Lloyd Cantley
The evolution of multicellular organisms necessitated the development of mechanisms to tightly coordinate the activities among cells. Such communication is fundamental to all biological processes, ranging from the induction of embryonic development to the integration of physiological responses in the face of environmental challenges.
As our understanding of cellular and molecular physiology has increased, it has become evident that all cells can receive and process information. External signals such as odorants, chemicals that reflect metabolic status, ions, hormones, growth factors, and neurotransmitters can all serve as chemical messengers linking neighboring or distant cells. Even external signals that are not considered chemical in nature (e.g., light and mechanical or thermal stimuli) may ultimately be transduced into a chemical messenger. Most chemical messengers interact with specific cell surface receptors and trigger a cascade of secondary events, including the mobilization of diffusible intracellular second-messenger systems that mediate the cell’s response to that stimulus. However, hydrophobic messengers, such as steroid hormones and some vitamins, can diffuse across the plasma membrane and interact with cytosolic or nuclear receptors. It is now clear that cells use a number of different, often intersecting intracellular signaling pathways to ensure that the cell’s response to a stimulus is tightly controlled.
MECHANISMS OF CELLULAR COMMUNICATION
Cells can communicate with one another by chemical signals
Early insight into signal transduction pathways was obtained from studies of the endocrine system. The classic definition of a hormone is a substance that is produced in one tissue or organ and released into the blood and carried to other organs (targets), where it acts to produce a specific response. The idea of endocrine or ductless glands developed from the recognition that certain organs—such as the pituitary, adrenal, and thyroid gland—can synthesize and release specific chemical messengers in response to particular physiological states. However, many other cells and tissues not classically thought of as endocrine in nature also produce hormones. For example, the kidney produces 1, 25-dihydroxyvitamin D3, and the salivary gland synthesizes nerve growth factor.
It is now recognized that intercellular communication can involve the production of a “hormone” or chemical signal by one cell type that acts in any (or all) of three ways, as illustrated in Figure 3-1: on distant tissues (endocrine), on a neighboring cell in the same tissue (paracrine), or on the same cell that released the signaling molecule (autocrine). For paracrine and autocrine signals to be delivered to their proper targets, their diffusion must be limited. This restriction can be accomplished by rapid endocytosis of the chemical signal by neighboring cells, its destruction by extracellular enzymes, or its immobilization by the extracellular matrix. The events that take place at the neuromuscular junction are excellent examples of paracrine signaling. When an electrical impulse travels down an axon and reaches the nerve terminal (Fig. 3-2), it stimulates release of the neurotransmitter acetylcholine (ACh). In turn, ACh transiently activates a ligand-gated cation channel on the muscle cell membrane. The resultant transient influx of Na+causes a localized positive shift of Vm (i.e., depolarization), initiating events that result in propagation of an action potential along the muscle cell. The ACh signal is rapidly terminated by the action of acetylcholinesterase, which is present in the synaptic cleft. This enzyme degrades the ACh that is released by the neuron.
Figure 3-1 Modes of cell communication.
Figure 3-2 Example of paracrine signaling. The release of ACh at the neuromuscular junction is a form of paracrine signaling because the nerve terminal releases a chemical (i.e., ACh) that acts on a neighboring cell (i.e., the muscle).
Soluble chemical signals interact with target cells by binding to surface or intracellular receptors
Four types of chemicals can serve as extracellular signaling molecules: amines, such as epinephrine; peptides and proteins, such as angiotensin II and insulin; steroids, including aldosterone, estrogens, and retinoic acid; and other small molecules, such as amino acids, nucleotides, ions (e.g., Ca2+), and gases (e.g., nitric oxide).
For a molecule to act as a signal, it must bind to a receptor. A receptor is a protein (or in some cases a lipoprotein) on the cell surface or within the cell that specifically binds a signaling molecule (the ligand). In some cases, the receptor is itself an ion channel, and ligand binding produces a change in Vm. Thus, the cell can transduce a signal with no machinery other than the receptor. In most cases, however, interaction of the ligand with one or more specific receptors results in an association of the receptor with an effector molecule that initiates a cellular response. Effectors include enzymes, channels, transport proteins, contractile elements, and transcription factors. The ability of a cell or tissue to respond to a specific signal is dictated by the complement of receptors it possesses and by the chain of intracellular reactions that are initiated by the binding of any one ligand to its receptor. Receptors can be divided into four categories on the basis of their associated mechanisms of signal transduction (Table 3-1).
Table 3-1 Classification of Receptors and Associated Signal Transduction Pathways
1. Ligand-gated ion channels. Integral membrane proteins, these hybrid receptor/channels are involved in signaling between electrically excitable cells. The binding of a neurotransmitter such as ACh to its receptor—which in fact is merely part of the channel—results in transient opening of the channel, thus altering the ion permeability of the cell.
2. G protein–coupled receptors. These integral plasma membrane proteins work indirectly—through an intermediary—to activate or to inactivate a separate membrane-associated enzyme or channel. The intermediary is a heterotrimeric guanosine triphosphate (GTP)–binding complex called a G protein.
3. Catalytic receptors. When activated by a ligand, these integral plasma membrane proteins are either enzymes themselves or part of an enzymatic complex.
4. Nuclear receptors. These proteins, located in the cytosol or nucleus, are ligand-activated transcription factors. These receptors link extracellular signals to gene transcription.
In addition to these four classes of membrane signaling molecules, some other transmembrane proteins act as messengers even though they do not fit the classic definition of a receptor. In response to certain physiological changes, they undergo regulated intramembrane proteolysis within the plane of the membrane, liberating cytosolic fragments that enter the nucleus to modulate gene expression. We discuss this process later in the chapter.
Signaling events initiated by plasma membrane receptors can generally be divided into six steps:
Step 1: Recognition of the signal by its receptor. The same signaling molecule can sometimes bind to more than one kind of receptor. For example, ACh can bind to both ligand-gated channels and G protein–coupled receptors. Binding of a ligand to its receptor involves the same three types of weak, noncovalent interactions that characterize substrate-enzyme interactions. Ionic bonds are formed between groups of opposite charge. In van der Waalsinteractions, a transient dipole in one atom generates the opposite dipole in an adjacent atom, thereby creating an electrostatic interaction. Hydrophobic interactions occur between nonpolar groups.
Step 2: Transduction of the extracellular message into an intracellular signal or second messenger. Ligand binding causes a conformational change in the receptor that triggers the catalytic activities intrinsic to the receptor or causes the receptor to interact with membrane or cytoplasmic enzymes. The final consequence is the generation of a second messenger or the activation of a catalytic cascade.
Step 3: Transmission of the second messenger’s signal to the appropriate effector. These effectors represent a diverse array of molecules, such as enzymes, ion channels, and transcription factors.
Step 4: Modulation of the effector. These events often result in the activation of protein kinases (which put phosphate groups on proteins) and phosphatases (which take them off), thereby altering the activity of other enzymes and proteins.
Step 5: Response of the cell to the initial stimulus. This collection of actions represents the summation and integration of input from multiple signaling pathways.
Step 6: Termination of the response by feedback mechanisms at any or all levels of the signaling pathway.
Cells can also communicate by direct interactions
Gap Junctions Neighboring cells can be electrically and metabolically coupled by means of gap junctions formed between apposing cell membranes. These water-filled channels facilitate the passage of inorganic ions and small molecules, such as Ca2+ and 3′, 5′-cyclic adenosine monophosphate (cAMP), from the cytoplasm of one cell into the cytoplasm of an adjacent cell. Mammalian gap junctions permit the passage of molecules that are less than ~1200 Da but restrict the movement of molecules that are greater than ~2000 Da. Gap junctions are also excellent pathways for the flow of electrical current between adjacent cells, playing a critical role in cardiac and smooth muscle.
The permeability of gap junctions can be rapidly regulated by changes in cytosolic concentrations of Ca2+, cAMP, and H+ as well as by the voltage across the cell membrane or membrane potential (Vm) (see Chapter 5). This type of modulation is physiologically important for cell-to-cell communication. For example, if a cell’s plasma membrane is damaged, Ca2+ passively moves into the cell and raises [Ca2+]i to toxic levels. Elevated intracellular [Ca2+] in the damaged cell triggers closure of the gap junctions, thus preventing the flow of excessive amounts of Ca2+ into the adjacent cell.
Adhering and Tight Junctions Adhering junctions form as the result of the Ca2+-dependent interactions of the extracellular domains of transmembrane proteins called cadherins (see Chapter 2). The clustering of cadherins at the site of interaction with an adjacent cell causes secondary clustering of intracellular proteins known as catenins, which in turn serve as sites of attachment for the intracellular actin cytoskeleton. Thus, adhering junctions provide important clues for the maintenance of normal cell architecture as well as the organization of groups of cells into tissues.
In addition to a homeostatic role, adhering junctions can serve a signaling role during organ development and remodeling. In a cell that is stably associated with its neighbors, a catenin known as β-catenin is mainly sequestered at the adhering junctions, minimizing concentration of free β-catenin. However, disruption of adhering junctions by certain growth factors, for example, causes β-catenin to disassociate from cadherin. The resulting rise in free β-catenin levels promotes the translocation of β-catenin to the nucleus. There, β-catenin regulates the transcription of multiple genes, including ones that promote cell proliferation and migration. (See Note: β-Catenins)
Similar to adhering junctions, tight junctions (see Chapter 2) comprise transmembrane proteins that link with their counterparts on adjacent cells as well as intracellular proteins that stabilize the complex and also have a signaling role. The transmembrane proteins—including claudins, occludin, and junctional adhesion molecule—and their extracellular domains create the diffusion barrier of the tight junction. One of the integral cytoplasmic proteins in tight junctions, zonula occludin 1 (ZO-1), colocalizes with a serine/threonine kinase known as WNK1, which is found in certain renal tubule epithelial cells that reabsorb Na+ and Cl− from the tubule lumen. Because WNK1 is important for determining the permeability of the tight junctions to Cl−, mutations in WNK1 can increase the movement of Cl− through the tight junctions (see Chapter 35) and thereby lead to hypertension. (See Note: WNK Kinases)
Membrane-Associated Ligands Another mechanism by which cells can directly communicate is by the interaction of a receptor in the plasma membrane with a ligand that is itself a membrane protein on an adjacent cell. Such membrane-associated ligands can provide spatial clues in migrating cells. For example, an ephrin ligand expressed on the surface of one cell can interact with an Eph receptor on a nearby cell. The resulting activation of the Eph receptor can in turn provide signals for regulating such developmental events as axonal guidance in the nervous system and endothelial cell guidance in the vasculature.
Second-messenger systems amplify signals and integrate responses among cell types
Once a signal has been received at the cell surface, it is typically amplified and transmitted to specific sites within the cells through second messengers. For a molecule to function as a second messenger, its concentration, or window of activity, must be finely regulated. The cell achieves this control by rapidly producing or activating the second messenger and then inactivating or degrading it. To ensure that the system returns to a resting state when the stimulus is removed, counterbalancing activities function at each step of the cascade.
The involvement of second messengers in catalytic cascades provides numerous opportunities to amplify a signal. For example, the binding of a ligand to its receptor can generate hundreds of second-messenger molecules, which can in turn alter the activity of thousands of downstream effectors. This modulation usually involves the conversion of an inactive species into an active molecule or vice versa. An example of such a cascade is the increased intracellular concentration of the second messenger cAMP. Receptor occupancy activates a G protein, which in turn stimulates a membrane-bound enzyme, adenylyl cyclase. This enzyme catalyzes the synthesis of cAMP from adenosine triphosphate (ATP), and a 5-fold increase in the intracellular concentration of cAMP is achieved in ~5 seconds. This sudden rise in cAMP levels is rapidly counteracted by its breakdown to adenosine 5′-monophosphate by cAMP phosphodiesterase.
Second-messenger systems also allow specificity and diversity. Ligands that activate the same signaling pathways in cells usually produce the same effect. For example, epinephrine, adrenocorticotropic hormone (ACTH), glucagon, and thyroid-stimulating hormone induce triglyceride breakdown through the cAMP messenger system. However, the same signaling molecule can produce distinct responses in different cells, depending on the complement of receptors and signal transduction pathways that are available in the cell as well as the specialized function that the cell carries out in the organism. For example, ACh induces contraction of skeletal muscle cells but inhibits contraction of heart muscle. It also facilitates the exocytosis of secretory granules in pancreatic acinar cells. This signaling molecule achieves these different endpoints by interacting with distinct receptors.
The diversity and specialization of second-messenger systems are important to a multicellular organism, as can be seen in the coordinated response of an organism to a stressful situation. Under these conditions, the adrenal gland releases epinephrine. Different organ systems respond to epinephrine in a distinct manner, such as activation of glycogen breakdown in the liver, constriction of the blood vessels of the skin, dilation of the blood vessels in skeletal muscle, and increased rate and force of heart contraction. The overall effect is an integrated response that readies the organism for attack, defense, or escape. In contrast, complex cell behaviors, such as proliferation and differentiation, are generally stimulated by combinations of signals rather than by a single signal. Integration of these stimuli requires crosstalkamong the various signaling cascades.
As discussed later, most signal transduction pathways use elaborate cascades of signaling proteins to relay information from the cell surface to effectors in the cell membrane, the cytoplasm, or the nucleus. In Chapter 4, we discuss how signal transduction pathways that lead to the nucleus can affect the cell by modulating gene transcription. These are genomic effects. Signal transduction systems that project to the cell membrane or to the cytoplasm produce nongenomic effects, the focus of this chapter.
RECEPTORS THAT ARE ION CHANNELS
Ligand-gated ion channels transduce a chemical signal into an electrical signal
The property that defines this class of multisubunit membrane-spanning receptors is that the signaling molecule itself controls the opening and closing of an ion channel by binding to a site on the receptor. Thus, these receptors are also called ionotropic receptors to distinguish them from the metabotropic receptors, which act through “metabolic” pathways. One superfamily of ligand-gated channels includes the ionotropic receptors for ACh, serotonin, γ-aminobutyric acid (GABA), and glycine. Most structural and functional information for ionotropic receptors comes from the nicotinic ACh receptor (AChR) present in skeletal muscle (Fig. 3-2). The nicotinic AChR is a cation channel that consists of four membrane-spanning subunits, α, β, γ, and δ, in a stoichiometry of 2 : 1 : 1 : 1. This receptor is called nicotinic because the nicotine contained in tobacco can activate or open the channel and thereby alter Vm. Note that the nicotinic AChR is very different from the muscarinic AChR discussed later, which is not a ligand-gated channel. Additional examples of ligand-gated channels are the IP3 receptor and the Ca2+ release channel (also known as the ryanodine receptor). Both receptors are tetrameric Ca2+ channels located in the membranes of intracellular organelles.
RECEPTORS COUPLED TO G PROTEINS
G protein–coupled receptors (GPCRs) constitute the largest family of receptors on the cell surface, with more than 1000 members. GPCRs mediate cellular responses to a diverse array of signaling molecules, such as hormones, neurotransmitters, vasoactive peptides, odorants, tastants, and other local mediators. Despite the chemical diversity of their ligands, most receptors of this class have a similar structure (Fig. 3-3). They consist of a single polypeptide chain with seven membrane-spanning α-helical segments, an extracellular N terminus that is glycosylated, a large cytoplasmic loop that is composed mainly of hydrophilic amino acids between helices 5 and 6, and a hydrophilic domain at the cytoplasmic C terminus. Most small ligands (e.g., epinephrine) bind in the plane of the membrane at a site that involves several membrane-spanning segments. In the case of larger protein ligands, a portion of the extracellular N terminus also participates in ligand binding. The 5, 6-cytoplasmic loop appears to be the major site of interaction with the intracellular G protein, although the 3, 4-cytoplasmic loop and the cytoplasmic C terminus also contribute to binding in some cases. Binding of the GPCR to its extracellular ligand regulates this interaction between the receptor and the G proteins, thus transmitting a signal to downstream effectors. In the next four sections of this subchapter, we discuss the general principles of how G proteins function; three major second-messenger systems that are triggered by G proteins are then considered.
Figure 3-3 Receptor coupled to a G protein.
GENERAL PROPERTIES OF G PROTEINS
G proteins are heterotrimers that exist in many combinations of different α, β, and γ subunits
G proteins are members of a superfamily of GTP-binding proteins. This superfamily includes the classic heterotrimeric G proteins that bind to GPCRs as well as the so-called small GTP-binding proteins, such as Ras. Both the heterotrimeric and small G proteins can hydrolyze GTP and switch between an active GTP-bound state and an inactive guanosine diphosphate (GDP)–bound state.
Heterotrimeric G proteins are composed of three subunits, α, β, and γ. At least 16 different α subunits (~42 to 50 kDa), 5 β subunits (~33 to 35 kDa), and 11 γ subunits (~8 to 10 kDa) are present in mammalian tissue. The α subunit binds and hydrolyzes GTP and also interacts with “downstream” effector proteins such as adenylyl cyclase. Historically, the α subunits were thought to provide the principal specificity to each type of G protein, with the βγ complex functioning to anchor the trimeric complex to the membrane. However, it is now clear that the βγ complex also functions in signal transduction by interacting with certain effector molecules. Moreover, both the α and γ subunits are involved in anchoring the complex to the membrane. The α subunit is held to the membrane by either a myristyl or a palmitoyl group; the γ subunit is held by a prenyl group.
The multiple α, β, and γ subunits demonstrate distinct tissue distributions and interact with different receptors and effectors (Table 3-2). Because of the potential for several hundred combinations of the known α, β, and γ subunits, G proteins are ideally suited to link a diversity of receptors to a diversity of effectors. The many classes of G proteins, in conjunction with the presence of several receptor types for a single ligand, provide a mechanism whereby a common signal can elicit the appropriate physiological changes in different tissues. For example, when epinephrine binds β1-adrenergic receptors in the heart, it stimulates adenylyl cyclase, which increases heart rate and the force of contraction. However, in the periphery, epinephrine acts on α2-adrenergic receptors coupled to a G protein that inhibits adenylyl cyclase, thereby increasing peripheral vascular resistance and consequently increasing venous return and blood pressure.
Table 3-2 Families of G Proteins
Among the first effectors found to be sensitive to G proteins was the enzyme adenylyl cyclase. The heterotrimeric G protein known as Gs was so named because it stimulates adenylyl cyclase. A separate class of G proteins was given the name Gi because it is responsible for the hormone-dependent inhibition of adenylyl cyclase. Identification of these classes of G proteins was greatly facilitated by the observation that the α subunits of individual G proteins are substrates for adenosine diphosphate (ADP) ribosylation catalyzed by bacterial toxins. The toxin from Vibrio cholerae activates Gs, whereas the toxin from Bordetella pertussis inactivates the cyclase-inhibiting Gi (see the box titled Action of Toxins on Heterotrimeric G Proteins).
For their work in identifying G proteins and elucidating the physiological role of these proteins, Alfred Gilman and Martin Rodbell received the 1994 Nobel Prize in Physiology or Medicine. (See Note: Alfred Gilman and Martin Rodbell)
G protein activation follows a cycle
In their inactive state, heterotrimeric G proteins are a complex of α, β, and γ subunits in which GDP occupies the guanine nucleotide–binding site of the α subunit. After ligand binding to the GPCR (Fig. 3-4, step 1), the activated receptor interacts with the αβγ heterotrimer to promote a conformational change that facilitates the release of bound GDP and simultaneous binding of GTP (step 2). This GDP-GTP exchange stimulates dissociation of the complex from the receptor (step 3) and causes disassembly of the trimer into a free α subunit and βγ complex (step 4). The free, active GTP-bound α subunit can now interact in the plane of the membrane with downstream effectors such as adenylyl cyclase and phospholipases (step 5). Similarly, the βγ subunit can now activate ion channels or other effectors.
Figure 3-4 Enzymatic cycle of heterotrimeric G proteins.
The α subunit terminates the signaling events that are mediated by the α and βγ subunits by hydrolyzing GTP to GDP and inorganic phosphate (Pi). The result is an inactive α-GDP complex that dissociates from its downstream effector and reassociates with a βγ subunit (Fig. 3-4, step 6), thus completing the cycle (step 1). The βγ subunit stabilizes α-GDP and thereby substantially slows the rate of GDP-GTP exchange (step 2) and dampens signal transmission in the resting state.
The RGS (for “regulation of G protein signaling”) family of proteins appears to enhance the intrinsic guanosine triphosphatase (GTPase) activity of some but not all α subunits. Investigators have identified at least 15 mammalian RGS proteins and shown that they interact with specific α subunits. RGS proteins bind the complex Gα/GDP/AlF4−, which is the structural analogue of the GTPase transition state. By stabilizing the transition state, RGS proteins may promote GTP hydrolysis and thus the termination of signaling.
As noted earlier, α subunits can be anchored to the cell membrane by myristyl or palmitoyl groups. Activation can result in the removal of these groups and the release of the α subunit into the cytosol. Loss of the α subunit from the membrane may decrease the interaction of G proteins with receptors and downstream effectors (e.g., adenylyl cyclase).
Activated α subunits couple to a variety of downstream effectors, including enzymes, ion channels, and membrane trafficking machinery
Activated α subunits can couple to a variety of enzymes. A major enzyme that acts as an effector downstream of activated α subunits is adenylyl cyclase (Fig. 3-5A). This enzyme can be either activated or inhibited by G protein signaling, depending on whether it associates with the GTP-bound form of Gαs (stimulatory) or Gαi (inhibitory). Thus, different hormones—acting through different G protein complexes—can have opposing effects on the same intracellular messenger. (See Note: Compartmentalization of Second Messenger Effects)
Figure 3-5 Downstream effects of activated G protein α subunits. A, When a ligand binds to a receptor coupled to αs, adenylyl cyclase (AC) is activated, whereas when a ligand binds to a receptor coupled to αi, the enzyme is inhibited. The activated enzyme converts ATP to cAMP, which then can activate protein kinase A (PKA). B, In phototransduction, a photon interacts with the receptor and activates the G protein transducin. The αt activates phosphodiesterase (PDE), which in turn hydrolyzes cGMP and lowers the intracellular concentrations of cGMP and therefore closes the cGMP-activated channels. C, In this example, the ligand binds to a receptor that is coupled to αq, which activates phospholipase C (PLC). This enzyme converts PIP2 to IP3 and diacylglycerol (DAG). The IP3 leads to the release of Ca2+ from intracellular stores, whereas the diacylglycerol activates protein kinase C (PKC). ER, endoplasmic reticulum.
G proteins can also activate enzymes that break down cyclic nucleotides. For example, the G protein called transducin, which plays a key role in phototransduction (see Chapter 15), activates the cyclic guanosine monophosphate (cGMP) phosphodiesterase, which catalyzes the breakdown of cGMP to GMP (Fig. 3-5B). Thus, in retinal cells expressing transducin, light leads to a decrease in [cGMP]i.
G proteins can also couple to phospholipases. These enzymes catabolize phospholipids, as discussed in detail later in the section on G protein second messengers. This super-family of phospholipases can be grouped into phospholipases A2, C, or D on the basis of the site at which the enzyme cleaves the phospholipid. The G protein αq subunit activates phospholipase C, which breaks phosphatidylinositol bisphosphate (PIP2) into two intracellular messengers, membrane-associated diacylglycerol and cytosolic IP3 (Fig. 3-5C). Diacylglycerol stimulates protein kinase C, whereas IP3 binds to a receptor on the endoplasmic reticulum membrane and triggers the release of Ca2+from intracellular stores.
Some G proteins interact with ion channels. Agonists that bind to the β-adrenergic receptor activate the L-type Ca2+ channel in the heart and skeletal muscle (see Chapter 7). The G protein Gs directly stimulates this channel as the α subunit of Gs binds to the channel, and Gs also indirectly stimulates this channel through a signal transduction cascade that involves cAMP-dependent protein kinase.
A clue that G proteins serve additional functions in membrane trafficking (see Chapter 2) in the cell comes from the observation that many cells contain intracellular pools of heterotrimeric G proteins, some bound to internal membranes and some free in the cytosol. Experiments involving toxins, inhibitors, and cell lines harboring mutations in G protein subunits have demonstrated that these intracellular G proteins are involved in vesicular transport. G proteins have been implicated in the budding of secretory vesicles from the trans-Golgi network, fusion of endosomes, recruitment of non–clathrin coat proteins, and transcytosis and apical secretion in polarized epithelial cells. The receptors and effectors that interact with these intracellular G proteins have not been determined.
Action of Toxins on Heterotrimeric G Proteins
Infectious diarrheal disease has a multitude of causes. Cholera toxin, a secretory product of the bacterium Vibrio cholerae, is responsible in part for the devastating characteristics of cholera. The toxin is an oligomeric protein composed of one A subunit and five B subunits (AB5). After cholera toxin enters intestinal epithelial cells, the A subunit separates from the B subunits and becomes activated by proteolytic cleavage. The resulting active A1 fragment catalyzes the ADP ribosylation of Gαs. This ribosylation, which involves transfer of the ADP-ribose moiety from the oxidized form of nicotinamide adenine dinucleotide (NAD+) to the α subunit, inhibits the GTPase activity of Gαs. As a result of this modification, Gαs remains in its activated, GTP-bound form and can activate adenylyl cyclase. In intestinal epithelial cells, the constitutively activated Gαs elevates levels of cAMP, which causes an increase in Cl− conductance and water flow and thereby contributes to the large fluid loss characteristic of this disease.
A related bacterial product is pertussis toxin, which is also an AB5 protein. It is produced by Bordetella pertussis, the causative agent of whooping cough. Pertussis toxin ADP-ribosylates Gαi. This ADP-ribosylated Gαi cannot exchange its bound GDP (inactive state) for GTP. Thus, αi remains in its GDP-bound inactive state. As a result, receptor occupancy can no longer release the active αi-GTP, so adenylyl cyclase cannot be inhibited. Thus, both cholera toxin and pertussis toxin increase the generation of cAMP.
The βγ subunits of G proteins can also activate downstream effectors
Considerable evidence now indicates that the βγ subunits can also interact with downstream effectors. The neurotransmitter ACh released from the vagus nerve reduces the rate and strength of heart contraction. This action in the atria of the heart is mediated by muscarinic M2 AChRs (see Chapter 14). These receptors can be activated by muscarine, an alkaloid found in certain poisonous mushrooms. Muscarinic AChRs are very different from the nicotinic AChRs discussed earlier, which are ligand-gated channels. Binding of ACh to the muscarinic M2 receptor in the atria activates a heterotrimeric G protein, resulting in the generation of both activated Gαi as well as a free βγ subunit complex. The βγ complex then interacts with a particular class of K+ channels, increasing their permeability. This increase in K+ permeability keeps the membrane potential relatively negative and thus renders the cell more resistant to excitation. The βγ subunit complex also modulates the activity of adenylyl cyclase and phospholipase C and stimulates phospholipase A2. Such effects of βγ can be independent of, synergize with, or antagonize the action of the α subunit. For example, studies using various isoforms of adenylyl cyclase have demonstrated that purified βγ stimulates some isoforms, inhibits others, and has no effect on still others. Different combinations of βγ isoforms may have different activities. For example, β1γ1 is one tenth as efficient at stimulating type II adenylyl cyclase as is β1γ2.
An interesting action of some βγ complexes is that they bind to a special protein kinase called the β-adrenergic receptor kinase (βARK). As a result of this interaction, βARK translocates to the plasma membrane, where it phosphorylates the ligand-receptor complex (but not the unbound receptor). This phosphorylation results in the recruitment of β-arrestin to the GPCR, which in turn mediates disassociation of the receptor-ligand complex and thus attenuates the activity of the same β-adrenergic receptors that gave rise to the βγ complex in the first place. This action is an example of receptor desensitization. These phosphorylated receptors eventually undergo endocytosis, which transiently reduces the number of receptors that are available on the cell surface. This endocytosis is an important step in resensitization of the receptor system.
Small GTP-binding proteins are involved in a vast number of cellular processes
A distinct group of proteins that are structurally related to the α subunit of the heterotrimeric G proteins are the small GTP-binding proteins. More than 100 of these have been identified to date, and they have been divided into five groups including the Ras, Rho, Rab, Arf, and Ran families. These 21-kDa proteins can be membrane associated (e.g., Ras) or may translocate between the membrane and the cytosol (e.g., Rho).
The three isoforms of Ras (N, Ha, and Ki) relay signals from the plasma membrane to the nucleus through an elaborate kinase cascade (see Chapter 4), thereby regulating gene transcription. In some tumors, mutation of the genes encoding Ras proteins results in constitutively active Ras. These mutated genes are called oncogenes because the altered Ras gene product promotes the malignant transformation of a cell and can contribute to the development of cancer (oncogenesis). In contrast, Rho family members are primarily involved in rearrangement of the actin cytoskeleton; Rab and Arf proteins regulate vesicle trafficking.
Similar to the α subunit of heterotrimeric G proteins, the small GTP-binding proteins switch between an inactive GDP-bound form and an active GTP-bound form. Two classes of regulatory proteins modulate the activity of these small GTP-binding proteins. The first of these includes the GTPase-activating proteins (GAPs) and neurofibromin (a product of the neurofibromatosis type 1 gene). GAPs increase the rate at which small GTP-binding proteins hydrolyze bound GTP and thus result in more rapid inactivation. Counteracting the activity of GAPs are guanine nucleotide exchange proteins (GEFs) such as “son of sevenless” or SOS, which promote the conversion of inactive Ras-GDP to active Ras-GTP. Interestingly, cAMP directly activates several GEFs, such as Epac (exchange protein activated by cAMP), demonstrating crosstalk between a classical heterotrimeric G protein signaling pathway and the small Ras-like G proteins.
G PROTEIN SECOND MESSENGERS: CYCLIC NUCLEOTIDES
cAMP usually exerts its effect by increasing the activity of protein kinase A
Activation of Gs-coupled receptors results in the stimulation of adenylyl cyclase and a rise in intracellular concentrations of cAMP (Fig. 3-5A). The downstream effects of this increase in [cAMP]i depend on the specialized functions that the responding cell carries out in the organism. For example, in the adrenal cortex, ACTH stimulation of cAMP production results in the secretion of aldosterone and cortisol; in the kidney, vasopressin-induced changes in cAMP levels facilitate water reabsorption (see Chapters 38 and 50). Excess cAMP is also responsible for certain pathologic conditions. One is cholera (see the box on page 57, titled Action of Toxins on Heterotrimeric G Proteins). Another pathologic process associated with excess cAMP is McCune-Albright syndrome, characterized by a triad of (1) variable hyperfunction of multiple endocrine glands, including precocious puberty in girls, (2) bone lesions, and (3) pigmented skin lesions (café au lait spots). This disorder is caused by a somatic mutation that constitutively activates the G protein αs subunit in a mosaic pattern.
cAMP exerts many of its effects through cAMP-dependent protein kinase A (PKA). This enzyme catalyzes transfer of the terminal phosphate of ATP to certain serine or threonine residues within selected proteins. PKA phosphorylation sites are present in a multitude of intracellular proteins, including ion channels, receptors, and signaling pathway proteins. Phosphorylation of these sites can influence either the localization or the activity of the substrate. For example, phosphorylation of the β2-adrenergic receptor causes receptor desensitization in neurons, whereas phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR) increases its Cl− channel activity.
To enhance regulation of phosphorylation events, the cell tightly controls the activity of PKA so that the enzyme can respond to subtle—and local—variations in cAMP levels. One important control mechanism is the use of regulatory subunits that constitutively inhibit PKA. In the absence of cAMP, two catalytic subunits of PKA associate with two of these regulatory subunits, resulting in a heterotetrameric protein complex that has a low level of catalytic activity (Fig. 3-6). Binding of cAMP to the regulatory subunits induces a conformational change that diminishes their affinity for the catalytic subunits, and the subsequent dissociation of the complex results in activation of kinase activity. In addition to the short-term effects of PKA activation noted before, the free catalytic subunit of PKA can also enter the nucleus, where substrate phosphorylation can activate the transcription of specific PKA-dependent genes (see Chapter 4). Although most cells use the same catalytic subunit, different regulatory subunits are found in different cell types.
Figure 3-6 Activation of protein kinase A by cAMP.
Another mechanism that contributes to regulation of PKA is the targeting of the enzyme to specific subcellular locations. Such targeting promotes the preferential phosphorylation of substrates that are confined to precise locations within the cell. PKA targeting is achieved by the association of a PKA regulatory subunit with an A kinase anchoring protein (AKAP), which in turn binds to cytoskeletal elements or to components of cellular subcompartments. More than 35 AKAPs are known. The specificity of PKA targeting is highlighted by the observation that in neurons, PKA is localized to postsynaptic densities through its association with AKAP79. This anchoring protein also targets calcineurin—a protein phosphatase—to the same site. This targeting of both PKA and calcineurin to the same postsynaptic site makes it possible for the cell to tightly regulate the phosphorylation state of important neuronal substrates.
The cAMP generated by adenylyl cyclase does not interact only with PKA. For example, olfactory receptors (see Chapter 15) interact with a member of the Gs family called Golf. The rise in [cAMP]i that results from activation of the olfactory receptor activates a cation channel, a member of the family of cyclic nucleotide–gated (CNG) ion channels. Na+ influx through this channel leads to membrane depolarization and the initiation of a nerve impulse.
For his work in elucidating the role played by cAMP as a second messenger in regulating glycogen metabolism, Earl Sutherland received the 1971 Nobel Prize in Physiology or Medicine. In 1992, Edmond Fischer and Edwin Krebs shared the prize for their part in demonstrating the role of protein phosphorylation in the signal transduction process. (See Note: Earl W. Sutherland, Jr.; Edmond H. Fischer and Edwin S. Krebs)
This coordinated set of phosphorylation and dephosphorylation reactions has several physiological advantages. First, it allows a single molecule (e.g., cAMP) to regulate a range of enzymatic reactions. Second, it affords a large amplification to a small signal. The concentration of epinephrine needed to stimulate glycogenolysis in muscle is ~10−10 M. This subnanomolar level of hormone can raise [cAMP]i to ~10−6 M. Thus, the catalytic cascades amplify the hormone signal 10,000-fold, resulting in the liberation of enough glucose to raise blood glucose levels from ~5 to ~8 mM. Although the effects of cAMP on the synthesis and degradation of glycogen are confined to muscle and liver, a wide variety of cells use cAMP-mediated activation cascades in the response to a wide variety of hormones.
Protein phosphatases reverse the action of kinases
As discussed, one way that the cell can terminate a cAMP signal is to use a phosphodiesterase to degrade cAMP. In this way, the subsequent steps along the signaling pathway can also be terminated. However, because the downstream effects of cAMP often involve phosphorylation of effector proteins at serine and threonine residues by kinases such as PKA, another powerful way to terminate the action of cAMP is to dephosphorylate these effector proteins. Such dephosphorylation events are mediated by enzymes called serine/threonine phosphoprotein phosphatases.
Four groups of serine/threonine phosphoprotein phosphatases (PP) are known, 1, 2a, 2b, and 2c. These enzymes themselves are regulated by phosphorylation at their serine, threonine, and tyrosine residues. The balance between kinase and phosphatase activity plays a major role in the control of signaling events.
PP1 dephosphorylates many proteins phosphorylated by PKA, including those phosphorylated in response to epinephrine (see Chapter 58). Another protein, phosphoprotein phosphatase inhibitor 1 (I-1), can bind to and inhibit PP1. Interestingly, PKA phosphorylates and thus activates I-1 (Fig. 3-7), thereby inhibiting PP1 and preserving the phosphate groups added by PKA in the first place.
Figure 3-7 Activation of phosphoprotein phosphatase 1 (PP1) by PKA. I-1, inhibitor of PP1.
PP2a, which is less specific than PP1, appears to be the main phosphatase responsible for reversing the action of other protein serine/threonine kinases. The Ca2+-dependent PP2b, also known as calcineurin, is prevalent in the brain, skeletal muscle, and cardiac muscle and is also the target of the immunosuppressive reagents FK-506 and cyclosporine. The importance of PP2c is presently unclear.
In addition to serine/threonine kinases such as PKA, a second group of kinases involved in regulating signaling pathways (discussed later in this chapter) are known as tyrosine kinases because they phosphorylate their substrate proteins on tyrosine residues. The enzymes that remove phosphates from these tyrosine residues are much more variable than the serine and threonine phosphatases. The first phosphotyrosine phosphatase (PTP) to be characterized was the cytosolic enzyme PTP1B from human placenta. PTP1B has a high degree of homology with CD45, a membrane protein that is both a receptor and a tyrosine phosphatase. cDNA sequence analysis has identified a large number of PTPs that can be divided into two classes: membrane-spanning receptor-like proteins such as CD45 and cytosolic forms such as PTP1B. A number of intracellular PTPs contain so-called Src homology 2 (SH2) domains, a peptide sequence or motif that interacts with phosphorylated tyrosine groups. Several of the PTPs are themselves regulated by phosphorylation.
cGMP exerts its effect by stimulating a nonselective cation channel in the retina
cGMP is another cyclic nucleotide that is involved in G protein signaling events. In the outer segments of rods and cones in the visual system, the G protein does not couple to an enzyme that generates cGMP but, as noted earlier, couples to an enzyme that breaks it down. As discussed further in Chapter 15, light activates a GPCR called rhodopsin, which activates the G protein transducin, which in turn activates the cGMP phosphodiesterase that lowers [cGMP]i. The fall in [cGMP]i closes cGMP-gated nonselective cation channels that are members of the same family of CNG ion channels that cAMP activates in olfactory signaling (see Chapter 15).
G PROTEIN SECOND MESSENGERS: PRODUCTS OF PHOSPHOINOSITIDE BREAKDOWN
Many messengers bind to receptors that activate phosphoinositide breakdown
Although the phosphatidylinositols (PIs) are minor constituents of cell membranes, they are largely distributed in the internal leaflet of the membrane and play an important role in signal transduction. The inositol sugar moiety of PI molecules (see Fig. 2-2A) can be phosphorylated to yield the two major phosphoinositides that are involved in signal transduction: phosphatidylinositol 4, 5-bisphosphate (PI4, 5P2 or PIP2) and phosphatidylinositol 3, 4, 5-trisphosphate (PI3, 4, 5P3). (See Note: Acyl Groups)
Certain membrane-associated receptors act though G proteins (e.g., Gq) that stimulate phospholipase C (PLC) to cleave PIP2 into inositol 1, 4, 5-trisphosphate (IP3) and diacylglycerol (DAG), as shown in Figure 3-8A. PLCs are classified into three families (β, γ, δ) that differ in their catalytic properties, cell type–specific expression, and modes of activation. PLCβ is typically activated downstream of certain G proteins (e.g., Gq), whereas PLCγ contains an SH2 domain and is activated downstream of certain tyrosine kinases. Stimulation of PLCβ results in a rapid increase in cytosolic IP3 levels as well as an early peak in DAG levels (Fig. 3-8B). Both products are second messengers. DAG remains in the plane of the membrane to activate protein kinase C, which migrates from the cytosol and binds to DAG in the membrane. The water-soluble IP3 travels through the cytosol to stimulate Ca2+ release from intracellular stores. It is within this system that Ca2+ was first identified as a messenger that mediates the stimulus-response coupling of endocrine cells.
Figure 3-8 Second messengers in the DAG/IP3 pathway. ER, endoplasmic reticulum; SERCA, sarcoplasmic and endoplasmic reticulum Ca2+-ATPase.
Phosphatidylcholines (PCs), which—unlike PI—are an abundant phospholipid in the cell membrane, are also a source of DAG. The cell can produce DAG from PC by either of two mechanisms (Fig. 3-8C). First, PLC can directly convert PC to phosphocholine and DAG. Second, phospholipase D (PLD), by cleaving the phosphoester bond on the other side of the phosphate, converts PC to choline and phosphatidic acid (PA; also phospho-DAG). This PA can then be converted to DAG by PA-phosphohydrolase. Production of DAG from PC, either directly (by PLC) or indirectly (by PLD), produces the slow wave of increasing cytosolic DAG shown in Figure 3-8B. Thus, in some systems, the formation of DAG is biphasic and consists of an early peak that is transient and parallels the formation of IP3, followed by a late phase that is slow in onset but sustained for several minutes.
Factors such as tumor necrosis factor α (TNF-α), interleukin 1 (IL-1), interleukin 3 (IL-3), interferon α (IFN-α), and colony-stimulating factor stimulate the production of DAG from PC. Once generated, some DAGs can be further cleaved by DAG lipase to arachidonic acid, which can have signaling activity itself or can be metabolized to other signaling molecules, the eicosanoids. We cover arachidonic acid metabolism later in this chapter.
Inositol triphosphate liberates Ca2+ from intracellular stores
As discussed earlier, IP3 is generated by the metabolism of membrane phospholipids and then travels through the cytosol to release Ca2+ from intracellular stores. The IP3 receptor (ITPR) is a ligand-gated Ca2+channel located in the membrane of the endoplasmic reticulum (Fig. 3-8A). This Ca2+ channel is structurally related to the Ca2+ release channel (or ryanodine receptor), which is responsible for releasing Ca2+from the sarcoplasmic reticulum of muscle and thereby switching on muscle contraction (see Chapter 9). The IP3 receptor is a tetramer composed of subunits of ~260 kDa. At least three genes encode the subunits of the receptor. These genes are subject to alternative splicing, which further increases the potential for receptor diversity. The receptor is a substrate for phosphorylation by protein kinases A and C and calcium-calmodulin (Ca2+-CaM)–dependent protein kinases. (See Note: IP3 Receptor Diversity)
Interaction of IP3 with its receptor results in passive efflux of Ca2+ from the endoplasmic reticulum and thus a rapid rise in the free cytosolic Ca2+ concentration. The IP3-induced changes in [Ca2+]i exhibit complex temporal and spatial patterns. The rise in [Ca2+]i can be brief or persistent and can oscillate repetitively, spread in spirals or waves within a cell, or spread across groups of cells that are coupled by gap junctions. In at least some systems, the frequency of [Ca2+]i oscillations seems to be physiologically important. For example, in isolated pancreatic acinar cells, graded increases in the concentration of ACh produce graded increases in the frequency—but not the magnitude—of repetitive [Ca2+]i spikes. The mechanisms responsible for [Ca2+]i oscillations and waves are complex. It appears that both propagation and oscillation depend on positive feedback mechanisms, in which low [Ca2+]i facilitates Ca2+release, as well as on negative feedback mechanisms, in which high [Ca2+]i inhibits further Ca2+ release.
The dephosphorylation of IP3 terminates the release of Ca2+ from intracellular stores; an ATP-fueled Ca2+ pump (SERCA; see Chapter 5) then moves the Ca2+ back into the endoplasmic reticulum. Some of the IP3 is further phosphorylated to IP4, which may mediate a slower and more prolonged response of the cell or may promote the refilling of intracellular stores. In addition to IP3, cyclic ADP ribose (cADPR) can mobilize Ca2+ from intracellular stores and augment a process known as calcium-induced Ca2+ release. Although the details of these interactions have not been fully elucidated, cADPR appears to bind to the Ca2+ release channel (ryanodine receptor) in a Ca2+-CaM–dependent manner.
In addition to the increase in [Ca2+]i produced by the release of Ca2+ from intracellular stores, [Ca2+]i can also rise as a result of enhanced influx of this ion through Ca2+ channels in the plasma membrane. For Ca2+ to function as a second messenger, it is critical that [Ca2+]i be normally maintained at relatively low levels (at or below ~100 nM). Leakage of Ca2+ into the cell through Ca2+ channels is opposed by the extrusion of Ca2+ across the plasma membrane by both an ATP-dependent Ca2+ pump and the Na-Ca exchanger (see Chapter 5).
As discussed later, increased [Ca2+]i exerts its effect by binding to cellular proteins and changing their activity. Some Ca2+-dependent signaling events are so sensitive to Ca2+ that a [Ca2+]i increase of as little as 100 nM can trigger a vast array of cellular responses. These responses include secretion of digestive enzymes by pancreatic acinar cells, release of insulin by β cells, contraction of vascular smooth muscle, conversion of glycogen to glucose in the liver, release of histamine by mast cells, aggregation of platelets, and DNA synthesis and cell division in fibroblasts.
Calcium activates calmodulin-dependent protein kinases
How does an increase in [Ca2+]i lead to downstream responses in the signal transduction cascade? The effects of changes in [Ca2+]i are mediated by Ca2+-binding proteins, the most important of which is calmodulin (CaM). CaM is a high-affinity cytoplasmic Ca2+-binding protein of 148 amino acids. Each molecule of CaM cooperatively binds four calcium ions. Ca2+ binding induces a major conformational change in CaM that allows it to bind to other proteins (Fig. 3-9). Although CaM does not have intrinsic enzymatic activity, it forms a complex with a number of enzymes and thereby confers a Ca2+ dependence on their activity. For example, binding of the Ca2+-CaM complex activates the enzyme that degrades cAMP, cAMP phosphodiesterase.
Figure 3-9 Calmodulin. After four intracellular Ca2+ ions bind to calmodulin, the Ca2+-CaM complex can bind to and activate another protein. In this example, the activated protein is a Ca2+-CaM–dependent kinase.
Many of the effects of CaM occur as the Ca2+-CaM complex binds to and activates a family of Ca2+-CaM–dependent kinases (CaM kinases). These kinases phosphorylate certain serine and threonine residues of a variety of proteins. An important CaM kinase in smooth muscle cells is myosin light chain kinase (MLCK) (see Chapter 9). Another CaM kinase is glycogen phosphorylase kinase (PK), which plays a role in glycogen degradation (see Chapter 58).
MLCK, PK, and some other CaM kinases have a rather narrow substrate specificity. The ubiquitous CaM kinase II, on the other hand, has a broad substrate specificity. Especially high levels of this multifunctional enzyme are present at the synaptic terminals of neurons. One of the actions of CaM kinase II is to phosphorylate and thereby activate the rate-limiting enzyme (tyrosine hydroxylase; see Fig. 13-8C) in the synthesis of catecholamine neurotransmitters. CaM kinase can also phosphorylate itself, which allows it to remain active in the absence of Ca2+.
Diacylglycerols and Ca2+ activate protein kinase C
As noted earlier, hydrolysis of PIP2 by PLC yields not only the IP3 that leads to Ca2+ release from internal stores but also DAG (Fig. 3-8A). The most important function of DAG is to activate protein kinase C (PKC), a serine/threonine kinase. In mammals, the PKC family comprises at least 10 members that differ in their tissue and cellular localization. This family is further subdivided into three groups that all require membrane-associated phosphatidylserine but have different requirements for Ca2+ and DAG. The classical PKC family members PKCα, PKCβ, and PKCγ require both DAG and Ca2+ for activation, whereas the novel PKCs (such as PKCδ, PKC
, and PKCη) are independent of Ca2+, and the atypical PKCs (PKCζ and PKCλ) appear to be independent of both DAG and Ca2+. As a consequence, the signals generated by the PKC pathway depend on the isoforms of the enzyme that a cell expresses as well as on the levels of Ca2+ and DAG at specific locations at the cell membrane. In its basal state, PKCα is an inactive, soluble cytosolic protein. When Ca2+ binds to cytosolic PKC, PKC can interact with DAG, which is located in the inner leaflet of the plasma membrane. This interaction with DAG activates PKCα by raising its affinity for Ca2+. This process is often referred to as translocation of PKC from the cytoplasm to the membrane. In most cells, the Ca2+ signal is transient, whereas the resulting physiological responses, such as proliferation and differentiation, often persist substantially longer. Sustained activation of PKCα may be essential for maintaining these responses. Elevated levels of active PKCα are maintained by a slow wave of elevated DAG (Fig. 3-8B), which is due to the hydrolysis of PC by PLC and PLD.
Physiological stimulation of the classical and novel PKCs by DAG can be mimicked by the exogenous application of a class of tumor promoters called phorbol esters. These plant products bind to these PKCs, cause them to translocate to the plasma membrane, and thus specifically activate them even in the absence of DAG.
Among the major substrates of PKC are the myristoylated, alanine-rich C kinase substrate (MARCKS) proteins. These acidic proteins contain consensus sites for PKC phosphorylation as well as CaM-and actin-binding sites. MARCKS proteins cross-link actin filaments and thus appear to play a role in translating extracellular signals into actin plasticity and changes in cell shape. Unphosphorylated MARCKS proteins are associated with the plasma membrane, and they cross-link actin. Phosphorylation of the MARCKS proteins causes them to translocate into the cytosol, where they are no longer able to cross-link actin. Thus, mitogenic growth factors that activate PKC may produce morphological changes and anchorage-independent cell proliferation, in part by modifying the activity of MARCKS proteins.
PKC can also directly or indirectly modulate transcription factors and thereby enhance the transcription of specific genes (see Chapter 4). Such genomic actions of PKC explain why phorbol esters are tumor promoters.
G PROTEIN SECOND MESSENGERS: ARACHIDONIC ACID METABOLITES
As previously discussed, PLC can hydrolyze PIP2 and thereby release two important signaling molecules, IP3 and DAG. In addition, both PLC and PLD can release DAG from PC. However, other hydrolysis products of membrane phospholipids can also act as signaling molecules. The best characterized of these hydrolysis products is arachidonic acid (AA), which is attached by an ester bond to the second carbon of the glycerol backbone of membrane phospholipids (Fig. 3-10). Phospholipase A2 initiates the cellular actions of AA by releasing this fatty acid from glycerol-based phospholipids. A series of enzymes subsequently convert AA into a family of biologically active metabolites that are collectively called eicosanoids (from the Greek eikosi for 20) because, like AA, they all have 20 carbon atoms. Three major pathways can convert AA into these eicosanoids (Fig. 3-11). In the first pathway, cyclooxygenase enzymes produce thromboxanes, prostaglandins, and prostacyclins. In the second pathway, 5-lipoxygenase enzymes produce leukotrienes and some hydroxyeicosatetraenoic acid (HETE) compounds. In the third pathway, the epoxygenase enzymes, which are members of the cytochrome P-450 class, produce other HETE compounds as well as cis-epoxyeicosatrienoic acid (EET) compounds. These three enzymes catalyze the stereospecific insertion of molecular O2 into various positions in AA. The cyclooxygenases, lipoxygenases, and epoxygenases are selectively distributed in different cell types, further increasing the complexity of eicosanoid biology. Eicosanoids have powerful biological activities, including effects on allergic and inflammatory processes, platelet aggregation, vascular smooth muscle, and gastric acid secretion. (See Note: Phospholipase A2)
Figure 3-10 Release of AA from membrane phospholipids by PLA2. AA is esterified to membrane phospholipids at the second carbon of the glycerol backbone. PLA2 cleaves the phospholipid at the indicated position and releases AA as well as a lysophospholipid.
Figure 3-11 AA signaling pathways. In the direct pathway, an agonist binds to a receptor that activates PLA2, which releases AA from a membrane phospholipid (see Fig. 3-10). In one of three indirect pathways, an agonist binds to a different receptor that activates PLC and thereby leads to the formation of DAG and IP3, as in Figure 3-8; DAG lipase then releases the AA from DAG. In a second indirect pathway, the IP3 releases Ca2+ from internal stores, which leads to the activation of PLA2 (see the direct pathway). In a third indirect pathway (not shown), mitogen-activated protein kinase stimulates PLA2. Regardless of its source, the AA may follow any of three pathways to form a wide array of eicosanoids. The cyclooxygenase pathway produces thromboxanes, prostacyclins, and prostaglandins. The 5-lipoxygenase pathway produces 5-HETE and the leukotrienes. The epoxygenase pathway leads to the production of other HETEs and EETs. ASA, acetylsalicylic acid; EET, cis-epoxyeicosatrienoic acid; ER, endoplasmic reticulum; HETE, hydroxyeicosatetraenoic acid; HPETE, hydroperoxyeicosatetraenoic acid; MAG, monoacylglycerol.
Phospholipase A2 is the primary enzyme responsible for releasing arachidonic acid
The first step in the phospholipase A2 (PLA2) signal transduction cascade is binding of an extracellular agonist to a membrane receptor (Fig. 3-11). These receptors include those for serotonin (5-HT2receptors), glutamate (mGLUR1 receptors), fibroblast growth factor-ß, IFN-α, and IFN-γ. Once the receptor is occupied by its agonist, it can activate a G protein that belongs to the Gi/Go family. The mechanism by which this activated G protein stimulates PLA2 is not well understood. It does not appear that a G protein α subunit is involved. The G protein ßγ dimer may stimulate PLA2 either directly or through mitogen-activated protein (MAP) kinase (see Chapter 4), which phosphorylates PLA2 at a serine residue. The result is rapid hydrolysis of phospholipids that contain AA.
In contrast to the direct pathway just mentioned, agonists acting on other receptors may promote AA release indirectly. First, a ligand may bind to a receptor coupled to PLC, which would lead to the release of DAG (Fig. 3-11). As noted earlier, DAG lipase can cleave DAG to yield AA and a monoacylglycerol. Agonists that act through this pathway include dopamine (D2 receptors), adenosine (A1 receptors), norepinephrine (α2-adrenergic receptors), and serotonin (5-HT1 receptors). Second, any agonist that raises [Ca2+]i can promote AA formation because Ca2+ can stimulate some cytosolic forms of PLA2. Third, any signal transduction pathway that activates MAP kinase can also enhance AA release because MAP kinase phosphorylates PLA2.
Cyclooxygenases, lipoxygenases, and epoxygenases mediate the formation of biologically active eicosanoids
Once it is released from the membrane, AA can diffuse out of the cell, be reincorporated into membrane phospholipids, or be metabolized (Fig. 3-11).
In the first pathway of AA metabolism (Fig. 3-11), cyclooxygenases catalyze the stepwise conversion of AA into the intermediates prostaglandin G2 (PGG2) and prostaglandin H2 (PGH2). PGH2 is the precursor of the other prostaglandins, the prostacyclins and the thromboxanes. As noted in the box titled Inhibition of Cyclooxygenase Isoforms by Aspirin, cyclooxygenase exists in two isoforms, COX-1 and COX-2. In many cells, COX-1 is expressed in a constitutive fashion, whereas COX-2 levels can be induced by specific stimuli. For example, in monocytes stimulated by inflammatory agents such as IL-1β, only levels of COX-2 increase. These observations have led to the concept that expression of COX-1 is important for homeostatic prostaglandin functions such as platelet aggregation and regulation of vascular tone, whereas upregulation of COX-2 is primarily important for mediating prostaglandin-dependent inflammatory responses. However, as selective inhibitors of COX-2 have become available, it has become clear that this is an oversimplification. (See Note: Cyclooxygenase)
In the second pathway of AA metabolism, 5-lipoxygenase initiates the conversion of AA into biologically active leukotrienes. For example, in myeloid cells, 5-lipoxygenase converts AA to 5-HPETE, which is short-lived and rapidly degraded by a peroxidase to the corresponding alcohol 5-HETE. Alternatively, a dehydrase can convert 5-HPETE to an unstable epoxide, LTA4, which can be either further metabolized by LTA4 hydrolase to LTB4 or coupled (“conjugated”) to the tripeptide glutathione (see Chapter 46). This conjugation—through the cysteine residue of glutathione—yields LTC4. Enzymes sequentially remove portions of the glutathione moiety to produce LTD4 and LTE4. LTC4, LTD4, and LTE4 are the “cysteinyl” leukotrienes; they participate in allergic and inflammatory responses and make up the mixture previously described as the slow-reacting substance of anaphylaxis. (See Note: Names of Arachidonic Acid Metabolites)
The third pathway of AA metabolism begins with the transformation of AA by epoxygenase (a cytochrome P-450 oxidase). Molecular O2 is a substrate in this reaction. The epoxygenase pathway converts AA into two major products, HETEs and EETs. Members of both groups display a diverse array of biological activities. Moreover, the cells of different tissues (e.g., liver, kidney, eye, and pituitary) use different biosynthetic pathways to generate different epoxygenase products. (See Note: Epoxygenase)
Prostaglandins, prostacyclins, and thromboxanes (cyclooxygenase products) are vasoactive, regulate platelet action, and modulate ion transport
The metabolism of PGH2 to generate selected prostanoid derivatives is cell specific. For example, platelets convert PGH2 to thromboxane A2 (TXA2), a short-lived compound that can aggregate platelets, bring about the platelet release reaction, and constrict small blood vessels. In contrast, endothelial cells convert PGH2 to prostacyclin I2 (also known as PGI2), which inhibits platelet aggregation and dilates blood vessels. Many cell types convert PGH2 to prostaglandins. Acting locally in a paracrine or autocrine fashion, prostaglandins are involved in such processes as platelet aggregation, airway constriction, renin release, and inflammation. Prostaglandin synthesis has also been implicated in the pathophysiological mechanisms of cardiovascular disease, cancer, and inflammatory diseases. NSAIDs such as aspirin, acetaminophen, ibuprofen, indomethacin, and naproxen directly target cyclooxygenase. NSAID inhibition of cyclooxygenase is a useful tool in the treatment of inflammation and fever and, at least in the case of aspirin, in the prevention of heart disease. (See Note: Actions of Prostanoids)
Eicosanoid Nomenclature
The nomenclature of the eicosanoids is not as arcane as it might first appear. The numerical subscript 2 (as in PGH2) or 4 (as in LTA4) refers to the number of double bonds in the eicosanoid backbone. For example, AA has four double bonds, as do the leukotrienes.
For the cyclooxygenase metabolites, the letter (A to I) immediately preceding the 2 refers to the structure of the 5-carbon ring that is formed about halfway along the 20-carbon chain of the eicosanoid. For the leukotrienes, the letters A and B that immediately precede the 4 refer to differences in the eicosanoid backbone. For the cysteinyl leukotrienes, the letter C refers to the full glutathione conjugate (see Fig. 46-8). Removal of glutamate from LTC4 yields LTD4, and removal of glycine from LTD4 yields LTE4, leaving behind only cysteine.
For 5-HPETE and 5-HETE, the fifth carbon atom (counting the carboxyl group as number 1) is derivatized with a hydroperoxy-or hydroxy-group, respectively.
Inhibition of Cyclooxygenase Isoforms by Aspirin
Cyclooxygenase is a bifunctional enzyme that first oxidizes AA to PGG2 through its cyclooxygenase activity and then peroxidizes this compound to PGH2. Cyclooxygenase exists in two forms, COX-1 and COX-2. X-ray crystallographic studies of COX-1 reveal that the sites for the two enzymatic activities (i.e., cyclooxygenase and peroxidase) are adjacent but spatially distinct. The cyclooxygenase site is a long hydrophobic channel. Aspirin (acetylsalicylic acid) irreversibly inhibits COX-1 by acetylating a serine residue at the top of this channel. Several of the other nonsteroidal anti-inflammatory drugs (NSAIDs) interact, through their carboxyl groups, with other amino acids in the same region.
COX-1 activation plays an important role in intravascular thrombosis as it leads to thromboxane A2 synthesis by platelets. Inhibition of this process by low-dose aspirin is a mainstay for prevention of coronary thrombosis in patients with atherosclerotic coronary artery disease. However, COX-1 activation is also important for producing cytoprotective prostacyclins in the gastric mucosa. It is the loss of these compounds that can lead to the unwanted side effect of gastrointestinal bleeding after chronic aspirin ingestion.
Inflammatory stimuli induce COX-2 in a number of cell types, and it is inhibition of COX-2 that provides the anti-inflammatory actions of high-dose aspirin (a weak COX-2 inhibitor) and other nonselective cyclooxygenase inhibitors such as ibuprofen. Because the two enzymes are only 60% homologous, pharmaceutical companies have now generated compounds that specifically inhibit COX-2, such as rofecoxib and celecoxib. These work well as anti-inflammatory agents and have a reduced likelihood of causing gastrointestinal bleeding because they do not inhibit COX-1–dependent prostacyclin production. At least one of the selective COX-2 inhibitors has been reported to increase the risk of thrombotic cardiovascular events when it is taken for long periods.
The diverse cellular responses to prostanoids are mediated by a family of G protein–coupled prostanoid receptors. This family currently has nine proposed members, including receptors for thromboxane/prostaglandin H2 (TP), PGI2 (IP), PGE2 (EP1-4), PGD2 (DP and CRTH2), and PGF2α (FP). These prostanoid receptors signal through Gq, Gi, or Gs, depending on cell type. These in turn regulate intracellular adenylyl cyclase and phospholipases.
The leukotrienes (5-lipoxygenase products) play a major role in inflammatory responses
The biological effects of many lipoxygenase metabolites of AA have led to the suggestion that they have a role in allergic and inflammatory diseases (Table 3-3). LTB4 is produced by inflammatory cells such as neutrophils and macrophages. The cysteinyl leukotrienes including LTC4 and LTE4 are synthesized by mast cells, basophils, and eosinophils, cells that are commonly associated with allergic inflammatory responses such as asthma and urticaria. (See Note: Actions of Leukotrienes)
Table 3-3 Involvement of Leukotrienes in Human Disease
|
Disease |
Evidence |
|
Asthma |
Bronchoconstriction from inhaled LTE4; identification of LTC4, LTD4, and LTE4 in the serum or urine or both of patients with asthma |
|
Psoriasis |
LTB4 and LTE4 found in fluids from psoriatic lesions |
|
Adult respiratory distress syndrome |
Elevated levels of LTB4 detected in the plasma of patients with ARDS |
|
Allergic rhinitis |
Elevated levels of LTB4 found in nasal fluids |
|
Gout |
LTB4 detected in joint fluid |
|
Rheumatoid arthritis |
Elevated LTB4 found in joint fluids and serum |
|
Inflammatory bowel disease (ulcerative colitis and Crohn disease) |
Identification of LTB4 in gastrointestinal fluids and LTE4 in urine |
The cysteinyl leukotriene receptors cysLT1 and cysLT2 are GPCRs found on airway smooth muscle cells as well as on eosinophils, mast cells, and lymphocytes. CysLT1, which couples to both pertussis toxin–sensitive and pertussis toxin–insensitive G proteins, mediates phospholipase-dependent increases in [Ca2+]i. In the airways, these events produce a potent bronchoconstriction, whereas activation of the receptor in mast cells and eosinophils causes release of the proinflammatory cytokines histamine and TNF-α.
In addition to their role in the inflammatory response, the lipoxygenase metabolites can also influence the activity of many ion channels, either directly or by regulating protein kinases. For example, in synaptic nerve endings, lipoxygenase metabolites decrease the excitability of cells by activating K+ channels. Lipoxygenase products may also regulate secretion. In pancreatic islet cells, free AA generated in response to glucose appears to be part of a negative feedback loop that prevents excess insulin secretion by inhibiting CaM kinase II.
The HETEs and EETs (epoxygenase products) tend to enhance Ca2+ release from intracellular stores and to enhance cell proliferation
The epoxygenase pathway leads to the production of HETEs other than 5-HETE as well as EETs. HETEs and EETs have been implicated in a wide variety of processes, some of which are summarized in Table 3-4. For example, in stimulated mononuclear leukocytes, HETEs enhance Ca2+ release from intracellular stores and promote cell proliferation. In smooth muscle cells, HETEs increase proliferation and migration; these AA metabolites may be one of the primary factors involved in the formation of atherosclerotic plaque. In blood vessels, HETEs can be potent vasoconstrictors. EETs enhance the release of Ca2+ from intracellular stores, increase Na-H exchange, and stimulate cell proliferation. In blood vessels, EETs primarily induce vasodilation and angiogenesis, although they have vasoconstrictive properties in the smaller pulmonary blood vessels.
Table 3-4 Actions of Epoxygenase Products
Role of Leukotrienes in Disease
Since the original description of the slow-reacting substance of anaphylaxis, which is generated during antigenic challenge of a sensitized lung, leukotrienes have been presumed to play a part in allergic disease of the airways (Table 3-3). The involvement of cells (mast cells, basophils, and eosinophils) that produce cysteinyl leukotrienes (LTC4 through LTF4) in these pathobiological processes supports this concept. In addition, the levels of LTC4, LTD4, and LTE4 are increased in lavage fluid from the nares of patients with allergic rhinitis after the application of specific antigens to the nasal airways. Introducing LTC4 or LTD4 into the airways as an aerosol (nebulizer concentration of only 10 μM) causes maximal expiratory airflow (a rough measure of airway resistance; see Chapter 27) to decline by ~30%. This bronchoconstrictor effect is 1000-fold more potent than that of histamine, the “reference” agonist. Leukotrienes affect both large and small airways; histamine affects relatively smaller airways. Activation of the cysLT1 receptor in mast cells and eosinophils results in the chemotaxis of these cells to sites of inflammation. Because antagonists of the cysLT1 receptor (e.g., montelukast sodium) can partially block these bronchoconstrictive and proinflammatory effects, these agents are useful in the treatment of allergen-induced asthma and rhinitis.
In addition to their involvement in allergic disease, several of the leukotrienes are associated with other inflammatory disorders. Synovial fluid from patients with rheumatoid arthritis contains 5-lipoxygenase products. Another example is the skin disease psoriasis. In patients with active psoriasis, LTB4, LTC4, and LTD4 have been recovered from skin chambers overlying abraded lesions. Leukotrienes also appear to be involved in inflammatory bowel disease. LTB4 and other leukotrienes are generated and released in vitro from intestinal mucosa obtained from patients with ulcerative colitis or Crohn disease.
EETs generally tend to enhance the release of Ca2+ from intracellular stores, Na-H exchange, and cell proliferation. In blood vessels, EETs cause vasodilation and angiogenesis.
Degradation of the eicosanoids terminates their activity
Inactivation of the products of eicosanoids is an important mechanism for terminating their biological action. In the case of cyclooxygenase products, the enzyme 15-hydroxyprostaglandin dehydrogenase catalyzes the initial reactions that convert biologically active prostaglandins into their inactive 15-keto metabolites. This enzyme also appears to be active in the catabolism of thromboxanes.
As far as the 5-lipoxygenase products are concerned, the specificity and cellular distribution of the enzymes that metabolize leukotrienes parallel the diversity of the enzymes involved in their synthesis. For example, 20-hydrolase-LTB4, a member of the P-450 family, catalyzes the ω oxidation of LTB4, thereby terminating its biological activity. LTC4 is metabolized through two pathways. One oxidizes the LTC4. The other pathway first removes the glutamic acid residue of the conjugated glutathione, which yields LTD4, and then removes the glycine residue, which yields LTE4, which is readily excreted into the urine. (See Note: The Electrochemical Potential Energy Difference for an Ion across a Cell Membrane)
In the case of epoxygenase (cytochrome P-450) products, it has been difficult to characterize their metabolic breakdown because the reactions are so rapid and complex. Both enzymatic and nonenzymatic hydration reactions convert these molecules to the corresponding vicinyl diols. Some members of this group can form conjugates with reduced glutathione (GSH).
Platelet-activating factor is a lipid mediator unrelated to arachidonic acid
Although it is not a member of the AA family, platelet-activating factor (PAF) is an important lipid signaling molecule. PAF is an ether lipid that the cell synthesizes either de novo or by remodeling of a membrane-bound precursor. PAF occurs in a wide variety of organisms and mediates many biological activities. In mammals, PAF is a potent inducer of platelet aggregation and stimulates the chemotaxis and degranulation of neutrophils, thereby facilitating the release of LTB4 and 5-HETE. PAF is involved in several aspects of allergic reactions; for example, it stimulates histamine release and enhances the secretion of IgE, IgA, and TNF. Endothelial cells are also an important target of PAF; PAF causes a negative shift of Vm in these cells by activating Ca2+-dependent K+ channels. PAF also enhances vascular permeability and the adhesion of neutrophils and platelets to endothelial cells.
PAF exerts its effects by binding to a specific receptor on the plasma membrane. A major consequence of PAF binding to its GPCR is formation of IP3 and stimulation of a group of MAP kinases. PAF acetylhydrolase terminates the action of this signaling lipid.
RECEPTORS THAT ARE CATALYTIC
A number of hormones and growth factors bind to cell surface proteins that have—or are associated with—enzymatic activity on the cytoplasmic side of the membrane. Here we discuss five classes of such catalytic receptors (Fig. 3-12):
Figure 3-12 Catalytic receptors. A, Receptor guanylyl cyclases have an extracellular ligand-binding domain. B, Receptor serine/threonine kinases have two subunits. The ligand binds only to the type II subunit. C, Receptor tyrosine kinases (RTKs) similar to the NGF receptor dimerize on binding a ligand. D, Tyrosine kinase–associated receptors have no intrinsic enzyme activity but associate noncovalently with soluble, nonreceptor tyrosine kinases. E, Receptor tyrosine phosphatases have intrinsic tyrosine phosphatase activity. ANP, atrial natriuretic peptide; JAK, Janus kinase (originally “just another kinase”); NGF, nerve growth factor; TGF-β, transforming growth factor β.
Receptor guanylyl cyclases catalyze the generation of cGMP from GTP.
Receptor serine/threonine kinases phosphorylate serine or threonine residues on cellular proteins.
Receptor tyrosine kinases (RTKs) phosphorylate tyrosine residues on themselves and other proteins.
Tyrosine kinase–associated receptors interact with cytosolic (i.e., non–membrane bound) tyrosine kinases.
Receptor tyrosine phosphatases cleave phosphate groups from tyrosine groups of cellular proteins.
The receptor guanylyl cyclase transduces the activity of atrial natriuretic peptide, whereas a soluble guanylyl cyclase transduces the activity of nitric oxide
Receptor (Membrane-Bound) Guanylyl Cyclase Some of the best characterized examples of a transmembrane protein with guanylyl cyclase activity (Fig. 3-12A) are the receptors for the natriuretic peptides. These are a family of related small proteins (~28 amino acids) including atrial natriuretic peptide (ANP), B-type or brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP). For example, in response to atrial stretch, cardiac myocytes release ANP and BNP. ANP and BNP have two major effects. First, they act on vascular smooth muscle to dilate blood vessels (see Chapter 23). Second, they enhance Na+ excretion into urine, which is termed natriuresis (see Chapter 40). Both activities contribute to lowering of blood pressure and effective circulating blood volume (see Chapter 5). (See Note: Atrial Natriuretic Peptide)
Natriuretic peptide receptors NPR-A and NPR-B are membrane proteins with a single membrane-spanning segment. The extracellular domain binds the ligand. The intracellular domain has two consensus catalytic domains for guanylyl cyclase activity. Binding of a natriuretic peptide induces a conformational change in the receptor that causes receptor dimerization and activation. Thus, binding of ANP to its receptor causes the conversion of GTP to cGMP and raises intracellular levels of cGMP. In turn, cGMP activates a cGMP-dependent kinase (PKG or cGK) that phosphorylates proteins at certain serine and threonine residues. In the renal medullary collecting duct, the cGMP generated in response to ANP may act not only through PKG but also by directly modulating ion channels (see Chapter 35).
Soluble Guanylyl Cyclase In contrast to the receptor for ANP, which is an intrinsic membrane protein with guanylyl cyclase activity, the receptor for nitric oxide (NO) is a soluble (i.e., cytosolic) guanylyl cyclase. This soluble guanylyl cyclase (sGC) is totally unrelated to the receptor guanylyl cyclase and contains a heme moiety that binds NO.
NO plays an important role in the control of blood flow and blood pressure. Vascular endothelial cells use the enzyme NO synthase (NOS) to cleave arginine into citrulline plus NO in response to stimuli such as ACh, bradykinin, substance P, thrombin, adenine nucleotides, and Ca2+. These agents trigger the entry of Ca2+, which binds to cytosolic CaM and then stimulates NOS. Activation of NOS also requires the cofactors tetrahydrobiopterin and NADPH. The newly synthesized NO rapidly diffuses out of the endothelial cell and crosses the membrane of a neighboring smooth muscle cell. In smooth muscle, NO stimulates its “receptor,” soluble guanylyl cyclase, which then converts GTP to cGMP. As a result, [cGMP]i may increase 50-fold and relax the smooth muscle.
The importance of NO in the control of blood flow had long been exploited unwittingly to treat angina pectoris. Angina is the classic chest pain that accompanies inadequate blood flow to the heart muscle, usually as a result of coronary artery atherosclerosis. Nitroglycerin relieves this pain by spontaneously breaking down and releasing NO, which relaxes the smooth muscles of peripheral arterioles, thereby reducing the work of the heart and relieving the associated pain.
In addition to its role as a chemical signal in blood vessels, NO appears to play an important role in the destruction of invading organisms by macrophages and neutrophils. NO also serves as a neurotransmitter and may play a role in learning and memory (see Chapter 13). Some of these actions may involve different forms of NOS.
The importance of the NO signaling pathway was recognized by the awarding of the 1998 Nobel Prize for Physiology or Medicine to R. F. Furchgott, L. J. Ignarro, and F. Murad for their discoveries concerning NO as a signaling molecule in the cardiovascular system.
Some catalytic receptors are serine/threonine kinases
Earlier in this chapter we discussed how activation of various G protein–linked receptors can initiate a cascade that eventually activates kinases (e.g., PKA, PKC) that phosphorylate proteins at serine and threonine residues. In addition, some receptors are themselves serine/threonine kinases—such as the one for transforming growth factor β (TGF-β)—and are thus catalytic receptors.
The TGF-β superfamily includes a large group of cytokines, including five TGF-βs, antimüllerian hormone, the inhibins, the activins, bone morphogenic proteins, and other glycoproteins, all of which control cell growth and differentiation. Members of this family participate in embryogenesis, suppress epithelial cell growth, promote wound repair, and influence immune and endocrine functions. Unchecked TGF-β signaling is important in progressive fibrotic disorders (e.g., liver cirrhosis, idiopathic pulmonary fibrosis) that result in replacement of normal organ tissue by deposits of collagen and other matrix components.
The receptors for TGF-β and related factors are glycoproteins with a single membrane-spanning segment and intrinsic serine/threonine kinase activity. Receptor types I and II (Fig. 3-12B) are required for ligand binding and catalytic activity. The type II receptor first binds the ligand, followed by the formation of a stable ternary complex of ligand, type II receptor, and type I receptor. Recruitment of the type I receptor into the complex results in phosphorylation of the type I receptor at serine and threonine residues, which in turn activates the kinase activity of the type I receptor and propagates the signal to downstream effectors.
Receptor tyrosine kinases produce phosphotyrosine motifs recognized by SH domains of downstream effectors
In addition to the class of receptors with intrinsic serine/threonine kinase activity, other plasma membrane receptors have intrinsic tyrosine kinase activity. All receptor tyrosine kinases discovered to date phosphorylate themselves in addition to other cellular proteins. Epidermal growth factor (EGF), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), insulin and insulin-related growth factor type 1 (IGF-1), fibroblast growth factor (FGF), and nerve growth factor (NGF) can all bind to receptors that possess intrinsic tyrosine kinase activity.
Creation of Phosphotyrosine (pY) Motifs Most RTKs are single-pass transmembrane proteins that contain a single intracellular kinase domain (Fig. 3-12C). Binding of a ligand, such as NGF, induces a conformational change in the receptor that facilitates the formation of receptor dimers. Dimerization allows the two cytoplasmic catalytic domains to phosphorylate each other (“autophosphorylation”) and thereby activate the receptor complex. The activated receptors also catalyze the addition of phosphate to tyrosine (Y) residues on specific cytoplasmic proteins. The resulting phosphotyrosine motifs of the receptor and other protein substrates serve as high-affinity binding sites for a number of intracellular signaling molecules. These interactions lead to the formation of a signaling complex and the activation of downstream effectors. Activation of insulin and IGF-1 receptors occurs by a somewhat different mechanism: the complex analogous to the dimeric NGF receptor exists even before ligand binding, as we will discuss in Chapter 51. (See Note: Insulin and IGF-1 Receptors)
Recognition of pY Motifs by SH2 and SH3 Domains The phosphotyrosine motifs created by tyrosine kinases serve as high-affinity binding sites for the recruitment of many cytoplasmic or membrane-associated proteins that contain a region such as an SH2 (Src homology 2), SH3 (Src homology 3), or PTB (phosphotyrosine-binding) domains. SH2 domains are ~100 amino acids in length. They are composed of relatively well conserved residues that form the binding pocket for pY motifs as well as more variable residues that are implicated in binding specificity. These residues that confer binding specificity primarily recognize the three amino acids located on the C-terminal side of the phosphotyrosine. For example, the activated PDGF receptor has five such pY motifs (Table 3-5), each of which interacts with a specific SH2-containing protein.
Table 3-5 Tyrosine Phosphopeptides of the PDGF Receptor That Are Recognized by SH2 Domains on Various Proteins
|
Tyrosine (Y) That Is Phosphorylated in the PDGF Receptor |
Phosphotyrosine (PY) Motif Recognized by the SH2-Containing Protein |
SH2-Containing Protein |
|
Y579 |
pYIYVD |
Src family kinases |
|
Y708 |
pYMDMS |
p85 |
|
Y719 |
pYVPML |
p85 |
|
Y739 |
pYNAPY |
GTPase-activating protein |
|
Y1021 |
pYIIPY |
PLCγ |
SH3 domains are ~50 amino acids in length and bind to proline-rich regions in other proteins. Although these interactions are typically constitutive, phosphorylation at distant sites can change protein conformation and thereby regulate the interaction. Like SH2 interactions, SH3 interactions appear to be responsible for targeting of signaling molecules to specific subcellular locations. SH2-or SH3-containing proteins include growth factor receptor-bound protein 2 (GRB2), PLCγ, and the receptor-associated tyrosine kinases of the Src family.
The MAPK Pathway A common pathway by which activated RTKs transduce their signal to cytosol and even to the nucleus is a cascade of events that increase the activity of the small GTP-binding protein Ras. This Ras-dependent signaling pathway involves the following steps (Fig. 3-13):
Step 1: A ligand binds to the extracellular domain of a specific RTK, thus causing receptor dimerization.
Step 2: The now-activated RTK phosphorylates itself on tyrosine residues of the cytoplasmic domain (autophosphorylation).
Step 3: GRB2 (growth factor receptor-bound protein 2), an SH2-containing protein, recognizes pY residues on the activated receptor.
Step 4: Binding of GRB2 recruits SOS (son of sevenless), a guanine nucleotide exchange protein.
Step 5: SOS activates Ras by causing GTP to replace GDP on Ras.
Step 6: The activated GTP-Ras complex activates other proteins by physically recruiting them to the plasma membrane. In particular, the active GTP-Ras complex interacts with the N-terminal portion of the serine/threonine kinase Raf-1 (also known as MAP kinase kinase kinase), which is the first in a series of sequentially activated protein kinases that ultimately transmits the activation signal.
Step 7: Raf-1 phosphorylates and activates a protein kinase called MEK (also known as MAP kinase kinase or MAPKK). MEK is a multifunctional protein kinase that phosphorylates substrates on bothtyrosine and serine/threonine residues. The JAK system (see next section) also activates MEK.
Step 8: MEK phosphorylates MAP kinase (MAPK), also called extracellular signal-regulated kinase (ERK1, ERK2). Activation of MAPK requires dual phosphorylation on neighboring serine and tyrosine residues.
Step 9: MAPK is an important effector molecule in Ras-dependent signal transduction because it phosphorylates many cellular proteins.
Step 10: Activated MAPK also translocates to the nucleus, where it phosphorylates a number of nuclear proteins that are transcription factors. Phosphorylation of a transcription factor by MAPK can enhance or inhibit binding to DNA and thereby enhance or suppress transcription. (See Note: Transcription Factors Phosphorylated by MAP Kinase)
Figure 3-13 Regulation of transcription by the Ras pathway. A ligand, such as a growth factor, binds to a specific RTK, leading to an increase in gene transcription in a 10-step process.
Two other signal transduction pathways (cAMP and Ca2+) can modulate the activity of some of the protein intermediates in this MAP kinase cascade, suggesting multiple points of integration for the various signaling systems.
Tyrosine kinase–associated receptors activate loosely associated tyrosine kinases such as Src and JAK
Some of the receptors for cytokines and growth factors that regulate cell proliferation and differentiation do not themselves have intrinsic tyrosine kinase activity but can associate with nonreceptor tyrosine kinases (Fig. 3-12D). Receptors in this class include those for several cytokines, including IL-2, IL-3, IL-4, IL-5, IL-6, leukemia inhibitory factor (LIF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and erythropoietin (EPO). The family also includes receptors for growth hormone (GH), prolactin (PRL), leptin, ciliary neurotrophin factor (CNTF), oncostatin M, and IFN-α, IFN-β, and IFN-γ.
The tyrosine kinase–associated receptors typically comprise multiple subunits that form homodimers (αα), heterodimers (αβ), or heterotrimers (αβγ). For example, the IL-3 and the GM-CSF receptors are heterodimers (αβ) that share common β subunits with transducing activity. However, none of the cytoplasmic portions of the receptor subunits contains kinase domains or other sequences with recognized catalytic function. Instead, tyrosine kinases of the Src family and Janus family (JAK or Janus kinases) associate noncovalently with the cytoplasmic domains of these receptors. Thus, these are receptor-associated tyrosine kinases. Ligand binding to these receptors results in receptor dimerization and tyrosine kinase activity. The activated kinase then phosphorylates tyrosines on both itself and the receptor. Thus, tyrosine kinase–associated receptors, together with their tyrosine kinases, function much like the RTKs discussed in the previous section. A key difference is that for the tyrosine kinase–associatedreceptors, the receptors and kinases are encoded by separate genes and the proteins are only loosely associated with one another. (See Note: Multimeric Composition of Tyrosine Kinase-Associated Receptors)
The Src family of receptor-associated tyrosine kinases includes at least nine members. Alternative initiation codons and tissue-specific splicing (see Chapter 4) result in at least 14 related gene products.
The conserved regions of Src-related proteins can be divided into five domains: (1) an N-terminal myristylation site, through which the kinase is tethered to the membrane; (2) an SH3 domain, which binds to proline-rich regions of the kinase itself or to other cytosolic proteins; (3) an SH2 domain, which binds phosphorylated tyrosines; (4) the catalytic domain, which has tyrosine kinase activity; and (5) a noncatalytic C terminus. Members of this family are kept in the inactive state by tyrosine phosphorylation at a conserved residue in the C terminus, causing this pY to bind to the amino-terminal SH2 domain of the same molecule, obscuring the intervening kinase domain. Dephosphorylation of the pY residue, after the activation of such phosphatases as RPTPα or SHP-2, releases this inhibition, and the kinase domain can then phosphorylate its intracellular substrates.
Many of the Src family members were first identified in transformed cells or tumors because of mutations that caused them to be constitutively active. When these mutations result in malignant transformation of the cell, the gene in question is designated an oncogene; the normal, unaltered physiological counterpart of an oncogene is called a proto-oncogene.
The Janus family of receptor-associated tyrosine kinases in mammals includes JAK1, JAK2, and Tyk2. JAK stands for “just another kinase.” Major downstream targets of the JAKs include one or more members of the STAT (signal transducers and activators of transcription) family. When phosphorylated, STATs interact with other STAT family members to form a complex that translocates to the nucleus (see Chapter 4). There, the complex facilitates the transcription of specific genes that are specialized for a rapid response, such as those that are characterized by the acute-phase response of inflammation (see Chapter 59). For example, after IL-6 binds to hepatocytes, the STAT pathway is responsible for producing acute-phase proteins. During inflammation, these acute-phase proteins function to limit tissue damage by inhibiting the proteases that attack healthy cells as well as diseased ones. The pattern of STAT activation provides a mechanism for cytokine individuality. For example, EPO activates STAT5a and STAT5b as part of the early events in erythropoiesis, whereas IL-4 or IL-12 activates STAT4 and STAT6.
Attenuation of the cytokine JAK-STAT signaling cascade involves the production of inhibitors that suppress tyrosine phosphorylation and activation of the STATs. For example, IL-6 and LIF both induce expression of the inhibitor SST-1, which contains an SH2 domain and prevents JAK2 or Tyk2 from activating STAT3 in M1 myeloid leukemia cells.
Receptor tyrosine phosphatases are required for lymphocyte activation
Tyrosine residues that are phosphorylated by the tyrosine kinases described in the preceding two sections are dephosphorylated by phosphotyrosine phosphatases (PTPs), which can be either cytosolic or membrane bound (i.e., the receptor tyrosine phosphatases). We discussed the cytosolic PTPs earlier. Both classes of tyrosine phosphatases have structures very different from the ones that dephosphorylate serine and threonine residues. Because the tyrosine phosphatases are highly active, pY groups tend to have brief life spans and are relatively few in number in unstimulated cells.
The CD45 protein, found at the cell surface of T and B lymphocytes, is an example of a receptor tyrosine phosphatase. CD45 makes a single pass through the membrane. Its glycosylated extracellular domain functions as a receptor for antibodies, whereas its cytoplasmic domain has tyrosine phosphatase activity (Fig. 3-12E). During their maturation, lymphocytes express several variants of CD45 characterized by different patterns of alternative splicing and glycosylation. CD45 plays a critical role in signal transduction in lymphocytes. For instance, CD45 dephosphorylates and thereby activates Lck and Fyn (two receptor-associated tyrosine kinases of the Src family) and triggers the phosphorylation of other proteins downstream in the signal transduction cascade. This interaction between receptor tyrosine phosphatases and tyrosine kinase–associated receptors is another example of crosstalk between signaling pathways.
Oncogenes
The ability of certain viral proteins (oncogenes) to transform a cell from a normal to a malignant phenotype was initially thought to occur because these viral proteins acted as transcriptional activators or repressors. However, during the last 20 years, only a few of these viral proteins have been found to work in this manner. The majority of oncogenes harbor mutations that transform them into constitutively active forms of normal cellular signaling proteins called proto-oncogenes. Most of these aberrant proteins (i.e., the oncogenes) encode proteins important in a key signal transduction pathway. For example, expression of the viral protein v-erb B is involved in fibrosarcomas, and both v-erb A and v-erb B are associated with leukemias. v-erb B resembles a constitutively activated receptor tyrosine kinase (epidermal growth factor receptor), and the retroviral v-erb A is derived from a cellular gene encoding a thyroid hormone receptor. Other receptors and signaling molecules implicated in cell transformation include Src, Ras, and platelet-derived growth factor receptor. A mutation in protein tyrosine phosphatase 1C results in abnormal hematopoiesis and an increased incidence of lymphoreticular tumors.
NUCLEAR RECEPTORS
Steroid and thyroid hormones enter the cell and bind to members of the nuclear receptor superfamily in the cytoplasm or nucleus
A number of important signaling molecules produce their effects not by binding to receptors on the cell membrane but by binding to nuclear receptors (also called intracellular receptors) that can act as transcription regulators, a concept that we will discuss in more depth in Chapter 4. This family includes receptors for steroid hormones, prostaglandins, vitamin D, thyroid hormones, and retinoic acid (Table 3-6). In addition, this family includes related receptors, known as orphan receptors, whose ligands have yet to be identified. Steroid hormones, vitamin D, and retinoic acid appear to enter the cell by diffusing through the lipid phase of the cell membrane. Thyroid hormones, which are charged amino acid derivatives, may cross the cell membrane either by diffusion or by carrier-mediated transport. Once inside the cell, these substances bind to intracellular receptors. The ligand-bound receptors are activated transcription factors that regulate the expression of target genes by binding to specific DNA sequences. In addition, steroid hormones can also have nongenomic effects (see Chapter 47). (See Note: Nongenomic Effects of Steroid Hormones)
Table 3-6 Nuclear Steroid and Thyroid Receptors
The family of nuclear receptors contains at least 32 genes and has been classically divided into two subfamilies based on structural homology. One subfamily consists of receptors for steroid hormones, including the glucocorticoids and mineralocorticoids (see Chapter 50), androgens (see Chapter 50), and estrogens and progesterone (see Chapter 55). These receptors function primarily as homodimers (Table 3-2). The other group includes receptors for retinoic acid (see Chapter 4), thyroid hormone (see Chapter 49), and vitamin D (see Chapter 52). These receptors appear to act as heterodimers (Table 3-2). As we will see in Chapters 4 and 47, other nuclear receptors recognize a wide range of xenobiotics and metabolites and respond by modulating the expression of genes that encode transporters and enzymes involved in drug metabolism (see Chapter 46).
The intracellular localization of the different unoccupied receptors varies. The glucocorticoid (GR) and mineralocorticoid (MR) receptors are mainly cytoplasmic, the estrogen (ER) and progesterone (PR) receptors are primarily nuclear, and the thyroid hormone (TR) and retinoic acid (RAR/RXR) receptors are bound to DNA in the nucleus. Cytoplasmic receptors are complexed to chaperone (or “heat shock”) proteins. Hormone binding induces a conformational change in these receptors that causes dissociation from the cytoplasmic chaperone and unmasks a nuclear transport signal that allows the hormone-receptor complex to translocate into the nucleus.
All nuclear receptors contain six functionally distinct domains, designated A to F from the N terminus to the C terminus (Fig. 3-14), that are differentially conserved among the various proteins. The N-terminal A/B region differs widely among receptors and contains the first of two transactivation domains. Transactivation is the process by which a ligand-induced conformational change of the receptor results in a change in conformation of the DNA, thus initiating transcription. The C region, the most highly conserved among receptor types, contains the DNA-binding domain and is also involved in dimerization(Table 3-6). It is composed of two “zinc finger” structures. The D, or hinge, region contains the “nuclear localization signal” and may also contain transactivation sequences. The E domain is responsible for hormone binding. Like the C region, it is involved in dimerization through its “basic zipper” region (see Chapter 4). Finally, like the A/B region, the E region contains a transactivation domain. The small C-terminal F domain is of unknown function.
Figure 3-14 Modular construction of intracellular (or nuclear) receptors. Members of this family exist in the cytoplasm or nucleus and include receptors for several ligands, including retinoic acid, vitamin D, thyroid hormones, and steroid hormones. These receptors have modular construction, with up to six elements. The percentages listed inside the A/B, C, and E domains refer to the degrees of amino acid identity, referenced to the glucocorticoid receptor. Thus, the DNA-binding or C domain of the retinoic acid receptor is 45% identical to the corresponding domain on the glucocorticoid receptor.
Activated nuclear receptors bind to sequence elements in the regulatory region of responsive genes and either activate or repress DNA transcription
One of the remarkable features of nuclear receptors is that they bind to specific DNA sequences—called hormone response elements—in the regulatory region of responsive genes. The various nuclear receptors display specific cell and tissue distributions. Thus, the battery of genes affected by a particular ligand depends on the complement of receptors in the cell, the ability of these receptors to form homodimers or heterodimers, and the affinity of these receptor-ligand complexes for a particular response element on the DNA.
In addition to their ability to affect transcription by directly binding to specific regulatory elements, several nuclear receptors modulate gene expression by acting as transcriptional repressors (see Chapter 4). For example, the glucocorticoids, acting through their receptor, can attenuate components of the inflammatory response by interacting with or “quenching” the transcription factor activator protein 1 (AP-1) and nuclear factor κB (NF-κB).
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