are cleaved from phosphoproteins by protein phosphatases.
(d) Calcium ions are pumped from the cell interior into the extracellular fluid or in tracellular storage organelles, for example, the endoplasmic reticulum .
(e) Inositol 1,4,5-trisphosphate can be degraded to inositol and inorganic phosphate by the sequential action of phosphatases or it can be phosphorylated by a kinase to form inositol 1,3,4,5-tetrakisphosphate.
(f) Diacylglycerol may be converted to phosphatidate, or hydrolyzed to glycerol and fatty acids.
8. The observation could be explained if the sulfate-containing analog of GTP bound to G protein, stimulating cyclic AMP production, but could not be hydrolyzed to GDP and sulfate by the GTPase activity of G. Thus the production of cyclic AMP would persist.
9. A homogenate of a tissue containing insulin receptors contains other molecules that may bind to insulin less specifically than do insulin receptors. Thus we could have a whole population of insulin-binding components, each with a different affinity for insulin. The result would be a Scatchard plot that is concave upward. The second reason for such behavior has its analog in allosteric enzyme behavior. Remember that binding of substrate to one subunit of an enzyme may increase or decrease the binding affinity of other subunits for substrate. (You may wish to review the discussion of the sequential and concerted models for allosterism on pp. 268–269 of the text.) In the case of the interaction of insulin with its receptor, we could postulate that binding of insulin to some receptors on a cell may inhibit binding of insulin to other receptor molecules, an instance of so-called negative cooperativity. This would also result in a Scatchard plot that is concave upward.
10. Energy requiring molecular pumps maintain a steep concentration gradient of Ca2; across the plasma membrane between the outside and inside of the cell and across the membrane between intracellular organelles and the cytoplasm. When the membrane, for instance, is rendered permeable to Ca2; as a result of the opening of a Ca2; channel, a flux of ions passes through the membrane raising the cytoplasmic Ca2; concentration. Such a sudden increase in Ca2; can act as a signal to Ca2;-sensing proteins within the cell.
Calmodulin binds Ca2; and interacts with several proteins and enzymes as a consequence of the binding. Because Ca2; can interact simultaneously with several anionic amino acid side chains, the carbonyls of the peptide backbone, or the carbonyls of Gln and Asn, it can cause large conformational changes in the protein to which it binds. Conformational changes in response to binding a ligand are the hallmarks of a molecular switch. Thus, the ability to rapidly change its concentration and to effect large conformational changes renders Ca2; an effective intracellular messenger. The cell avoids precipitating the Ca2; salts of its intracellular components by maintaining the Ca2; concentration below the solubility product for various compounds. Endergonic pumps and exchangers maintain the low intracellular Ca2; concentrations.
11. SH2 domains bind to peptides or sections of proteins that contain phosphotyrosine residues in particular sequence contexts. The formation of phosphorylated tyrosine residues in a receptor often results from hormone activation of the receptor. The phosphorylated tyrosine-containing peptides in the receptor can be recognized and bound by other proteins that have SH2 domains. The SH2 domain allows different proteins to respond to and be affected by the phosphorylated tyrosines that arise in proteins as a result of a signal transduction event.
12. The simplest interpretation of the observation is that this particular protein kinase B iso form is directly involved in mediating the ability of insulin to lower blood glucose concentrations by facilitating its entry into muscle cells. The kinase presumably acts by phosphorylating a target molecule that, in turn, facilitates the movement of a glucose transporter (GULT4) to the surface of the cell. An alternative explanation could be that the lack of the Akt2 kinase during the growth of the knockout mouse led to the failure to synthesize a molecule that was, itself, the active component in the insulin-signaling pathway. The mutation-induced lack of the protein in some other tissue could also have caused the effect if that tissue normally supplied a compound needed in the muscle cells for the insulin response. For instance, adipose tissue is known to affect glucose uptake by muscle cells. The gene deletion could have affected the ability of adipose tissue to make that compound. Further experiments would be required to verify the simplest conclusion. (This problem is based on Cho, H., Mu, J., Kim, J. K., Thorvaldsen, J. L., et al.
(2001). Insulin resistance and a diabetes mellitus-like syndrome in mice lacking the protein kinase Akt2 (PKBb). Science 292: 1728–1731.)
EXPANDED SOLUTIONS TO TEXT PROBLEMS 1. epinephrine to cAMP: There are two significant amplification stages. The binding of one epinephrine to a receptor stimulates the formation of many molecules of Gas. In turn, each molecule of Gas (when bound to adenylate cyclase) stimulates the formation of many molecules of cAMP.
human growth hormone to STAT5: There is one amplification event in this part of the pathway. Each hormone-/receptor-binding event activates a kinase (JAK2), which then can phosphorylate many molecules of STAT5.
EGF to Ras: All of the reactions up to Ras are stoichiometric (no amplification). An EGF/receptor complex autoactivates its own tyrosine kinase. The receptor’s phosphotyrosine then recruits Grb-2, which recruits Sos, which binds and activates Ras.
(Downstream from Ras, amplification does occur through a chain of protein phosphorylations [Figure 15.32].)
18
CHAPTER 15
2. The common feature between glutamate and phospho-Ser (or phospho-Thr) is the pres ence of a negative charge (at physiological pH). The negative charge on the glutatmate side chain therefore may sometimes fulfill the role of the negative charge on the phosphate.
3. No. Phospho-Ser and phospho-Thr are significantly smaller than phospho-Tyr. The phos phate of these smaller side chains probably will not reach sufficiently far into the deep binding pocket to make a favorable electrostatic interaction with a counter (+) charge.
4. In the dark the catalytic subunits (a and b) of cGMP phosphodiesterase (PDE) are in hibited by a pair of g subunits. Ta-GTP, the active form of transducin, activates PDE by prying away its g subunits.
The R2C2 complex of protein kinase A (PKA) is inactive. The binding of cAMP to the R (regulatory) chains releases catalytically active C chains.
The regulatory domain of protein kinase C (PKC) inhibits the catalytic domain by occupying the substrate binding site. The binding of diacylglycerol in the presence of Ca2; disrupts this interaction and enables a protein substrate to enter.
5. In the pseudosubstrate sequence A-R-K-G-A*-L-R-Q-K (Section 15.2.2), the central ala nine (A*) is critical in preventing activity. A* replaces the serine or threonine of a substrate sequence and is not phosphorylated. The other underlined residues are identical or similar to the consensus substrate sequence: X-R-XX (S,T)-Hyd-R-X, where Hyd refers to “hydrophobic” (e.g., Leu in the pseudosequence). The mutations therefore should be expected not to be at the underlined positions, but rather at any three of the four positions corresponding to X, that is either the K, G, Q, or initial A in the pseudosubstrate sequence.
6. Some growth factors act by binding two receptor molecules and causing the receptor to dimerize. Antibodies, with two identical binding sites, could similarly cause receptor dimerization and initiate the signaling process.
7. The mutated a-subunit would be defective for signaling because it would be turned “on” at all times, even in the absence of an activated receptor. The inability to turn “off” the signaling pathway would be a serious flaw.
8. The mutated hormone would bind to only one of its receptors. Receptor dimerization and signaling would be blocked. The mutated hormone would nevertheless be useful for studies of the interactions at the binding interface that remains active. For example, one would expect that it should be easier to co-crystallize the receptor with the mutant hormone (in a single binding motif) than with the native hormone (making two different binding interfaces).
9. Calcium is slowed because its intracellular concentration is low and it binds tightly to larger molecules, including proteins. The effective molecular weight of the diffusing complex therefore is large.
10. Epinephrine initiates a pathway that raises the level of cAMP within the muscle cell. The higher level of cAMP ultimately will mobilize glucose (make more glucose available).
Inhibitors of cAMP phosphodiesterase also will raise the level of cAMP within the cell.
Therefore the phosphodiesterase inhibitors will act similarly to epinephrine to increase
the mobilization of glucose.
11. It is reasonable to propose that the nerve growth factor will cause dimerization, au tophosphorylation, and activation of its receptor protein tyrosine kinase upon binding.
The active tyrosine kinase then should phosphorylate and activate a gamma (non-beta) isoform of phospholipase C. Active PLC then would release both diacylglycerol and inositol 1,4,5-trisphosphate from phosphatidyl inositol 4,5-bisphosphate (PIP2). Therefore, the concentration of the second messenger inositol 1,4,5-trisphosphate, as well as of diacylglycerol, would be expected to increase.
SIGNAL TRANSDUCTION PATHWAYS: AN INTRODUCTION TO INFORMATION METABOLISM
19
12. There are several similarities. Both adenylate cyclase and DNA polymerases use ATP as a substrate. In addition, both enzymes release pyrophosphate while forming a new phosphodiester bond. The key difference is that the adenylate cyclase forms a new intramolecular bond, whereas DNA polymerases join molecules by forming new intermolecular bonds.
13. (a) From the graphs, approximately 10:7 M of X, 3 * 10:6 M of Y, or 10:3 M of Z.
(b) Hormone X achieves maximal binding in the lowest concentration range and there fore has the highest binding affinity.
(c) For each hormone, the trend for the activation of adenylate cyclase is similar to the trend for hormone/receptor binding. Therefore, it is likely that the hormone/receptor complex plays a direct role in the mechanism of activation of adenylate cyclase.
(d) A requirement for GTP in addition to hormone would suggest that a Gs protein may be required. The trend for Gas activity should then be measured as a function of the concentration of hormones X, Y, and Z. This could be done by monitoring GTP/GDP exchange activity. Gs protein, the receptor, and unlabeled GDP should be preincubated in the absence of GTP and hormone. Then labeled GTP could be added together with varying amounts of a hormone X, Y, or Z. One would then test for the association of labeled GTP with protein when the proteins are subjected to precipitation, electrophoresis, or chromatography.
14. (a) The ligand X may “stick” to a few sites other than the specific receptor. These sites should not be counted.
(b) The experiment allows the background nonspecific binding to be determined. The large excess of nonradioactive ligand will bind to all of the authentic receptor sites.
The remaining (residual) background binding of the labeled ligand will be revealed as nonspecific binding (line labeled “nonspecific binding”).
(c) The plateau indicates that the ligand binding sites can be saturated. The sites can be saturated because in fact there exist only a discrete number of receptor molecules per cell. (Alternatively, if the cell uptake of ligand were to continue to increase without reaching a plateau, the result would indicate the absence of a specific receptor, and a different uptake mechanism would be operating.)
15. Paying attention to the units, we set up an equation to divide the binding activity by the specific activity and by the number of cells, and finally multiply by Avogadro’s number to convert moles to molecules: 104 cpm
1
protein
mg
6*1023
molecules
protein
mg 1010 cells 103 mmole 600 molecules .
1012 cpm
cell
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