Goodman and Gilman Manual of Pharmacology and Therapeutics

Section I
General Principles

chapter 3
Pharmacodynamics: Molecular Mechanisms of Drug Action

Pharmacodynamic Concepts

Pharmacodynamics is the study of the biochemical and physiological effects of drugs and their mechanisms of action. The effects of most drugs result from their interaction with macromolecular components of the organism. The term drug receptor or drug target denotes the cellular macromolecule or macromolecular complex with which the drug interacts to elicit a cellular response. Drugs commonly alter the rate or magnitude of an intrinsic cellular response rather than create new responses. Drug receptors are often located on the surface of cells, but may also be located in specific intracellular compartments such as the nucleus.

Many drugs also interact with acceptors (e.g., serum albumin) within the body. Acceptors are entities that do not directly cause any change in biochemical or physiological response. However, interactions of drugs with acceptors can alter the pharmacokinetics of a drug’s actions.

PHYSIOLOGICAL RECEPTORS

Many drug receptors are proteins that normally serve as receptors for endogenous regulatory ligands. These drug targets are termed physiological receptors. Drugs that bind to physiological receptors and mimic the regulatory effects of the endogenous signaling compounds are termed agonists. If the drug binds to the same recognition site as the endogenous agonist, the drug is said to be a primary agonist. Allosteric (or allotopic) agonists bind to a different region on the receptor referred to as an allosteric or allotopic site. Drugs that block or reduce the action of an agonist are termed antagonists. Antagonism generally results from competition with an agonist for the same or overlapping site on the receptor (a syntopicinteraction), but can also occur by interacting with other sites on the receptor (allosteric antagonism), by combining with the agonist (chemical antagonism), or by functional antagonism by indirectly inhibiting the cellular or physiological effects of the agonist. Agents that are only partly as effective as agonists are termed partial agonists. Many receptors exhibit some constitutive activity in the absence of a regulatory ligand; drugs that stabilize such receptors in an inactive conformation are termed inverse agonists (Figure 3–1). In the presence of a full agonist, partial and inverse agonists will behave as competitive antagonists.

images

Figure 3–1 Regulation of the activity of a receptor with conformation-selective drugs. The ordinate is the activity of the receptor produced by Ra, the active receptor conformation (e.g., stimulation of adenylyl cyclase by a β adrenergic receptor). If a drug L selectively binds to Ra, it will produce a maximal response. If L has equal affinity for Ri and Ra, it will not perturb the equilibrium between them and will have no effect on net activity; L would appear as an inactive compound. If the drug selectively binds to Ri, then the net amount of Ra will be diminished. If L can bind to receptor in an active conformation Ra but also bind to inactive receptor Ri with lower affinity, the drug will produce a partial response; L will be a partial agonist. If there is sufficient Ra to produce an elevated basal response in the absence of ligand (agonist-independent constitutive activity), then activity will be inhibited; L will then be an inverse agonist. Inverse agonists selectively bind to the inactive form of the receptor and shift the conformational equilibrium toward the inactive state. In systems that are not constitutively active, inverse agonists will behave like competitive antagonists, which helps explain why the properties of inverse agonists and the number of such agents previously described as competitive antagonists were only recently appreciated. Receptors that have constitutive activity and are sensitive to inverse agonists include benzodiazepine, histamine, opioid, cannabinoid, dopamine, bradykinin, and adenosine receptors.

SPECIFICITY OF DRUG RESPONSES

The strength of the reversible interaction between a drug and its receptor, as measured by the dissociation constant, is defined as the affinity of one for the other. Both the affinity of a drug for its receptor and its intrinsic activity are determined by its chemical structure. The chemical structure of a drug also contributes to the drug’s specificity. A drug that interacts with a single type of receptor that is expressed on only a limited number of differentiated cells will exhibit high specificity. Conversely, drugs acting on a receptor expressed ubiquitously throughout the body will exhibit widespread effects.

Many clinically important drugs exhibit a broad (low) specificity because they interact with multiple receptors in different tissues. Such broad specificity might enhance the clinical utility of a drug, but also contribute to a spectrum of adverse side effects because of off-target interactions. One example of a drug that interacts with multiple receptors is amiodarone, an agent used to treat cardiac arrhythmias. Amiodarone also has a number of serious toxicities, some of which are caused by the drug’s structural similarity to thyroid hormone and its ability to interact with nuclear thyroid receptors. Amiodarone’s salutary effects and toxicities may also be mediated through interactions with receptors that are poorly characterized or unknown. Some drugs are administered as racemic mixtures of stereoisomers. The stereoisomers can exhibit different pharmacodynamic as well as pharmacokinetic properties. For example, the anti-arrhythmic drug sotalol is prescribed as a racemic mixture; the D- and L-enantiomers are equipotent as K+ channel blockers, but the L-enantiomer is a much more potent - adrenergic antagonist (see Chapter 29). A drug may have multiple mechanisms of action that depend on receptor specificity, the tissue-specific expression of the receptor(s), drug access to target tissues, drug concentration in different tissues, pharmacogenetics, and interactions with other drugs.

Chronic administration of a drug may cause a downregulation of receptors or desensitization of response that can require dose adjustments to maintain adequate therapy. Chronic administration of nitrovasodilators to treat angina results in the rapid development of complete tolerance, a process known as tachyphylaxis. Drug resistance may also develop because of pharmacokinetic mechanisms (i.e., the drug is metabolized more rapidly with chronic exposure), the development of mechanisms that prevent the drug from reaching its receptor (i.e., increased expression of the multidrug resistance transporter in drug-resistant cancer cells; see Chapter 5), or the clonal expansion of cancer cells containing drug-resistant mutations in the drug receptor.

Some drug effects do not occur by means of macromolecular receptors; aluminum and magnesium hydroxides [Al(OH)3 and Mg(OH)2] reduce gastric acid chemically, neutralizing H+ with OH and raising gastric pH. Mannitol acts osmotically to cause changes in the distribution of water to promote diuresis, catharsis, expansion of circulating volume in the vascular compartment, or reduction of cerebral edema (see Chapter 25). Anti-infective drugs such as antibiotics, antivirals, and antiparasitics target receptors or cell processes that are critical for the growth or survival of the infective agent but are nonessential or lacking in the host organism. Resistance to antibiotics, antivirals, and other drugs can result through a variety of mechanisms including mutation of the target receptor, increased expression of enzymes that degrade or increase efflux of the drug from the infective agent, and development of alternative biochemical pathways that circumvent the drug’s effects on the infective agent.

STRUCTURE-ACTIVITY RELATIONSHIPS AND DRUG DESIGN

The receptors responsible for the clinical effects of many drugs have yet to be identified. Conversely, sequencing of the entire human genome has identified novel genes related by sequence to known receptors, for which endogenous and exogenous ligands are unknown; these are called orphan receptors.

Both the affinity of a drug for its receptor and its intrinsic activity are determined by its chemical structure. This relationship frequently is quite stringent. Relatively minor modifications in the drug molecule may result in major changes in its pharmacological properties based on altered affinity for 1 or more receptors. Exploitation of structure-activity relationships has frequently led to the synthesis of valuable therapeutic agents. Because changes in molecular configuration need not alter all actions and effects of a drug equally, it is sometimes possible to develop a congener with a more favorable ratio of therapeutic to adverse effects, enhanced selectivity among different cells or tissues, or more acceptable secondary characteristics than those of the parent drug. Therapeutically useful antagonists of hormones or neurotransmitters have been developed by chemical modification of the structure of the physiological agonist.

With information about the molecular structures and pharmacological activities of a relatively large group of congeners, it is possible to use computer analysis to identify the chemical properties (i.e., the pharmacophore) required for optimal action at the receptor: size, shape, position, and orientation of charged groups or hydrogen bond donors, and so on. Advances in molecular modeling of organic compounds and the methods for drug target (receptor) discovery and biochemical measurement of the primary actions of drugs at their receptors have enriched the quantitation of structure-activity relationships and its use in drug design. Such information increasingly is allowing the optimization or design of chemicals that can bind to a receptor with improved affinity, selectivity, or regulatory effect. Similar structure-based approaches also are used to improve pharmacokinetic properties of drugs, particularly if knowledge of their metabolism is known. Knowledge of the structures of receptors and of drug-receptor complexes, determined at atomic resolution by X-ray crystallography, is even more helpful in the design of ligands and in understanding the molecular basis of drug resistance and circumventing it. Emerging technology in the field of pharmacogenetics (see Chapter 7) is improving our understanding of the nature of and variation in receptors.

QUANTITATIVE ASPECTS OF DRUG INTERACTIONS WITH RECEPTORS

Receptor occupancy theory assumes that response emanates from a receptor occupied by a drug, a concept that has its basis in the law of mass action. The dose-response curve depicts the observed effect of a drug as a function of its concentration in the receptor compartment. Figure 3–2 shows a typical dose-response curve.

images

Figure 3–2 Graded responses (y axis as a percentage of maximal response) expressed as a function of the concentration of drug A present at the receptor. The hyperbolic shape of the curve in panel Abecomes sigmoid when plotted semi-logarithmically, as in panel B. The concentration of drug that produces 50% of the maximal response quantifies drug activity and is referred to as the EC50 (effective concentration for 50% response). The range of concentrations needed to fully depict the dose-response relationship (~3 log10 [10] units) is too wide to be useful in the linear format of Figure 3–2A; thus, most dose-response curves use log [Drug] on the x axis, as in Figure 3–2B. Dose-response curves presented in this way are sigmoidal in shape and have 3 properties: threshold, slope, and maximal asymptote. These 3 parameters quantitate the activity of the drug.

Some drugs cause low-dose stimulation and high-dose inhibition of response. These U-shaped relationships for some receptor systems are said to display hormesis. Several drug-receptor systems can display this property (e.g., prostaglandins, endothelin, and purinergic and serotonergic agonists), which may be at the root of some drug toxicities.

AFFINITY, EFFICACY, AND POTENCY. In general, the drug-receptor interaction is characterized by (1) binding of drug to receptor and (2) generation of a response in a biological system, as illustrated in Equation 3–1 where the drug or ligand is denoted as L and the inactive receptor as R. The first reaction, the reversible formation of the ligand-receptor complex LR, is governed by the chemical property of affinity.

images

LR* is produced in proportion to [LR] and leads to a response. This simple relationship illustrates the reliance of the affinity of the ligand (L) with receptor (R) on both the forward orassociation rate (k+1) and the reverse or dissociation rate (k–1). At any given time, the concentration of ligand-receptor complex [LR] is equal to the product of k+1[L][R], the rate of formation of the bimolecular complex LR, minus the product k–1[LR], the rate dissociation of LR into L and R. At equilibrium (i.e., when δ[LR]/δt = 0), k+1[L][R] = [k–1[LR]. Theequilibrium dissociation constant (KD) is then described by ratio of the off and on rate constants (k–1/k+1).

Thus, at equilibrium,

images

The affinity constant or equilibrium association constant (KA) is the reciprocal of the equilibrium dissociation constant (i.e., KA = 1/KD); thus, a high-affinity drug has a low KD and will bind a greater number of a particular receptor at a low concentration than a low-affinity drug. As a practical matter, the affinity of a drug is influenced most often by changes in its off-rate (k–1) rather than its on-rate (k+1).

Equation 3–2 permits us to write an expression of the fractional occupancy (f) of receptors by agonist:

images

This can be expressed in terms of KA (or KD) and [L]:

images

From this relationship, it follows that when the concentration of drug equals the KD (or 1/KA), f = 0.5, that is, the drug will occupy 50% of the receptors. This relationship describes only receptor occupancy, not the eventual response that may be amplified by the cell. Many signaling systems reach a full biological response with only a fraction of receptors occupied.

Potency is defined by example in Figure 3–3. Basically, when 2 drugs produce equivalent responses, the drug whose dose-response curve (plotted as in Figure 3–3A) lies to the left of the other (i.e., the concentration producing a half-maximal effect [EC50] is smaller) is said to be the more potent.

images

Figure 3–3 Two ways of quantifying agonismA. The relative potency of 2 agonists (Drug X, red line; Drug Y, purple line) obtained in the same tissue is a function of their relative affinities and intrinsic efficacies. The EC50 of Drug X occurs at a concentration that is one-tenth the EC50 of Drug Y. Thus, Drug X is more potent than Drug Y. B. In systems where the 2 drugs do not both produce the maximal response characteristic of the tissue, the observed maximal response is a nonlinear function of their relative intrinsic efficacies. Drug X is more efficacious than Drug Y; their asymptotic fractional responses are 100% (Drug X) and 50% (Drug Y).

Efficacy reflects the capacity of a drug to activate a receptor and generate a cellular response. Thus, a drug with high efficacy may be a full agonist, eliciting, at some concentration, a full response. A drug with a lower efficacy at the same receptor may not elicit a full response at any dose (see Figure 3–1). A drug with a low intrinsic efficacy will be a partial agonist. A drug that binds to a receptor and exhibits zero efficacy is an antagonist.

QUANTIFYING AGONISM. When the relative potency of 2 agonists of equal efficacy is measured in the same biological system, and downstream signaling events are the same for both drugs, the comparison yields a relative measure of the affinity and efficacy of the 2 agonists (see Figure 3–3). We often describe agonist response by determining the half-maximally effective concentration (EC50) for producing a given effect. We can also compare maximal asymptotes in systems where the agonists do not produce maximal response (Figure 3–3B). The advantage of using maxima is that this property depends solely on efficacy, whereas drug potency is a mixed function of both affinity and efficacy.

QUANTIFYING ANTAGONISM. Characteristic patterns of antagonism are associated with certain mechanisms of blockade of receptors. One is straightforward competitive antagonism, whereby a drug with affinity for a receptor but lacking intrinsic efficacy competes with the agonist for the primary binding site on the receptor. The characteristic pattern of such antagonism is the concentration-dependent production of a parallel shift to the right of the agonist dose-response curve with no change in the maximal response (Figure 3–4A). The magnitude of the rightward shift of the curve depends on the concentration of the antagonist and its affinity for the receptor. A competitive antagonist will reduce the response to zero.

images

Figure 3–4 Mechanisms of receptor antagonismA. Competitive antagonism occurs when the agonist A and antagonist I compete for the same binding site on the receptor. Response curves for the agonist are shifted to the right in a concentration-related manner by the antagonist such that the EC50 for the agonist increases (e.g., L versus L′, L″, and L″′) with the concentration of the antagonist. B. If the antagonist binds to the same site as the agonist but does so irreversibly or pseudo-irreversibly (slow dissociation but no covalent bond), it causes a shift of the dose-response curve to the right, with further depression of the maximal response. Allosteric effects occur when an allosteric ligand I or P binds to a different site on the receptor to either inhibit (I) the response (see panel C) or potentiate (P) the response (see panel D). This effect is saturable; inhibition or potentiation reaches a limiting value when the allosteric site is fully occupied.

partial agonist similarly can compete with a “full” agonist for binding to the receptor. However, increasing concentrations of a partial agonist will inhibit response to a finite level characteristic of the drug’s intrinsic efficacy. Partial agonists may be used therapeutically to buffer a response by inhibiting excessive receptor stimulation without totally abolishing receptor stimulation.

An antagonist may dissociate so slowly from the receptor that its action is exceedingly prolonged. In the presence of a slowly dissociating antagonist, the maximal response to the agonist will be depressed at some antagonist concentrations (Figure 3–4B). Operationally, this is referred to as noncompetitive antagonism, although the molecular mechanism of action cannot be inferred unequivocally from the effect. An irreversible antagonist competing for the same binding site as the agonist can produce the pattern of antagonism shown in Figure 3–4B. Noncompetitive antagonism can be produced by anallosteric or allotopic antagonist, which binds to a site on the receptor distinct from that of the primary agonist, thereby changing the affinity of the receptor for the agonist. In the case of an allosteric antagonist, the affinity of the receptor for the agonist is decreased by the antagonist (Figure 3–4C). In contrast, a drug binding at an allosteric site could potentiate the effects of primary agonists (Figure 3–4D); such a drug would be referred to as an allosteric agonist or co-agonist.

The affinity of a competitive antagonist (Ki) for its receptor can be determined in radioligand binding assays or by measuring the functional response of a system to a drug in the presence of the antagonist. Concentration curves are run with the agonist alone and with the agonist plus an effective concentration of the antagonist (see Figure 3–4A). As more antagonist (I) is added, a higher concentration of the agonist (A) is needed to produce an equivalent response (the half-maximal or 50%, response is a convenient and accurately determined level of response). The extent of the rightward shift of the concentration-dependence curve is a measure of the affinity of the inhibitor, and a higher-affinity inhibitor will cause a greater rightward shift than a lower-affinity inhibitor at the same inhibitor concentration. Using Equations 3–3 and 3–4, one may write mathematical expressions of fractional occupancy (f) of the receptor by agonist for the agonist alone (control) and agonist in the presence of inhibitor.

For the agonist drug (L) alone,

images

For the case of agonist plus antagonist (I),

images

Assuming that equal responses result from equal fractional receptor occupancies in both the absence and presence of antagonist, one can set the fractional occupancies equal at agonist concentrations (L andL’) that generate equivalent responses in Figure 3–4A. Thus,

images

Simplifying, one gets:

images

where all values are known except Ki. Thus, one can determine the Ki for a reversible, competitive antagonist without knowing the KD for the agonist and without needing to define the precise relationship between receptor and response.

PHARMACODYNAMIC VARIABILITY: INDIVIDUAL AND POPULATION PHARMACODYNAMICS

Individuals vary in the magnitude of their response to the same concentration of a single drug, and a given individual may not always respond in the same way to the same drug concentration. Drug responsiveness may change because of disease or because of previous drug administration. Receptors are dynamic, and their concentration and function may be up- or downregulated by endogenous and exogenous factors.

Data on the correlation of drug levels with efficacy and toxicity must be interpreted in the context of the pharmacodynamic variability in the population (e.g., genetics, age, disease, and the presence of coadministered drugs). The variability in pharmacodynamic response in the population may be analyzed by constructing a quantal concentration-effect curve (Figure 3–5A). The dose of a drug required to produce a specified effect in 50% of the population is the median effective dose (ED50see Figure 3–5A). In preclinical studies of drugs, the median lethal dose (LD50) is determined in experimental animals (Figure 3–5B). The LD50/ED50 ratio is an indication of the therapeutic index, which is a statement of how selective the drug is in producing its desired effects versus its adverse effects. A similar term, thetherapeutic window, is the range of steady-state concentrations of drug that provides therapeutic efficacy with minimal toxicity (Figures 2–6 and 3–6). In clinical studies, the dose, or preferably the concentration, of a drug required to produce toxic effects can be compared with the concentration required for therapeutic effects in the population to evaluate the clinical therapeutic index. The concentration or dose of drug required to produce a therapeutic effect in most of the population usually will overlap the concentration required to produce toxicity in some of the population, even though the drug’s therapeutic index in an individual patient may be large. Thus, a population therapeutic window expresses a range of concentrations at which the likelihood of efficacy is high and the probability of adverse effects is low (see Figure 3–6); it does not guarantee efficacy or safety. Therefore, use of the population therapeutic window to adjust dosage of a drug should be complemented by monitoring appropriate clinical and surrogate markers for drug effect(s).

images

Figure 3–5 Frequency distribution curves and quantal concentration-effect and dose-effect curvesA. Frequency distribution curves. An experiment was performed on 100 subjects, and the effective plasma concentration that produced a quantal response was determined for each individual. The number of subjects who required each dose was plotted, giving a log-normal frequency distribution (purple bars). The normal frequency distribution, when summated, yields the cumulative frequency distribution—a sigmoidal curve that is a quantal concentration-effect curve (red bars, red line). B. Quantal dose-effect curves. Animals were injected with varying doses of a drug and the responses were determined and plotted. The calculation of the therapeutic index, the ratio of the LD50 to the ED50, is an indication of how selective a drug is in producing its desired effects relative to its toxicity. See text for additional explanation.

images

Figure 3–6 Relation of the therapeutic window of drug concentrations to therapeutic and adverse effects in the population. The ordinate is linear; the abscissa is logarithmic.

FACTORS MODIFYING DRUG ACTION. Numerous factors contribute to the wide patient-to-patient variability in the dose required for optimal therapy observed with many drugs (Figure 3–7). The effects of these factors on variability of drug pharmacokinetics are described more thoroughly in Chapters 256, and 7.

images

Figure 3–7 Factors influencing the response to a prescribed drug dose.

COMBINATION THERAPY. Marked alterations in the effects of some drugs can result from coadministration with other agents, including prescription and nonprescription drugs, as well as supplements and nutraceuticals. Such interactions can cause toxicity, or inhibit the drug effect and the therapeutic benefit. Drug interactions always should be considered when unexpected responses to drugs occur. Understanding the mechanisms of drug interactions provides a framework for preventing them. Drug interactions may be pharmacokinetic (the delivery of a drug to its site of action is altered by a second drug) or pharmacodynamic (the response of the drug target is modified by a second drug). Examples of pharmacokinetic interactions that can enhance or diminish the delivery of drug to its site of action are provided in Chapter 2. In a patient with multiple comorbidities requiring a variety of medications, it may be difficult to identify adverse effects due to medication interactions, and to determine whether these are pharmacokinetic, pharmacodynamic, or some combination of interactions.

Combination therapy constitutes optimal treatment for many conditions, including heart failure (see Chapter 28), hypertension (see Chapter 27), and cancer (see Chapters 60-63). However, some drug combinations produce pharmacodynamic interactions that result in adverse effects. For example, nitrovasodilators produce relaxation of vascular smooth muscle (vasodilation) via NO-dependent elevation of cyclic GMP in vascular smooth muscle. The pharmacologic effects of sildenafil, tadalafil, and vardenafil result from inhibition of the type 5 cyclic nucleotide phosphodiesterase (PDE5) that hydrolyzes cyclic GMP to 5′GMP in the vasculature. Thus, coadministration of an NO donor (e.g., nitroglycerin) with a PDE5 inhibitor can cause potentially catastrophic hypotension. The oral anticoagulant warfarin has a narrow margin between therapeutic inhibition of clot formation and bleeding complications and is subject to numerous important pharmacokinetic and pharmacodynamic drug interactions. Alterations in dietary vitamin K intake may significantly affect the pharmacodynamics of warfarin and mandate altered dosing; antibiotics that alter the intestinal flora reduce the bacterial synthesis of vitamin K, thereby enhancing the effect of warfarin; concurrent administration of nonsteroidal anti-inflammatory drugs (NSAIDs) with warfarin increases the risk of GI bleeding almost 4-fold compared with warfarin alone. By inhibiting platelet aggregation, aspirin increases the incidence of bleeding in warfarin-treated patients.

Most drugs are evaluated in young and middle-aged adults, and data on their use in children and the elderly are sparse. At the extremes of age, drug pharmacokinetics and pharmacodynamics can be altered, possibly requiring substantial alteration in the dose or dosing regimen to safely produce the desired clinical effect.

Mechanisms of Drug Action

RECEPTORS THAT AFFECT CONCENTRATIONS OF ENDOGENOUS LIGANDS

A large number of drugs act by altering the synthesis, storage, release, transport, or metabolism of endogenous ligands such as neurotransmitters, hormones, and other intercellular mediators. For example, some of the drugs acting on adrenergic neurotransmission (see Chapters 8 and 12) include α-methyltyrosine [inhibits synthesis of norepinephrine (NE)], cocaine (blocks NE reuptake), amphetamine (promotes NE release), and selegiline (inhibits NE breakdown by MAO). There are similar examples for other neurotransmitter systems including acetylcholine (see Chapters 8 and 10), dopamine (DA), and serotonin (5HT) (see Chapters 13-15). Drugs that affect the synthesis of circulating mediators such as vasoactive peptides (e.g., angiotensin-converting enzyme inhibitors; see Chapter 26) and lipid-derived autocoids (e.g., cyclooxygenase inhibitors; see Chapter 33) are also widely used in the treatment of hypertension, inflammation, and myocardial ischemia.

RECEPTORS THAT REGULATE THE IONIC MILIEU

A relatively small number of drugs act by affecting the ionic milieu of blood, urine, and the GI tract. The receptors for these drugs are ion pumps and transporters, many of which are expressed only in specialized cells of the kidney and GI system. Most of the diuretics (e.g., furosemide, chlorothiazide, amiloride) act by directly affecting ion pumps and transporters in epithelial cells of the nephron that increase the movement of Na+ into the urine, or by altering the expression of ion pumps in these cells (e.g., aldosterone). Another therapeutically important target is the H+, K+-ATPase (proton pump) of gastric parietal cells. Irreversible inhibition of this proton pump by drugs such as esomeprazole reduces gastric acid secretion by 80-95% (see Chapter 45).

CELLULAR PATHWAYS ACTIVATED BY PHYSIOLOGICAL RECEPTORS

SIGNAL TRANSDUCTION PATHWAYS. Physiological receptors have 2 major functions, ligand binding and message propagation (i.e., signaling). These functions imply the existence of at least 2 functional domains within the receptor: a ligand-binding domain and an effector domain.

The regulatory actions of a receptor may be exerted directly on its cellular target(s), on effector protein(s), or may be conveyed by intermediary cellular signaling molecules called transducers. The receptor, its cellular target, and any intermediary molecules are referred to as a receptor-effector system or signal transduction pathway. Frequently, the proximal cellular effector protein is not the ultimate physiological target but rather is an enzyme, ion channel, or transport protein that creates, moves, or degrades a small molecule (e.g., a cyclic nucleotide, inositol trisphosphate, or NO) or ion (e.g., Ca2+) termed a second messenger. Second messengers can diffuse in the proximity of their synthesis or release and convey information to a variety of targets, which may integrate multiple signals. Even though these second messengers originally were thought of as freely diffusible molecules within the cell, imaging studies show that their diffusion and intracellular actions are constrained by compartmentation—selective localization of receptor-transducer-effector-signal termination complexes—established by protein-lipid and protein-protein interactions. All cells express multiple forms of proteins designed to localize signaling pathways by protein-protein interactions; these proteins are termed scaffolds or anchoring proteins.

Receptors and their associated effector and transducer proteins also act as integrators of information as they coordinate signals from multiple ligands with each other and with the differentiated activity of the target cell. For example, signal transduction systems regulated by changes in cyclic AMP (cAMP) and intracellular Ca2+ are integrated in many excitable tissues. In cardiac myocytes, an increase in cellular cAMP caused by activation of β adrenergic receptors enhances cardiac contractility by augmenting the rate and amount of Ca2+ delivered to the contractile apparatus; thus, cAMP and Ca2+ are positive contractile signals in cardiac myocytes. By contrast, cAMP and Ca2+ produce opposing effects on the contractility of smooth muscle cells: as usual, Ca2+ is a contractile signal, however, activation of βadrenergic receptors on these cells activates the cAMP-PKA pathway, which leads to relaxation through the phosphorylation of proteins that mediate Ca2+ signaling, such as myosin light chain kinase and ion channels that hyperpolarize the cell membrane.

Another important property of physiological receptors is their capacity to significantly amplify a physiological signal. Neurotransmitters, hormones, and other extracellular ligands are often present at the ligand-binding domain of a receptor in very low concentrations (nM to μM levels). However, the effector domain or the signal transduction pathway often contains enzymes and enzyme cascades that catalytically amplify the intended signal. These signaling systems are excellent targets for drugs.

STRUCTURAL AND FUNCTIONAL FAMILIES OF PHYSIOLOGICAL RECEPTORS

Receptors for physiological regulatory molecules can be assigned to functional families that share common molecular structures and biochemical mechanisms. Table 3–1 outlines 6 major families of receptors with examples of their physiological ligands, signal transduction systems, and drugs that affect these systems.

Table 3–1

Physiological Receptors

images

G PROTEIN–COUPLED RECEPTORS (GPCRS)

GPCRS span the plasma membrane as a bundle of 7 α-helices (Figure 3–8). Among the ligands for GPCRs are neurotransmitters such as ACh, biogenic amines such as NE, all eicosanoids and other lipid-signaling molecules, peptide hormones, opioids, amino acids such as GABA, and many other peptide and protein ligands. GPCRs are important regulators of nerve activity in the CNS and are the receptors for the neurotransmitters of the peripheral autonomic nervous system. Because of their number and physiological importance, GPCRs are the targets for many drugs.

images

Figure 3–8 Stimulation of a G–protein coupled receptor by ligand, the activation of the G protein, and stimulation of selected effectors. In the absence of ligand, the receptor and G protein heterotrimer form a complex in the membrane with the G subunit bound to GDP. Following binding of ligand, the receptor and G protein subunit undergo a conformational change leading to release of GDP, binding of GTP, and dissociation of the complex. The activated GTP-bound G subunit and the freed dimer bind to and regulate effectors. The system is returned to the basal state by hydrolysis of the GTP on the subunit; a reaction that is markedly enhanced by the regulators of G protein signaling (RGS) proteins. Prolonged stimulation of the receptor can lead to downregulation of the receptor. This event is initiated by G protein receptor kinases (GRKs) that phosphorylate the C terminal tail of the receptor, leading to recruitment of proteins termed arrestins; arrestins bind to the receptor on the internal surface, displacing G proteins and inhibiting signaling. Detailed descriptions of these signaling pathways are given throughout the text in relation to the therapeutic actions of drugs affecting these pathways.

GPCR Subtypes. There are multiple receptor subtypes within families of receptors. Ligand-binding studies initially identified receptor subtypes; molecular cloning has greatly accelerated the discovery and definition of additional receptor subtypes; their expression as recombinant proteins has facilitated the discovery of subtype-selective drugs. The distinction between classes and subtypes of receptors, however, is often arbitrary or historical. The α1, α2, and β adrenergic receptors differ from each other both in ligand selectivity and in coupling to G proteins (Gq, Gi, and Gs, respectively), yet s and are considered receptor classes and α1 and α2 are considered subtypes. Pharmacological differences among receptor subtypes are exploited therapeutically through the development and use of receptor-selective drugs. For example, β2 adrenergic agonists such as terbutaline are used for bronchodilation in the treatment of asthma in the hope of minimizing cardiac side effects caused by stimulation of the β1adrenergic receptor (see Chapter 12). Conversely, the use of β1-selective antagonists minimizes the chance of bronchoconstriction in patients being treated for hypertension or angina (see Chapters 12 and27).

Receptor Dimerization. GPCRs undergo both homo- and heterodimerization and possibly oligomerization. Dimerization of receptors may regulate the affinity and specificity of the complex for G proteins and the sensitivity of the receptor to phosphorylation by receptor kinases and the binding of arrestin, events important in termination of the action of agonists and removal of receptors from the cell surface. Dimerization also may permit binding of receptors to other regulatory proteins such as transcription factors.

G PROTEINS. GPCRs couple to a family of heterotrimeric GTP-binding regulatory proteins termed G proteins. G proteins are signal transducers that convey the information from the agonist-bound receptor to 1 or more effector proteins. G protein–regulated effectors include enzymes such as adenylyl cyclase, phospholipase C, cyclic GMP phosphodiesterase (PDE6), and membrane ion channels selective for Ca2+ and K+ (see Table 3–1 and Figure 3–8).

The G protein heterotrimer consists of a guanine nucleotide-binding α subunit, which confers specific recognition to both receptors and effectors, and an associated dimer of β and γ subunits that helps confer membrane localization of the G protein heterotrimer by prenylation of the γ subunit. In the basal state of the receptor-heterotrimer complex, the α subunit contains bound GDP and the α-GDP:βγ complex is bound to the unliganded receptor (see Figure 3–8). The α subunits fall into 4 families (Gs, Gi, Gq, and G12/13) which are responsible for coupling GPCRs to relatively distinct effectors. The Gssubunit uniformly activates adenylyl cyclase; the Gi subunit inhibits certain isoforms of adenylyl cyclase; the Gq subunit activates all forms of phospholipase C-β (PLCβ); and the G12/13 α subunits couple to guanine nucleotide exchange factors (GEFs), such as p115RhoGEF for the small GTP-binding proteins Rho and Rac. The signaling specificity of the large number of possible βγ combinations is not yet clear; nonetheless, it is known that K+ channels, Ca2+ channels, and PI-3 kinase (PI3K) are some of the effectors of free βγ dimer. Figure 3–8 and its legend summarize the basic activation/inactivation scheme for GPCR-linked systems.

SECOND MESSENGER SYSTEMS

CYCLIC AMP. Cyclic AMP is synthesized by adenylyl cyclase; stimulation is mediated by the Gs α subunit, inhibition by the Gi α subunit. There are 9 membrane-bound isoforms of adenylyl cyclase (AC) and 1 soluble isoform found in mammals. Cyclic AMP generated by adenylyl cyclases has 3 major targets in most cells, the cyclic AMP dependent protein kinase (PKA), cAMP-regulated guanine nucleotide exchange factors termed EPACs (exchange factors directly activated by cAMP), and via PKA phosphorylation, a transcription factor termed CREB (cAMP response element binding protein). In cells with specialized functions, cAMP can have additional targets such as cyclic nucleotide-gated ion channels, cyclic nucleotide-regulated phosphodiesterases (PDEs), and several ABC transporters (MRP4 and MRP5) (see Chapter 7).

PKA. The PKA holoenzyme consists of 2 catalytic (C) subunits reversibly bound to a regulatory (R) subunit dimer to form a heterotetramer complex (R2C2). When AC is activated and cAMP concentrations are increased, 4 cyclic AMP molecules bind to the R2C2 complex, 2 to each R subunit, causing a conformational change in the R subunits that lowers their affinity for the C subunits, causing their activation. The active C subunits phosphorylate serine and threonine residues on specific protein substrates. There are multiple isoforms of PKA; molecular cloning has revealed α and β isoforms of both the regulatory subunits (RI and RII), as well as 3 C subunit isoforms Cα, Cβ, and Cγ. The R subunits exhibit different subcellular localization and binding affinities for cAMP, giving rise to PKA holoenzymes with different thresholds for activation. PKA function also is modulated by subcellular localization mediated by A-kinase anchoring proteins (AKAPs).

PKG. Stimulation of receptors that raise intracellular cyclic GMP concentrations (see Figure 3–11) leads to the activation of the cyclic GMP-dependent protein kinase G (PKG) that phosphorylates some of the same substrates as PKA and some that are PKG-specific. Unlike the heterotetramer (R2C2) structure of the PKA holoenzyme, the catalytic domain and cyclic nucleotide-binding domains of PKG are expressed as a single polypeptide, which dimerizes to form the PKG holoenzyme.

images

Figure 3–11 Cyclic GMP signaling pathways. Formation of cyclic GMP is regulated by cell surface receptors with intrinsic guanylyl cyclase (GC) activity and by soluble forms of GC. The cell surface receptors respond to natriuretic peptides such as atrial natriuretic peptide (ANP) with an increase in cyclic GMP. Soluble GC responds to nitric oxide (NO) generated from L-arginine by NO synthase (NOS). Cellular effects of cyclic GMP are carried out by PKG and cyclic GMP-regulated phosphodiesterases (PDEs). In this diagram, NO is produced by a Ca2+/calmodulin-dependent NOS in an adjacent endothelial cell. Detailed descriptions of these signaling pathways are given throughout the text in relation to the therapeutic actions of drugs affecting these pathways.

PKG exists in 2 homologous forms, PKG-I and PKG-II. PKG-I has an acetylated N terminus, is associated with the cytoplasm, and has 2 isoforms (Iα and Iβ) that arise from alternate splicing. PKG-II has a myristylated N terminus, is membrane-associated, and can be localized by PKG-anchoring proteins in a manner analogous to that known for PKA, although the docking domains of PKA and PKG are very different structurally. Pharmacologically important effects of elevated cyclic GMP include modulation of platelet activation and relaxation of smooth muscle. Receptors linked to cGMP synthesis are covered below in a separate section.

PDEs. Cyclic nucleotide PDEs form another family of important signaling proteins whose activities are regulated via the rate of gene transcription as well as by second messengers (cyclic nucleotides or Ca2+) and interactions with other signaling proteins such as β arrestin and protein kinases. PDEs hydrolyze the cyclic 3’,5’-phosphodiester bond in cAMP and cGMP, thereby terminating their action. The enzymes comprise a superfamily with >50 different PDE proteins. The substrate specificities of the different PDEs include those specific for cAMP hydrolysis, cGMP hydrolysis, and some that hydrolyze both cyclic nucleotides. PDEs (mainly PDE3 forms) are drug targets for treatment of diseases such as asthma, cardiovascular diseases such as heart failure, atherosclerotic coronary and peripheral arterial disease, and neurological disorders. PDE5 inhibitors (e.g., sildenafil) are used in treating chronic obstructive pulmonary disease and erectile dysfunction.

Gq-PLC-DAG/IP3-CA2+ PATHWAY. Calcium is an important messenger in all cells and can regulate diverse responses including gene expression, contraction, secretion, metabolism, and electrical activity. Ca2+ can enter the cell through Ca2+ channels in the plasma membrane (see “Ion Channels,” below) or be released by hormones or growth factors from intracellular stores. In keeping with its role as a signal, the basal Ca2+ level in cells is maintained in the 100 nM range by membrane Ca2+ pumps that extrude Ca2+ to the extracellular space and a sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA) in the membrane of the endoplasmic reticulum (ER) that accumulates Ca2+ into its storage site in the ER/SR.

Hormones and growth factors release Ca2+ from its intracellular storage site, the ER, via a signaling pathway that begins with activation of phospholipase C (PLC) at the plasma membrane, of which there are 2 primary forms, PLCβ and PLCγ. GPCRs that couple to Gq or Gi activate PLCβ by activating the G protein α subunit (see Figure 3–8) and releasing the βγ dimer. Both the active, Gq-GTP bound α subunit and the βγ dimer can activate certain isoforms of PLCβ. PLCγ isoforms are activated by tyrosine phosphorylation, including phosphorylation by receptor and nonreceptor tyrosine kinases.

PLCs are cytosolic enzymes that translocate to the plasma membrane upon receptor stimulation. When activated they hydrolyze a minor membrane phospholipid, phosphatidylinositol-4, 5-bisphosphate, to generate 2 intracellular signals, inositol-1,4,5-trisphosphate (IP3) and the lipid diacylglycerol (DAG). DAG directly activates members of the protein kinase C (PKC) family. IP3 diffuses to the ER where it activates the IP3 receptor in the ER membrane causing release of stored Ca2+ from the ER. Release of Ca2+ from these intracellular stores raises Ca2+ levels in the cytoplasm manyfold within seconds and activates calmodulin-sensitive enzymes such as cyclic AMP PDE and a family of Ca2+/calmodulin-sensitive protein kinases (e.g., phosphorylase kinase, myosin light chain kinase, and CaM kinases II and IV). Depending on the cell’s differentiated function, the Ca2+/calmodulin kinases and PKC may regulate the bulk of the downstream events in the activated cells.

ION CHANNELS

Changes in the flux of ions across the plasma membrane are critical regulatory events in both excitable and nonexcitable cells. To establish the electrochemical gradients required to maintain a membrane potential, all cells express ion transporters for Na+, K+, Ca2+, and Cl. For example, the Na+, K+-ATPase expends cellular ATP to pump Na+ out of the cell and K+ into the cell. The electrochemical gradients thus established are used by excitable tissues such as nerve and muscle to generate and transmit electrical impulses, by nonexcitable cells to trigger biochemical and secretory events, and by all cells to support a variety of secondary symport and antiport processes (see Chapter 5). Because of their roles as regulators of cell function, these proteins are important drug targets. The diverse ion channel family can be divided into subfamilies based on the mechanisms that open the channels, their architecture, and the ions they conduct. They can also be classified as voltage-activated, ligand-activated, store-activated, stretch-activated, and temperature-activated channels.

VOLTAGE-GATED CHANNELS. Humans express multiple isoforms of voltage-gated channels for Na+, K+, Ca2+, and Cl ions. In nerve and muscle cells, voltage-gated Na+ channels are responsible for the generation of robust action potentials that depolarize the membrane from its resting potential of –70 mV up to a potential of +20 mV within a few msec. These Na+ channels are composed of 3 subunits, a pore-forming α subunit and 2 regulatory β subunits (Figure 3–9A). The voltage-activated Na+ channels in pain neurons are targets for local anesthetics such as lidocaine and tetracaine, which block the pore, inhibit depolarization, and thus block the sensation of pain. They are also the targets of the naturally occurring marine toxins, tetrodotoxin and saxitoxin (see Chapter 20). Voltage-activated Na+channels are also important targets of many drugs used to treat cardiac arrhythmias (see Chapter 29).

images

Figure 3–9 Two types of ion channels regulated by receptors and drugsA. Diagram of a voltage-activated Na+ channel with the pore in the open and closed state. The pore-forming P loops are shown in blue, angled into the pore to form the selectivity filter. The S4 helices forming the voltage sensor are shown in orange, with the positively charged amino acids displayed as red dots. B. Ligand-gated nicotinic acetylcholine receptor expressed in the skeletal muscle neuromuscular junction. The pore is made up of 5 subunits, each with a large extracellular domain and 4 transmembrane helices (1 of these subunits is shown at the left of panel B). The helix that lines the pore is shown in blue. The receptor is composed of 2 α subunits, and β, γ, and δ subunits. See text for discussion of other ligand-gated ion channels. Detailed descriptions of specific channels are given throughout the text in relation to the therapeutic actions of drugs affecting these channels (see especially Chapters 1114, and 20). (Adapted with permission from Purves D, Augustine GJ, Fitzpatrick D, Hall WC, LaMantia AS, McNamara JO, and White LE, eds. Neuroscience, 4th ed. Sunderland, MA: Sinauer Associates, Inc., 2008.)

Voltage-gated Ca2+ channels have a similar architecture to voltage-gated Na+ channels with a large α subunit (4 domains of 5 membrane-spanning helices) and 3 regulatory subunits (the β, δ, and γ subunits). Ca2+ channels can be responsible for initiating an action potential (as in the pacemaker cells of the heart), but are more commonly responsible for modifying the shape and duration of an action potential initiated by fast voltage-gated Na+ channels. These channels initiate the influx of Ca2+ that stimulates the release of neurotransmitters in the central, enteric, and autonomic nervous systems, and that control heart rate and impulse conduction in cardiac tissue (see Chapters 814, and 27). The L-type voltage-gated Ca2+ channels are subject to additional regulation via phosphorylation by PKA. Voltage-gated Ca2+ channels expressed in smooth muscle regulate vascular tone; the intracellular concentration of Ca2+ is critical to regulating the phosphorylation state of the contractile apparatus via the activity of the Ca2+/calmodulin-sensitive myosin light chain kinase. Ca2+ channel antagonists such as nifedipine, diltiazem, and verapamil are effective vasodilators and are widely used to treat angina, cardiac arrhythmias, and hypertension.

Voltage-gated K+ channels are the most numerous and structurally diverse members of the voltage-gated channel family and include the voltage-gated Kv channels, the inwardly rectifying K+ channel, and the tandem or 2-pore domain “leak” K+ channels. The inwardly rectifying channels and the 2-pore channels are voltage insensitive, regulated by G proteins and H+ ions, and greatly stimulated by general anesthetics. Increasing K+ conductance through these channels drives the membrane potential more negative (closer to the equilibrium potential for K+); thus, these channels are important in regulating resting membrane potential and restoring the resting membrane at ~70 to ~90 mV following depolarization.

LIGAND-GATED CHANNELS. Channels activated by the binding of a ligand to a specific site in the channel protein have a diverse architecture and set of ligands. Major ligand-gated channels in the nervous system are those that respond to excitatory neurotransmitters such as acetylcholine (Figure 3–9B) or glutamate (or agonists such as AMPA and NMDA) and inhibitory neurotransmitters such as glycine or γ-aminobutyric acid (GABA). Activation of these channels is responsible for the majority of synaptic transmission by neurons both in the CNS and in the periphery (see Chapters 811, and 14). In addition, there are a variety of more specialized ion channels that are activated by intracellular small molecules, and are structurally distinct from conventional ligand-gated ion channels. These include ion channels that are formally members of the Kv family, such as the hyperpolarization and cyclic nucleotide-gated (HCN) channel expressed in the heart (see Chapter 29), and the cyclic nucleotide-gated (CNG) channels important for vision (see Chapter 64). The intracellular small molecule category of ion channels also includes the IP3-sensitive Ca2+ channel responsible for release of Ca2+ from the ER and the sulfonylurea “receptor” (SUR1) that associates with the Kir6.2 channel to regulate the ATP-dependent K+ channel (KATP) in pancreatic beta cells. The KATP channel is the target of oral hypoglycemic drugs such as sulfonylureas and meglitinides that stimulate insulin release from pancreatic β cells and are used to treat type 2 diabetes (see Chapter 43).

TRANSMEMBRANE RECEPTORS LINKED TO INTRACELLULAR ENZYMES

RECEPTOR TYROSINE KINASES. The receptor tyrosine kinases include receptors for hormones such as insulin, for multiple growth factors such EGF, platelet-derived growth factor (PDGF), nerve growth factor (NGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), and ephrins. With the exception of the insulin receptor, which has α and β chains (see Chapter 43), these macromolecules consist of single polypeptide chains with large, cysteine-rich extracellular domains, short transmembrane domains, and an intracellular region containing 1 or 2 protein tyrosine kinase domains. Activation of growth factor receptors leads to cell survival, cell proliferation, and differentiation. Activation of the ephrin receptors leads to neuronal angiogenesis, axonal migration, and guidance.

The inactive state of growth factor receptors is monomeric; binding of ligand induces dimerization of the receptor and cross-phosphorylation of the kinase domains on multiple tyrosine residues (Figure 3–10A). The phosphorylation of other tyrosine residues forms docking sites for the SH2 domains contained in a large number of signaling proteins. Molecules recruited to phosphotyrosine-containing proteins by their SH2 domains include PLCγ, which raises intracellular levels of Ca2+ and activates PKC. The α and β isoforms of PI3K contain SH2 domains, dock at the phosphorylated receptor, are activated, and increase the level of phosphatidylinositol 3,4,5 trisphosphate (PIP3) and protein kinase B (PKB, also known as Akt). PKB can regulate the mammalian target of rapamycin (mTOR) in the various signaling pathways and the bad protein that is important in apoptosis.

images

Figure 3–10 Mechanism of activation of a receptor tyrosine kinase and a cytokine receptorA. Activation of the EGF receptor. The extracellular structure of the unliganded receptor (a) contains 4 domains (I-IV), which rearrange significantly upon binding 2 EGF molecules. (b). The conformational changes lead to activation of the cytoplasmic tyrosine kinase domains and tyrosine phosphorylation of intracellular regions to form SH2 binding sites. (c). The adapter molecule Grb2 binds to the phosphorylated tyrosine residues and activates the Ras-MAP kinase cascade. B. Activation of a cytokine receptor. Binding of the cytokine causes dimerization of the receptor and recruits the Janus kinases (JAKs) to the cytoplasmic tails of the receptor. JAKs transphosphorylate and lead to the phosphorylation of the signal transducers and activators of transcription (STATs). The phosphorylated STATS translocate to the nucleus and regulate transcription. There are proteins termed suppressors of cytokine signaling (SOCS) that inhibit the JAK-STAT pathway.

In addition to recruiting enzymes, phosphotyrosine-presenting proteins can interact with SH2 domain-containing adaptor molecules without activity (e.g., Grb2), which in turn attract guanine nucleotide exchange factors (GEFs) such as Sos that can activate the small GTP-binding protein, Ras. The small GTP-binding proteins Ras and Rho belong to a large family of small monomeric GTPases. All of the small GTPases are activated by GEFs that are regulated by a variety of mechanisms and inhibited by GTPase-activating proteins (GAPs). Activation of members of the Ras family leads in turn to activation of a protein kinase cascade termed the mitogen-activated protein kinase (MAP kinase or MAPK) pathway. Activation of the MAPK pathway is one of the major routes used by growth factor receptors to signal to the nucleus and stimulate cell growth.

JAK-STAT RECEPTOR PATHWAY. Cells express a family of receptors for cytokines such as C-interferon and hormones like growth hormone and prolactin, which signal to the nucleus by a more direct manner than the receptor tyrosine kinases. These receptors have no intrinsic enzymatic activity, rather the intracellular domain binds a separate, intracellular tryosine kinase termed a Janus kinase (JAK). Upon dimerization induced by ligand binding, JAKs phosphorylate other proteins termed signal transducers and activators of transcription (STATs), which translocate to the nucleus and regulate transcription (Figure 3–10B). The entire pathway is termed the JAK-STAT pathway. There are 4 JAKs and 5 STATs in mammals that, depending on the cell type and signal, combine differently to activate gene transcription.

RECEPTOR SERINE-THREONINE KINASES. Protein ligands such as TGF-β activate a family of receptors that are analogous to the receptor tyrosine kinases except that they have a serine/threonine kinase domain in the cytoplasmic region of the protein. There are 2 isoforms of the monomeric receptor protein, type I (7 forms) and type II (5 forms). In the basal state, these proteins exist as monomers; upon binding an agonist ligand, they dimerize, leading to phosphorylation of the kinase domain of the type I monomer, which activates the receptor. The activated receptor then phosphorylates a gene regulatory protein termed a Smad. Once phosphorylated by the activated receptor on a serine residue, Smad dissociates from the receptor, migrates to the nucleus, associates with transcription factors, and regulates genes leading to morphogenesis and transformation. There are also inhibitory Smads (the Smad6 or Smad7 isoforms) that compete with the phosphorylated Smads to terminate signaling.

TOLL-LIKE RECEPTORS. Signaling related to the innate immune system is carried out by a family of >10 single membrane-spanning receptors termed Toll-like receptors (TLR), which are highly expressed in hematopoietic cells. In a single polypeptide chain, these receptors contain a large extracellular ligand-binding domain, a short membrane-spanning domain, and a cytoplasmic region termed the TIR domain that lacks intrinsic enzymatic activity. Ligands for TLR are comprised of a multitude of pathogen products including lipids, peptidoglycans, lipopeptides, and viruses. Activation of these receptors produces an inflammatory response to the pathogenic microorganisms.

The first step in activation of TLR by ligands is dimerization, which in turn causes signaling proteins to bind to the receptor to form a signaling complex. Ligand-induced dimerization recruits a series of adaptor proteins including Mal and the myeloid differentiation protein 88 (MyD88) to the intracellular TIR domain, which in turn recruit the interleukin-associated kinases termed IRAKs. The IRAKs autophosphorylate in the complex and subsequently form a more stable complex with MyD88. The phosphorylation event also recruits TRAF6 to the complex, which facilitates interaction with a ubiquitin ligase that attaches a polyubiquitin molecule to TRAF6. This complex can now interact with the protein kinase TAK1 and the adaptor TAB1. TAK1 is a member of the MAP kinase family, which activates the NF-κB kinases; phosphorylation of the NF-κB transcription factors causes their translocation to the nucleus and transcriptional activation of a variety of inflammatory genes.

TNF-α RECEPTORS. The mechanism of action of tumor necrosis factor α (TNF-α) signaling to the NF-κB transcription factors is very similar to that used by Toll-like receptors in that the intracellular domain of the receptor has no enzymatic activity. The TNF-α receptor is another single membrane-spanning receptor with an extracellular ligand-binding domain, a transmembrane domain, and a cytoplasmic domain termed the death domain. TNF-α binds a complex composed of TNF-receptor 1 and TNF-receptor 2. On trimerization, the death domains bind the adaptor protein TRADD, which recruits the receptor interacting protein 1 (RIP1) to form a receptor-adapto r complex at the membrane. RIP1 is polyubiquinated, resulting in recruitment of the TAK1 kinase and the IκB kinase (IKK) complex to the ubiquinated molecules. The activation loop of IKK is phosphorylated in the complex eventually resulting in IκBα being released from the complex allowing the p50/p65 heterodimer of the complex to translocate to the nucleus and activate the transcription of inflammatory genes. Humanized monoclonal antibodies to TNF-α itself, such as infliximab and adalimumab, are important for the treatment of rheumatoid arthritis and Crohn disease (see Chapters 35 and 47).

RECEPTORS THAT STIMULATE SYNTHESIS OF CYCLIC GMP. The signaling pathways that regulate the synthesis of cyclic GMP in cells include hormonal regulation of transmembrane guanylyl cyclases such as the atrial natriuretic peptide (ANP) receptor and the activation of soluble forms of guanylyl cyclase by NO. The downstream effects of cyclic GMP are carried out by multiple isoforms of PKG, cyclic GMP-gated ion channels, and cyclic GMP-modulated PDEs that degrade cyclic AMP.

Natriuretic Peptide Receptors: Ligand-Activated Guanylyl Cyclases. The class of membrane receptors with intrinsic enzymatic activity includes the receptors for 3 small peptide ligands released from cells in cardiac tissues and the vascular system, the natriuretic peptides: atrial natriuretic peptide (ANP), released from atrial storage granules following expansion of intravascular volume or stimulation with pressor hormones; brain natriuretic peptide (BNP), synthesized and released in large amounts from ventricular tissue in response to volume overload; and C-type natriuretic peptide (CNP), synthesized in the brain and endothelial cells. Like BNP, CNP is not stored in granules; rather, its synthesis and release are increased by growth factors and sheer stress on vascular endothelial cells. The major physiological effects of these hormones are to decrease blood pressure (ANP, BNP), to reduce cardiac hypertrophy and fibrosis (BNP), and to stimulate long bone growth (CNP). The transmembrane receptors for ANP, BNP, and CNP are ligand-activated guanylyl cyclases. The ANP receptor (NPR-A) is the molecule that responds to ANP and BNP. The NPR-B receptor responds to CNP. The natriuretic peptide C receptor (NPR-C) has an extracellular domain similar to those of NPR-A and NPR-B but does not contain the intracellular kinase or guanylyl cyclase domains. It has no enzymatic activity and is thought to function as a clearance receptor, removing excess natriuretic peptide from the circulation.

NO Synthase and Soluble Guanylyl Cyclase. NO is produced locally in cells by the enzyme NO synthase (NOS). NO stimulates the soluble form of guanylyl cyclase to produce cyclic GMP. There are 3 forms of NO synthase, neuronal NOS (nNOS or NOS1), endothelial NOS (eNOS or NOS3), and inducible NOS (iNOS or NOS2). All 3 forms of this enzyme are widely expressed but are especially important in the cardiovascular system, where they are found in myocytes, vascular smooth muscle cells, endothelial cells, hematopoietic cells, and platelets. Elevated cell Ca2+, acting via calmodulin, markedly activates nNOS and eNOS; the inducible form is less sensitive to Ca2+ but synthesis of iNOS protein in cells can be induced > 1000-fold by inflammatory stimuli such as endotoxin, TNF-α, interleukin-1β, and interferon-γ,.

NOS produces NO by catalyzing the oxidation of the guanido nitrogen of L-arginine, producing L-citrulline and NO. NO activates the soluble guanylyl cyclase (sGC), a heterodimer that contains a protoporphyrin-IX heme domain. NO binds to this domain at low nM concentrations and produces a 200- to 400-fold increase in the Vmax of guanylyl cyclase, leading to an elevation of cellular cyclic GMP. The cellular effects of cyclic GMP on the vascular system are mediated by a number of mechanisms, but especially by PKG. In vascular smooth muscle, activation of PKG leads to vasodilation by:

• Inhibiting IP3-mediated Ca2+ release from intracellular stores

• Phosphorylating voltage-gated Ca2+ channels to inhibit Ca2+ influx

• Phosphorylating phospholamban, a modulator of the sarcoplasmic Ca2+ pump, leading to a more rapid reuptake of Ca2+ into intracellular stores

• Phosphorylating and opening the Ca2+-activated K+ channel leading to hyperpolarization of the cell membrane, which closes L-type Ca2+ channels and reduces the flux of Ca2+ into the cell.

NUCLEAR HORMONE RECEPTORS AND TRANSCRIPTION FACTORS

Nuclear hormone receptors comprise a superfamily of 48 receptors that respond to a diverse set of ligands. The receptor proteins are transcription factors able to regulate the expression of genes controlling numerous physiological processes such as reproduction, development, and metabolism. Members of the family include receptors for circulating steroid hormones such as androgens, estrogens, glucocorticoids, thyroid hormone, and vitamin D. Other members of the family are receptors for a diverse group of fatty acids, bile acids, lipids, and lipid metabolites.

Examples include the retinoic acid receptor (RXR); the liver X receptor (LXR—the ligand is 22-OH cholesterol); the farnesoid X receptor (FXR—the ligand is chenodeoxycholic acid); and the peroxisomeproliferator-activated receptors (PPARs α, β, and γ; 15 deoxy prostaglandin J2 is a possible ligand for PPARγ; the cholesterol-lowering fibrates bind to and regulate PPARγ). In the inactive state, receptors for steroids such as glucocorticoids reside in the cytoplasm and translocate to the nucleus upon binding ligand. Other members of the family such as the LXR and FXR receptors reside in the nucleus and are activated by changes in the concentration of hydrophobic lipid molecules.

Nuclear hormone receptors contain 4 major domains in a single polypeptide chain. The N-terminal domain can contain an activation region (AF-1) essential for transcriptional regulation followed by a very conserved region with 2 zinc fingers that bind to DNA (the DNA-binding domain). The N-terminal activation region (AF-1) is subject to regulation by phosphorylation and other mechanisms that stimulate or inhibit transcription. The C terminal half of the molecule contains a hinge region (which can be involved in binding DNA), the domain responsible for binding the hormone or ligand (the ligand-binding domain or LBD), and specific sets of amino acid residues for binding coactivators and corepressors in a second activation region (AF-2). The LBD is formed from a bundle of 12 helices; ligand binding induces a major conformational change in helix 12 that affects the binding of the coregulatory proteins essential for activation of the receptor-DNA complex (Figure 3–12).

images

Figure 3–12 Activation of nuclear hormone receptors. A nuclear hormone receptor (OR) is shown in complex with the retinoic acid receptor (RXR). When an agonist (yellow triangle) and coactivator bind, a conformational change occurs in helix 12 (black bar) and gene transcription is stimulated. If corepressors are bound, activation does not occur. See text for details; see also Figure 6–13.

When bound to DNA, most of the nuclear hormone receptors act as dimers—some as homodimers, others as heterodimers. Steroid hormone receptors such as the glucocorticoid receptor are commonly homodimers, whereas those for lipids are heterodimers with the RXR receptor. The receptor dimers bind to repetitive DNA sequences, either direct repeat sequences or an inverted repeat termed hormone response elements (HRE) that are specific for each type of receptor. The hormone response elements in DNA are found upstream of the regulated genes or in some cases within the regulated genes. An agonist-bound nuclear hormone receptor often activates a large number of genes to carry out a program of cellular differentiation or metabolic regulation. An important property of these receptors is that they must bind their ligand, the appropriate HRE, and a coregulator to regulate their target genes. The activity of the nuclear hormone receptors in a given cell depends not only on the ligand, but the ratio of coactivators and corepressors recruited to the complex. Coactivators recruit enzymes to the transcription complex that modify chromatin, such as histone acetylase that serves to unravel DNA for transcription. Corepressors recruit proteins such as histone deacetylase that keeps DNA tightly packed and inhibits transcription.

APOPTOSIS

Organ development and renewal requires a balance between survival and expansion of the cell population, and cell death and removal. The process by which cells are genetically programmed for death is termed apoptosis.

Apoptosis is a highly regulated program of biochemical reactions that leads to cell rounding, shrinking of the cytoplasm, condensation of the nucleus and nuclear material, and changes in the cell membrane that eventually lead to presentation of phosphatidylserine on the outer surface of the cell. Phosphatidylserine is recognized as a sign of apoptosis by macrophages, which engulf and phagocytize the dying cell. During this process, the membrane of the apoptotic cell remains intact and the cell does not release its cytoplasm or nuclear material. Thus, unlike necrotic cell death, the apoptotic process does not initiate an inflammatory response. Alterations in apoptotic pathways are implicated in a variety of diseases such as cancer, and neurodegenerative and autoimmune diseases.

Two major signaling pathways induce apoptosis. Apoptosis can be initiated by external signals that have features in common with those used by ligands such as TNF-α or by an internal pathway activated by DNA damage, improperly folded proteins, or withdrawal of cell survival factors (Figure 3–13). The apoptotic program is carried out by a large family of cysteine proteases termed caspases. The caspases are highly specific cytoplasmic proteases that are inactive in normal cells but become activated by apoptotic signals.

images

Figure 3–13 Two pathways leading to apoptosis. Apoptosis can be initiated by external ligands such as TNF, Fas, or TNF-related apoptosis-inducing ligand (TRAIL) at specific transmembrane receptors (left half of figure). Activation leads to trimerization of the receptor, and binding of adaptor molecules such as TRADD, to the intracellular death domain. The adaptors recruit caspase 8, activate it leading to cleavage and activation of the effector caspase, caspase 3, which activates the caspase pathway, leading to apoptosis. Apoptosis can also be initiated by an intrinsic pathway regulated by Bcl-2 family members such as Bax and Bcl-2. Bax is activated by DNA damage or malformed proteins via p53 (right half of figure). Activation of this pathway leads to release of cytochrome c from the mitochondria, formation of a complex with Apaf-1 and caspase 9. Caspase 9 is activated in the complex and initiates apoptosis thru activation of caspase 3. Either the extrinsic or the intrinsic pathway can overwhelm the inhibitors of apoptosis proteins (IAPs) that otherwise keep apoptosis in check.

The external apoptosis signaling pathway can be activated by ligands such as TNF, Fas (also called Apo-1), or the TNF-related apoptosis-inducing ligand (TRAIL). The receptors for Fas and TRAIL are transmembrane receptors with no enzymatic activity, similar to the organization of the TNF receptor described above. Upon binding TNF, Fas ligand, or TRAIL, these receptors form a receptor dimer, undergo a conformational change, and recruit adapter proteins to the death domain. The adaptor proteins then recruit the receptor-interacting protein kinase (RIP1) and caspase 8 to form a complex that results in the activation of caspase 8. Activation of caspase 8 leads to the activation of caspase 3, which initiates the apoptotic program. The final steps of apoptosis are carried out by caspase 6 and 7, leading to degradation of enzymes, structural proteins, and DNA fragmentation characteristic of cell death (see Figure 3–13).

The internal apoptosis pathway can be activated by signals such as DNA damage leading to increased transcription of the p53 gene and involves damage to the mitochondria by pro-apoptotic members of the Bcl-2 family of proteins. This family includes proapoptotic members such as Bax, Bak, and Bad, which induce damage at the mitochondrial membrane. There are also anti-apoptotic Bcl-2 members, such as Bcl-2, Bcl-X, and Bcl-W, which serve to inhibit mitochondrial damage and are negative regulators of the system. When DNA damage occurs, p53 transcription is activated and holds the cell at a cell cycle checkpoint until the damage is repaired. If the damage cannot be repaired, apoptosis is initiated through the pro-apoptotic Bcl-2 members such as Bax. Bax is activated, translocates to the mitochondria, overcomes the anti-apoptotic proteins, and induces the release of cytochrome c and a protein termed the second mitochondria-derived activator of caspase (SMAC). SMAC binds to and inactivates the inhibitors of apoptosis proteins (IAPs) that normally prevent caspase activation. Cytochrome C combines in the cytosol with another protein, apoptotic activating protease factor -1 (Apaf-1), and with caspase 9. This complex leads to activation of caspase 9 and ultimately to the activation of caspase 3. Once activated, caspase 3 activates the same downstream pathways as the external pathway described above, leading to the cleavage of proteins, cytoskeletal elements, DNA repair proteins, with subsequent DNA condensation and membrane blebbing that eventually lead to cell death and engulfment by macrophages (see Figure 3–13).

RECEPTOR DESENSITIZATION AND REGULATION OF RECEPTORS

Receptors are almost always subject to feedback regulation by their own signaling outputs. Continued stimulation of cells with agonists generally results in a state of desensitization (also referred to asadaptation, refractoriness, or downregulation) such that the effect that follows continued or subsequent exposure to the same concentration of drug is diminished. This phenomenon, called tachyphylaxis, occurs rapidly and is important therapeutically; an example is attenuated response to the repeated use of β adrenergic receptor agonists as bronchodilators for the treatment of asthma (see Chapters 12 and36).

Desensitization can result from temporary inaccessibility of the receptor to agonist or from fewer receptors being synthesized (e.g., downregulation of receptor number). Phosphorylation of GPCR receptors by specific GPCR kinases (GRKs) plays a key role in triggering rapid desensitization. Phosphorylation of agonist-occupied GPCRs by GRKs facilitates the binding of cytosolic proteins termed arrestins to the receptor, resulting in the uncoupling of G protein from the receptor. The β arrestins recruit proteins, such as PDE4, that limit cyclic AMP signaling, and clathrin and β 2-adaptin, that promote sequestration of receptor from the membrane (internalization), thereby providing a scaffold that permits additional signaling steps.

Conversely, supersensitivity to agonists also frequently follows chronic reduction of receptor stimulation. Such situations can result, e.g., following withdrawal from prolonged receptor blockade (e.g., the long-term administration of a adrenergic receptor antagonists such as metoprolol) or in the case where chronic denervation of a preganglionic fiber induces an increase in neurotransmitter release per pulse, indicating post-ganglionic neuronal supersensitivity.

DISEASES RESULTING FROM RECEPTOR MALFUNCTION. Alteration in receptors and their immediate signaling effectors can be the cause of disease. The loss of a receptor in a highly specialized signaling system may cause a phenotypic disorder (e.g., deficiency of the androgen receptor and testicular feminization syndrome; see Chapter 41). The expression of constitutively active, aberrant, or ectopic receptors, effectors, and coupling proteins potentially can lead to supersensitivity, subsensitivity, or other untoward responses.

PHYSIOLOGICAL SYSTEMS INTEGRATE MULTIPLE SIGNALS

Consider the vascular wall of an arteriole (Figure 3–14). Several cell types interact at this site, including vascular smooth muscle cells (SMCs), endothelial cells (ECs), platelets, and post-ganglionic sympathetic neurons. A variety of physiological receptors and ligands are present, including ligands that cause SMCs to contract (angiotensin II [AngII], norepinephrine [NE]) and relax (nitric oxide [NO], B-type natriuretic peptide [BNP], and epinephrine), as well as ligands that alter SMC gene expression (platelet-derived growth factor [PDGF], AngII, NE, and eicosanoids).

images

Figure 3–14 Interaction of multiple signaling systems regulating vascular smooth muscle cells. See text for explanation of signaling and contractile pathways and abbreviations.

AngII has both acute and chronic effects on SMC. Interaction of AngII with AT1 receptors (AT1R) mobilizes stored Ca2+ via Gq-PLC-IP3-Ca2+ pathway. The Ca2+ binds and activates calmodulin and its target protein, myosin light-chain kinase (MLCK). The activation of MLCK results in the phosphorylation of myosin, leading to smooth muscle cell contraction. Activation of the sympathetic nervous system also regulates SMC tone through release of NE from post-ganglionic sympathetic neurons. NE binds α1 adrenergic receptors that also activate the Gq-PLC-IP3-Ca2+ pathway, resulting in SMC contraction, an effect that is additive to that of AngII.

The contraction of SMCs is opposed by mediators that promote relaxation, including NO, BNP, and catecholamines acting at β2 receptors. NO is formed in ECs by eNOS when the Gq-PLC-IP3-Ca2+pathway is activated, and by iNOS when that isoform is induced. The NO formed in ECs diffuses into SMCs, and activates the soluble form of guanylyl cyclase (sGC), which catalyzes the formation of cyclic GMP, which leads to activation of PKG and phosphorylation of proteins in SMCs that reduce intracellular concentrations of Ca2+ and thereby promote relaxation. Intracellular concentrations of cyclic GMP are also increased by activation of the transmembrane BNP receptor (BNP-R), whose guanylyl cyclase activity is increased when BNP binds.

As a consequence of the variety of pathways that affect arteriolar tone, a patient with hypertension may be treated with 1 or several drugs that alter signaling through these pathways. Drugs commonly used to treat hypertension include β1 antagonists to reduce secretion of renin (the rate-limiting first step in AngII synthesis), a direct renin inhibitor (aliskiren) to block the rate-limiting step in AngII production, angiotensin-converting enzyme (ACE) inhibitors (e.g., enalapril) to reduce the concentrations of circulating AngII, AT1 receptor blockers (e.g., losartan) to block AngII binding to AT1Rs on SMCs, R1adrenergic blockers to block NE binding to SMCs, sodium nitroprusside to increase the quantities of NO produced, or a Ca2+ channel blocker (e.g., nifedipine) to block Ca2+ entry into SMCs. β1 antagonists would also block the baroreceptor reflex increase in heart rate and blood pressure elicited by a drop in blood pressure induced by the therapy. ACE inhibitors also inhibit the degradation of a vasodilating peptide, bradykinin (see Chapter 26). Thus, the choices and mechanisms are complex, and the appropriate therapy in a given patient depends on many considerations, including the diagnosed causes of hypertension in the patient, possible side effects of the drug, efficacy in a given patient, and cost.