Many hormones are water soluble, have no transport proteins (and therefore have a short plasma half-life), and initiate a response by binding to a receptor located in the plasma membrane (see Tables 41–3 and 41–4). The mechanism of action of this group of hormones can best be discussed in terms of the intracellular signals they generate. These signals include cAMP (cyclic AMP; 3′, 5′-adenylic acid; see Figure 18–5), a nucleotide derived from ATP through the action of adenylyl cyclase; cGMP, a nucleotide formed by guanylyl cyclase; Ca2+; and phosphatidylinositides; such small molecules are termed second messengers as their synthesis is triggered by the presence of the primary hormone (molecule) binding its receptor. Many of these second messengers affect gene transcription, as described in the previous paragraph; but they also influence a variety of other biologic processes, as shown in Figure 42–3, but see also Figures 42–6 and 42–8.
G Protein–Coupled Receptors
Many of the group II hormones bind to receptors that couple to effectors through a GTP-binding protein (G proteins) intermediary. These receptors typically have seven hydrophobic plasma membrane-spanning domains. This is illustrated by the seven interconnected helices extending through the lipid bilayer in Figure 42–4. Receptors of this class, which signal through guanine nucleotide-bound protein intermediates, are known as G protein–coupled receptors (GPCRs). To date, hundreds of G protein-linked receptor genes have been identified. GPCRs represent the largest family of cell surface receptors in humans. A wide variety of responses are mediated by the GPCRs.
cAMP Is the Intracellular Signal for Many Responses
Cyclic AMP was the first intracellular second messenger signal identified in mammalian cells. Several components comprise a system for the generation, degradation, and action of cAMP (Table 42–2).
Different peptide hormones can either stimulate (s) or inhibit (i) the production of cAMP from adenylyl cyclase through the action of the G proteins. G proteins are encoded by at least ten different genes (Table 42–3). Two parallel systems, a stimulatory (s) one and an inhibitory (i) one, converge upon a catalytic molecule (C). Each consists of a receptor, Rs or Ri, and a regulatory complex, Gs and Gi. Gs and Gi are each heterotrimeric G protein composed of α, β, and γ subunits. Since the α subunit in Gs differs from that in Gi, the proteins, which are distinct gene products, are designated αs and αi. The α subunits bind guanine nucleotides. The β and γ subunits are always associated (βγ) and appear to function as a heterodimer. The binding of a hormone to Rs or Ri results in a receptor-mediated activation of G protein, which entails the exchange of GDP by GTP on α and the concomitant dissociation of βγ from α.
TABLE 42–3Classes and Functions of Selected G Proteinsa ||Download (.pdf) TABLE 42–3 Classes and Functions of Selected G Proteinsa
|Class or Type ||Stimulus ||Effector ||Effect |
|Gs || || || |
| αs ||Glucagon, β-adrenergics ||↑Adenylyl cyclase ||Glyconeogenesis, lipolysis, glycogenolysis |
| || ||↑Cardiac Ca2+, Cl–, and Na+ channels ||Olfaction |
| αolf ||Odorant ||↑Adenylyl cyclase || |
|Gi || || || |
| αi-1,2,3 ||Acetylcholine, α2-adrenergics ||↓Adenylyl cyclase ||Slowed heart rate |
| || ||↑Potassium channels || |
| ||M2 cholinergics ||↓Calcium channels || |
| αo ||Opioids, endorphins ||↑Potassium channels ||Neuronal electrical activity |
| αt ||Light ||↑cGMP phosphodiesterase ||Vision |
|Gq || || || |
| αq ||M1 cholinergics || || |
| ||α1-Adrenergics ||↑Phospholipase C-β1 ||↓Muscle contraction and |
| α11 ||α 1-Adrenergics ||↑Phospholipase C-β2 ||↓Blood pressure |
|G12 || || || |
| α12 ||Thrombin ||Rho ||Cell shape changes |
The αs protein has intrinsic GTPase activity. The active form, αs·GTP, is inactivated upon hydrolysis of the GTP to GDP; the trimeric Gs complex (αβγ) is then re-formed and is ready for another cycle of activation. Cholera and pertussis toxins catalyze the ADP ribosylation of as and αi−2 (Table 42–3), respectively. In the case of αs, this modification disrupts the intrinsic GTPase activity; thus, αs cannot reassociate with βγ and is therefore irreversibly activated. ADP ribosylation of αi−2 prevents the dissociation of αi−2 from βγ, and free αi−2 thus cannot be formed. αs activity in such cells is therefore unopposed.
There is a large family of G proteins, and these are part of the superfamily of GTPases. The G protein family is classified according to sequence homology into four subfamilies, as illustrated in Table 42–3. There are 21 α, 5 β, and 8 γ subunit genes. Various combinations of these subunits provide a large number of possible αβγ and cyclase complexes.
The α subunits and the βγ complex have actions independent of those on adenylyl cyclase (see Figure 42–4 and Table 42–3). Some forms of αi stimulate K+ channels and inhibit Ca2+ channels, and some αs molecules have the opposite effects. Members of the Gq family activate the phospholipase C group of enzymes. The βγ complexes have been associated with K+ channel stimulation and phospholipase C activation. G proteins are involved in many important biologic processes in addition to hormone action. Notable examples include olfaction (αOLF) and vision (αt). Some examples are listed in Table 42–3. GPCRs are implicated in a number of diseases and are major targets for pharmaceutical agents.
Components of the hormone receptor–G protein effector system. Receptors that couple to effectors through G proteins, the G protein coupled receptors (GPCRs), typically have seven α-helical membrane-spanning domains (here shown as long cylinders). In the absence of hormone (left), the heterotrimeric G-protein complex (α,β,γ) is in an inactive guanosine diphosphate (GDP)-bound form and is probably not associated with the receptor. This complex is anchored to the plasma membrane through prenylated groups on the βγ subunits (wavy lines) and perhaps by myristoylated groups on α subunits (not shown). On binding of hormone (H) to the receptor, there are conformational changes within the receptor—as indicated by the tilted membrane spanning domains—and activation of the G-protein complex. This results from the exchange of GDP with guanosine triphosphate (GTP) on the α subunit, after which α and βγ dissociate. The α subunit binds to and activates the effector (E). E can be adenylyl cyclase, Ca2+, Na+, or Cl– channels (αs), or it could be a K+ channel (αi), phospholipase Cβ (αq), or cGMP phosphodiesterase (αt); see Table 42–3. The βγ subunit can also have direct actions on E. (Modified and reproduced, with permission, from Granner DK. In: Principles and Practice of Endocrinology and Metabolism, 2nd ed. Becker KL (editor). Lippincott, 1995.)
As discussed in Chapter 38, in prokaryotic cells, cAMP binds to a specific protein called catabolite regulatory protein (CRP) that binds directly to DNA and influences gene expression. By contrast, in eukaryotic cells, cAMP binds to a protein kinase called protein kinase A (PKA), a heterotetrameric molecule consisting of two regulatory subunits (R) that inhibit the activity of the two catalytic subunits (C) when bound as a tetrameric complex. cAMP binding to the R2C2 tetramer results in the following reaction:
The R2C2 complex has no enzymatic activity, but the binding of cAMP by R induces dissociation of the R–C complex, thereby activating the latter (Figure 42–5). The active C subunit catalyzes the transfer of the γ phosphate of ATP to a serine or threonine residue in a variety of proteins. The consensus PKA phosphorylation sites are -ArgArg/Lys-X-Ser/Thr- and -Arg-Lys-X-X-Ser-, where X can be any amino acid.
Hormonal regulation of cellular processes through cAMP-dependent protein kinase (PKA). PKA exists in an inactive form as an R2C2 heterotetramer consisting of two regulatory (R) and two catalytic (C) subunits. The cAMP generated by the action of adenylyl cyclase (activated as shown in Figure 42–4) binds to the regulatory subunit of PKA. This results in dissociation of the regulatory and catalytic subunits and activation of the latter. The active catalytic subunits phosphorylate a number of target proteins on serine and threonine residues. Phosphatases remove phosphate from these residues and thus terminate the physiologic response. A phosphodiesterase can also terminate the response by converting cAMP to 5′-AMP.
Historically protein kinase activities were described as being “cAMP-dependent” or “cAMP-independent.” This classification has changed, as protein phosphorylation is now recognized as being a major and ubiquitous regulatory mechanism. Several hundred protein kinases have now been described. These kinases are related in sequence and structure within the catalytic domain, but each is a unique molecule with considerable variability with respect to subunit composition, molecular weight, autophosphorylation, Km for ATP, and substrate specificity. Both kinase and protein phosphatase activities can be targeted by interaction with specific kinase binding proteins. In the case of PKA, such targeting proteins are termed A kinase anchoring proteins, or AKAPs. AKAPs serve as scaffolds, which localize PKA near to substrates thereby focusing PKA activity toward physiological substrates and facilitating spatiotemporal biological regulation while also allowing for common, shared proteins to elicit specific physiological responses. Multiple AKAPs have been described and importantly they can bind PKA and other kinases as well as phosphatases, phosphodiesterases (which hydrolyze cAMP), and protein kinase substrates. The multifunctionality of AKAPs facilitates signaling localization, rate (production and destruction of signals), specificity and dynamics.
The effects of cAMP in eukaryotic cells are all thought to be mediated by protein phosphorylation-dephosphorylation, principally on serine and threonine residues. The control of any of the effects of cAMP, including such diverse processes as steroidogenesis, secretion, ion transport, carbohydrate and fat metabolism, enzyme induction, gene regulation, synaptic transmission, and cell growth and replication, could be conferred by a specific protein kinase, by a specific phosphatase, or by specific substrates for phosphorylation. The array of specific substrates define a target tissue, and are involved in defining the extent of a particular response within a given cell. For example, the effects of cAMP on gene transcription are mediated by CREB, the cyclic AMP response element binding protein. CREB binds to a cAMP responsive DNA enhancer element (CRE) (see Table 42–1) in its nonphosphorylated state and is a weak activator of transcription. When phosphorylated by PKA, CREB binds the coactivator CREB-binding protein CBP/p300 (see below) and as a result is a much more potent transcription activator. CBP and the related p300 contain histone acetyltransferase activities, and hence serve as chromatin-active transcriptional coregulators (see Chapters 36, 38). Interestingly, CBP/p300 can also acetylate certain transcription factors thereby stimulating their ability to bind DNA and modulate transcription.
Actions caused by hormones that increase cAMP concentration can be terminated in a number of ways, including the hydrolysis of cAMP to 5′-AMP by phosphodiesterases (see Figure 42–5). The presence of these hydrolytic enzymes ensures a rapid turnover of the signal (cAMP) and hence a rapid termination of the biologic process once the hormonal stimulus is removed. There are at least 11 known members of the phosphodiesterase family of enzymes. These are subject to regulation by their substrates, cAMP and cGMP; by hormones; and by intracellular messengers such as calcium, probably acting through calmodulin. Inhibitors of phosphodiesterase, most notably methylated xanthine derivatives such as caffeine, increase intracellular cAMP and mimic or prolong the actions of hormones through this signal.
Given the importance of protein phosphorylation, it is not surprising that regulation of the protein dephosphorylation reaction is another important control mechanism (see Figure 42–5). The phosphoprotein phosphatases are themselves subject to regulation by phosphorylation-dephosphorylation reactions and by a variety of other mechanisms, such as protein-protein interactions. In fact, the substrate specificity of the phosphoserine-phosphothreonine phosphatases may be dictated by distinct regulatory subunits whose binding is regulated hormonally. One of the best-studied roles of regulation by the dephosphorylation of proteins is that of glycogen metabolism in muscle (see Figures 18–6 to 18–8). Two major types of phosphoserine-phosphothreonine phosphatases have been described. Type I preferentially dephosphorylates the β subunit of phosphorylase kinase, whereas type II dephosphorylates the α subunit. Type I phosphatase is implicated in the regulation of glycogen synthase, phosphorylase, and phosphorylase kinase. This phosphatase is itself regulated by phosphorylation of certain of its subunits, and these reactions are reversed by the action of one of the type II phosphatases. In addition, two heat-stable protein inhibitors regulate type I phosphatase activity. Inhibitor-1 is phosphorylated and activated by cAMP-dependent protein kinases, and inhibitor-2, which may be a subunit of the inactive phosphatase, is also phosphorylated, possibly by glycogen synthase kinase-3. Phosphatases that target phosphotyrosine are also important in signal transduction (see Figure 42–8).
Certain hormone-receptor interactions result in the activation of phospholipase C (PLC). PLC activation appears to involve a specific G protein, which also may activate a calcium channel. Phospholipase C generates inositol trisphosphate (IP3), which liberates stored intracellular Ca2+, and diacylglycerol (DAG), a potent activator of protein kinase C (PKC). In this scheme, the activated PKC phosphorylates specific substrates, which then alter physiologic processes. Likewise, the Ca2+–calmodulin complex can activate specific kinases, two of which are shown here. These actions result in phosphorylation of substrates, and this leads to altered physiologic responses. This figure also shows that Ca2+ can enter cells through voltage- or ligand-gated Ca2+ channels. The intracellular Ca2+ is also regulated through storage and release by the mitochondria and endoplasmic reticulum. (Reprinted with permission from JH Exton.)
Phospholipase C cleaves PIP2 into diacylglycerol and inositol trisphosphate. R1 generally is stearate, and R2 is usually arachidonate. IP3 can be dephosphorylated (to the inactive I-1,4-P2) or phosphorylated (to the potentially active I-1,3,4,5-P4).
Insulin signaling pathways. The insulin signaling pathways provide an excellent example of the “recognition → hormone release → signal generation → effects” paradigm outlined in Figure 42–1. Insulin is released into the bloodstream from pancreatic β-cells in response to hyperglycemia. Binding of insulin to a target cell-specific plasma membrane heterotetrameric insulin receptor (IR) results in a cascade of intracellular events. First, the intrinsic tyrosine kinase activity of the insulin receptor is activated, and marks the initial event. Receptor activation results in increased tyrosine phosphorylation (conversion of specific Y residues → Y-P) within the receptor. One or more of the insulin receptor substrate (IRS) molecules (IRS 1-4) then bind to the tyrosine-phosphorylated receptor and themselves are specifically tyrosine phosphorylated. IRS proteins interact with the activated IR via N-terminal PH (pleckstrin homology) and PTB (phosphotyrosine binding) domains. IR-docked IRS proteins are tyrosine phosphorylated and the resulting P-Y-residues form the docking sites for several additional signaling proteins (ie, PI-3 kinase, GRB2, and mTOR). GRB2 and PI3K bind to IRS P-Y residues via their SH (Src Homology) domains, Binding to IRS-Y-P residues leads to activation of the activity of many intracellular signaling molecules such as GTPases, protein kinases, and lipid kinases, all of which play key roles in certain metabolic actions of insulin. The two best-described pathways are shown. In detail, phosphorylation of an IRS molecule (probably IRS-2) results in docking and activation of the lipid kinase, PI-3 kinase; PI-3K generates novel inositol lipids that act as “second messenger” molecules. These, in turn, activate PDK1 and then a variety of downstream signaling molecules, including protein kinase B (PKB/AKT), SGK, and aPKC. An alternative pathway involves the activation of p70S6K and perhaps other as yet unidentified kinases. Next, phosphorylation of IRS (probably IRS-1) results in docking of GRB2/mSOS and activation of the small GTPase, p21Ras, which initiates a protein kinase cascade that activates Raf-1, MEK, and the p42/p44 MAP kinase isoforms. These protein kinases are important in the regulation of proliferation and differentiation of many cell types. The mTOR pathway provides an alternative way of activating p70S6K and appears to be involved in nutrient signaling as well as insulin action. Each of these cascades may influence different biological processes, as shown (protein translocation, protein/enzyme activity, gene transcription, cell growth). All of the phosphorylation events are reversible through the action of specific phosphatases. As an example, the lipid phosphatase PTEN dephosphorylates the product of the PI-3 kinase reaction, thereby antagonizing the pathway and terminating the signal. Representative effects of major actions of insulin are shown in each of the boxes. The asterisk after phosphodiesterase indicates that insulin indirectly affects the activity of many enzymes by activating phosphodiesterases and reducing intracellular cAMP levels. (aPKC, atypical protein kinase C; GRB2, growth factor receptor binding protein 2; IGFBP, insulin-like growth factor binding protein; IRS 1–4, insulin receptor substrate isoforms 1–4; MAP kinase, mitogen-activated protein kinase; MEK, MAP kinase kinase and ERK kinase; mSOS, mammalian son of sevenless; mTOR, mammalian target of rapamycin; p70S6K, p70 ribosomal protein S6 kinase; PDK1, phosphoinositide-dependent kinase; PI-3 kinase, phosphatidylinositol 3-kinase; PKB, protein kinase B; PTEN, phosphatase and tensin homolog deleted on chromosome 10; SGK, serum and glucocorticoid-regulated kinase.)
cGMP Is Also an Intracellular Signal
Cyclic GMP is made from GTP by the enzyme guanylyl cyclase, which exists in soluble and membrane-bound forms. Each of these enzyme forms has unique physiologic properties. The atriopeptins, a family of peptides produced in cardiac atrial tissues, cause natriuresis, diuresis, vasodilation, and inhibition of aldosterone secretion. These peptides (eg, atrial natriuretic factor) bind to and activate the membrane-bound form of guanylyl cyclase. This results in an increase of cGMP by as much as 50-fold in some cases, and this is thought to mediate the effects mentioned above. Other evidence links cGMP to vasodilation. A series of compounds, including nitroprusside, nitroglycerin, nitric oxide, sodium nitrite, and sodium azide, all cause smooth muscle relaxation and are potent vasodilators. These agents increase cGMP by activating the soluble form of guanylyl cyclase, and inhibitors of cGMP phosphodiesterase (the drug sildenafil [Viagra], for example) enhance and prolong these responses. The increased cGMP activates cGMP-dependent protein kinase (PKG), which in turn phosphorylates a number of smooth muscle proteins. Presumably, this is involved in relaxation of smooth muscle and vasodilation.
Several Hormones Act Through Calcium or Phosphatidylinositols
Ionized calcium, Ca2+, is an important regulator of a variety of cellular processes, including muscle contraction, stimulus-secretion coupling, blood clotting cascade, enzyme activity, and membrane excitability. Ca2+ is also an intracellular messenger of hormone action.
The extracellular Ca2+ concentration is ∼5 mmol/L and is very rigidly controlled. Although substantial amounts of calcium are associated with intracellular organelles such as mitochondria and the endoplasmic reticulum, the intracellular concentration of free or ionized calcium (Ca2+) is very low: 0.05 to 10 μmol/L. In spite of this large concentration gradient and a favorable transmembrane electrical gradient, Ca2+ is restrained from entering the cell. A considerable amount of energy is expended to ensure that the intracellular Ca2+ is controlled, as a prolonged elevation of Ca2+ in the cell is very toxic. A Na+/Ca2+ exchange mechanism that has a high-capacity but low-affinity pumps Ca2+ out of cells. There also is a Ca2+/proton ATPase-dependent pump that extrudes Ca2+ in exchange for H+. This has a high affinity for Ca2+ but a low capacity and is probably responsible for fine-tuning cytosolic Ca2+. Furthermore, Ca2+-ATPases pump Ca2+ from the cytosol to the lumen of the endoplasmic reticulum. There are three ways of changing cytosolic Ca2+ levels: (1) Certain hormones (class II.C, Table 41–3) by binding to receptors that are themselves Ca2+ channels, enhance membrane permeability to Ca2+, and thereby increase Ca2+ influx. (2) Hormones also indirectly promote Ca2+ influx by modulating the membrane potential at the plasma membrane. Membrane depolarization opens voltage-gated Ca2+ channels and allows for Ca2+ influx. (3) Ca2+ can be mobilized from the endoplasmic reticulum, and possibly from mitochondrial pools.
An important observation linking Ca2+ to hormone action involved the definition of the intracellular targets of Ca2+ action. The discovery of a Ca2+-dependent regulator of phosphodiesterase activity provided the basis for a broad understanding of how Ca2+ and cAMP interact within cells.
The calcium-dependent regulatory protein is calmodulin, a 17-kDa protein that is homologous to the muscle protein troponin C in structure and function. Calmodulin has four Ca2+ binding sites, and full occupancy of these sites leads to a marked conformational change, which allows calmodulin to activate enzymes and ion channels. The interaction of Ca2+ with calmodulin (with the resultant change of activity of the latter) is conceptually similar to the binding of cAMP to PKA and the subsequent activation of this molecule. Calmodulin can be one of numerous subunits of complex proteins and is particularly involved in regulating various kinases and enzymes of cyclic nucleotide generation and degradation. A partial list of the enzymes regulated directly or indirectly by Ca2+, probably through calmodulin, is presented in Table 42–4.
TABLE 42–4Some Enzymes and Proteins Regulated by Calcium or Calmodulin ||Download (.pdf) TABLE 42–4 Some Enzymes and Proteins Regulated by Calcium or Calmodulin
Ca2+-dependent protein kinases
Ca2+-phospholipid-dependent protein kinase
Cyclic nucleotide phosphodiesterase
Some cytoskeletal proteins
Some ion channels (eg, l-type calcium channels)
Nitric oxide synthase
Phosphoprotein phosphatase 2B
Some receptors (eg, NMDA-type glutamate receptor)
In addition to its effects on enzymes and ion transport, Ca2+/calmodulin regulates the activity of many structural elements in cells. These include the actin-myosin complex of smooth muscle, which is under β-adrenergic control, and various microfilament-mediated processes in noncontractile cells, including cell motility, cell conformation changes, mitosis, granule release, and endocytosis.
Calcium Is a Mediator of Hormone Action
A role for Ca2+ in hormone action is suggested by the observations that the effect of many hormones is (1) blunted by Ca2+-free media or when intracellular calcium is depleted; (2) can be mimicked by agents that increase cytosolic Ca2+, such as the Ca2+ ionophore A23187; and (3) influences cellular calcium flux. Again, the regulation of glycogen metabolism in liver (by vasopressin and β-adrenergic catecholamines; Figures 18–6 and 18–7.)
A number of critical metabolic enzymes are regulated by Ca2+, phosphorylation, or both. These include glycogen synthase, pyruvate kinase, pyruvate carboxylase, glycerol-3-phosphate dehydrogenase, and pyruvate dehydrogenase among others (see Figure 19–1).
Phosphatidylinositide Metabolism Affects Ca2+-Dependent Hormone Action
Some signal must provide communication between the hormone receptor on the plasma membrane and the intracellular Ca2+ reservoirs. This is accomplished by products of phosphatidylinositol metabolism. Cell surface receptors such as those for acetylcholine, antidiuretic hormone, and α1-type catecholamines are, when occupied by their respective ligands, potent activators of phospholipase C. Receptor binding and activation of phospholipase C are coupled by the Gq isoforms (Table 42–3 and Figure 42–6). Phospholipase C catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate to inositol trisphosphate (IP3) and 1,2-diacylglycerol (Figure 42–7). Diacylglycerol (DAG) is itself capable of activating protein kinase C (PKC), the activity of which also depends upon Ca2+ (see Figures 21–10; 24–1, 24–2, and 55–7). IP3, by interacting with a specific intracellular receptor, is an effective releaser of Ca2+ from intracellular storage sites in the endoplasmic reticulum. Thus, the hydrolysis of phosphatidylinositol 4,5-bisphosphate leads to activation of PKC and promotes an increase of cytoplasmic Ca2+. As shown in Figure 42–4, the activation of G proteins can also have a direct action on Ca2+ channels. The resulting elevations of cytosolic Ca2+ activate Ca2+-calmodulin-dependent kinases and many other Ca2+-calmodulin-dependent enzymes.
Steroidogenic agents—including ACTH and cAMP in the adrenal cortex; angiotensin II, K+, serotonin, ACTH, and cAMP in the zona glomerulosa of the adrenal; LH in the ovary; and LH and cAMP in the Leydig cells of the testes—have been associated with increased amounts of phosphatidic acid, phosphatidylinositol, and polyphosphoinositides (see Chapter 21) in the respective target tissues. Several other examples could be cited.
The roles that Ca2+ and polyphosphoinositide breakdown products might play in hormone action are presented in Figure 42–6. In this scheme, the activated protein kinase C can phosphorylate specific substrates, which then alter physiologic processes. Likewise, the Ca2+-calmodulin complex can activate specific kinases. These then modify substrates and thereby alter physiologic responses.
Some Hormones Act Through a Protein Kinase Cascade
Single protein kinases such as PKA, PKC, and Ca2+-calmodulin (CaM)-kinases, which result in the phosphorylation of serine and threonine residues in target proteins, play a very important role in hormone action. The discovery that the EGF receptor contains an intrinsic tyrosine kinase activity that is activated by the binding of the ligand EGF was an important breakthrough. The insulin and IGF-I receptors also contain intrinsic ligand-activated tyrosine kinase activity. Several receptors—generally those involved in binding ligands involved in growth control, differentiation, and the inflammatory response—either have intrinsic tyrosine kinase activity or are associated with proteins that are tyrosine kinases. Another distinguishing feature of this class of hormone action is that these kinases preferentially phosphorylate tyrosine residues, and tyrosine phosphorylation is infrequent (<0.03% of total amino acid phosphorylation) in mammalian cells. A third distinguishing feature is that the ligand-receptor interaction that results in a tyrosine phosphorylation event initiates a cascade that may involve several protein kinases, phosphatases, and other regulatory proteins.
Insulin Transmits Signals by Several Kinase Cascades
The insulin, epidermal growth factor (EGF), and IGF-I receptors have intrinsic protein tyrosine kinase activities located in their cytoplasmic domains. These activities are stimulated when their ligands bind to the cognate receptor. The receptors are then autophosphorylated on tyrosine residues, and this initiates a complex series of events (summarized in simplified fashion in Figure 42–8). The phosphorylated insulin receptor next phosphorylates insulin receptor substrates (there are at least four of these molecules, called IRS 1–4) on tyrosine residues. Phosphorylated IRS binds to the Src homology 2 (SH2) domains of a variety of proteins that are directly involved in mediating different effects of insulin. One of these proteins, PI-3 kinase, links insulin receptor activation to insulin action through activation of a number of molecules, including the kinase PDK1 (phosphoinositide-dependent kinase-1). This enzyme propagates the signal through several other kinases, including PKB (also known as AKT), SKG, and aPKC (see legend to Figure 42–8 for definitions and expanded abbreviations). An alternative pathway downstream from PDK1 involves p70S6K and perhaps other as yet unidentified kinases. A second major pathway involves mTOR. This enzyme is directly regulated by amino acid levels and insulin and is essential for p70S6K activity. This pathway provides a distinction between the PKB and p70S6K branches downstream from PKD1. These pathways are involved in protein translocation, enzyme activity, and the regulation, by insulin, of genes involved in metabolism (Figure 42–8). Another SH2 domain-containing protein is GRB2, which binds to IRS-1 and links tyrosine phosphorylation to several proteins, the result of which is activation of a cascade of threonine and serine kinases. A pathway showing how this insulin-receptor interaction activates the mitogen-activated protein kinase (MAPK) pathway and the anabolic effects of insulin is illustrated in Figure 42–8. The exact roles of many of these docking proteins, kinases, and phosphatases remain to be established.
The Jak/STAT Pathway Is Used by Hormones and Cytokines
Tyrosine kinase activation can also initiate a phosphorylation and dephosphorylation cascade that involves the action of several other protein kinases and the counterbalancing actions of phosphatases. Two mechanisms are employed to initiate this cascade. Some hormones, such as growth hormone, prolactin, erythropoietin, and the cytokines, initiate their action by activating a tyrosine kinase, but this activity is not an integral part of the hormone receptor. The hormone-receptor interaction promotes binding and activation of cytoplasmic protein tyrosine kinases, such as Tyk-2, Jak1, or Jak2.
These kinases phosphorylate one or more cytoplasmic proteins, which then associate with other docking proteins through binding to SH2 domains. One such interaction results in the activation of a family of cytosolic proteins called STATs, or signal transducers and activators of transcription. The phosphorylated STAT protein dimerizes and translocates into the nucleus, binds to a specific DNA element such as the interferon response element (IRE), and activates transcription. This is illustrated in Figure 42–9. Other SH2 docking events may result in the activation of PI-3 kinase, the MAP kinase pathway (through SHC or GRB2), or G protein–mediated activation of phospholipase C (PLCγ) with the attendant production of diacylglycerol and activation of protein kinase C. It is apparent that there is a potential for “cross-talk” when different hormones activate these various signal transduction pathways.
Initiation of signal transduction by receptors linked to Jak kinases. The receptors (R) that bind prolactin, growth hormone, interferons, and cytokines lack endogenous tyrosine kinase. Upon ligand binding, these receptors dimerize and an associated protein (Jak1, Jak2, or TYK) is phosphorylated. Jak-P, an active kinase, phosphorylates the receptor on tyrosine residues. The STAT proteins associate with the phosphorylated receptor and then are themselves phosphorylated by Jak-P. The phosphorylated STAT protein, STAT
dimerizes, translocates to the nucleus, binds to specific DNA elements, and regulates transcription. The phosphotyrosine residues of the receptor also bind to several SH2 domain-containing proteins (X-SH2). This results in activation of the MAP kinase pathway (through SHC or GRB2), PLCγ, or PI-3 kinase.
The NF-κB Pathway Is Regulated by Glucocorticoids
The transcription factor NF-κB is a heterodimeric complex typically composed of two subunits termed p50 and p65 (Figure 42–10). Normally NF-κβ is sequestered in the cytoplasm, in a transcriptionally inactive form by members of the IκB (inhibitor of NF-κβ) family of proteins. Extracellular stimuli such as proinflammatory cytokines, reactive oxygen species, and mitogens lead to activation of the IKK (IκB kinase) complex, which is a heterohexameric structure consisting of α, β, and γ subunits. IKK phosphorylates IκB on two serine residues, and this targets IκB for polyubiquitylation and subsequent degradation by the proteasome. Following IκB degradation, free NF-κB translocates to the nucleus, where it binds to a number of gene promoters and activates transcription, particularly of genes involved in the inflammatory response. Transcriptional regulation by NF-κB is mediated by a variety of coactivators such as CREB binding protein (CBP), as described below (Figure 42–13).
Regulation of the NF-κβ pathway. NF-κβ consists of two subunits, p50 and p65, which when present in the nucleus regulate transcription of the multitude of genes important for the inflammatory response. NF-κB is restricted from entering the nucleus by IκB, an inhibitor of NF-κB. IκB binds to—and masks—the nuclear localization signal of NF-κB. This cytoplasmic protein is phosphorylated by an IKK complex which is activated by cytokines, reactive oxygen species, and mitogens. Phosphorylated IκB can be ubiquitinylated and degraded, thus releasing its hold on NF-κB, and allowing for nuclear translocation. Glucocorticoids, potent anti-inflammatory agents, are thought to affect at least three steps in this process (1, 2, 3), as described in the text.
Glucocorticoid hormones are therapeutically useful agents for the treatment of a variety of inflammatory and immune diseases. Their anti-inflammatory and immunomodulatory actions are explained in part by the inhibition of NF-κB and its subsequent actions. Evidence for three mechanisms for the inhibition of NF-κB by glucocorticoids has been described: (1) glucocorticoids increase IκB mRNA, which leads to an increase of IκB protein and more efficient sequestration of NF-κB in the cytoplasm. (2) The glucocorticoid receptor competes with NF-κB for binding to coactivators. (3) The glucocorticoid receptor directly binds to the p65 subunit of NF-κB and inhibits its activation (Figure 42–10).