Signal Generation

The Ligand-Receptor Complex Is the Signal for Group I Hormones

The lipophilic group I hormones diffuse through the plasma membrane of all cells but only encounter their specific, high-affinity intracellular receptors in target cells. These receptors can be located in the cytoplasm or in the nucleus of target cells. The hormone-receptor complex first undergoes an activation reaction. As shown in Figure 43-2, receptor activation occurs by at least two mechanisms. For example, glucocorticoids diffuse across the plasma membrane and encounter their cognate receptor in the cytoplasm of target cells. Ligand-receptor binding results in the dissociation of heat shock protein 90 (hsp90) from the receptor. This step appears to be necessary for subsequent nuclear localization of the glucocorticoid receptor. This receptor also contains nuclear localization sequences that assist in the translocation from cytoplasm to nucleus. The now activated receptor moves into the nucleus (Figure 43-2) and binds with high affinity to a specific DNA sequence called the hormone response element (HRE). In the case illustrated, this is a glucocorticoid response element, or GRE. Consensus sequences for HREs are shown in Table 43-1. The DNA-bound, lig-anded receptor serves as a high-affinity binding site for

STIMULUS

Recognition

Group I hormones

Group II hormones

Hormone«receptor complex

Many different signals

Hormone release

Signal generation

Effects

Gene transcription

Transporters Channels

Protein translocation

Protein modification

COORDINATED RESPONSE TO STIMULUS

COORDINATED RESPONSE TO STIMULUS

Figure 43-1. Hormonal involvement in responses to a stimulus. A challenge to the integrity of the organism elicits a response that includes the release of one or more hormones. These hormones generate signals at or within target cells, and these signals regulate a variety of biologic processes which provide for a coordinated response to the stimulus or challenge. See Figure 43-8 for a specific example.

one or more coactivator proteins, and accelerated gene transcription typically ensues when this occurs. By contrast, certain hormones such as the thyroid hormones and retinoids diffUse from the extracellular fluid across the plasma membrane and go directly into the nucleus. In this case, the cognate receptor is already bound to the HRE (the thyroid hormone response element [TRE], in this example). However, this DNA-bound receptor fails to activate transcription because it is com-plexed with a corepressor. Indeed, this receptor-corepressor complex serves as an active repressor of gene transcription. The association of ligand with these receptors results in dissociation of the corepressor. The liganded receptor is now capable of binding one or more coactivators with high affinity, resulting in the activation of gene transcription. The relationship of hormone receptors to other nuclear receptors and to coreg-ulators is discussed in more detail below.

By selectively affecting gene transcription and the consequent production of appropriate target mRNAs, the amounts of specific proteins are changed and metabolic processes are influenced. The influence of each of these hormones is quite specific; generally, the hormone affects less than 1% of the genes, mRNA, or proteins in a target cell; sometimes only a few are affected. The nuclear actions of steroid, thyroid, and retinoid hormones are quite well defined. Most evidence sug gests that these hormones exert their dominant effect on modulating gene transcription, but they—and many of the hormones in the other classes discussed below— can act at any step of the "information pathway" illustrated in Figure 43-3. Direct actions of steroids in the cytoplasm and on various organelles and membranes have also been described.

GROUP II (PEPTIDE & CATECHOLAMINE) HORMONES HAVE MEMBRANE RECEPTORS & USE INTRACELLULAR MESSENGERS

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 42-3 and 42-4). 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 nu-cleotide derived from ATP through the action of adenylyl cyclase; cGMP, a nucleotide formed by gua-nylyl cyclase; Ca2+; and phosphatidylinositides. Many of these second messengers affect gene transcription, as described in the previous paragraph; but they also influ-

Figure 43-2. Regulation of gene expression by class I hormones. Steroid hormones readily gain access to the cytoplasmic compartment of target cells. Glucocorticoid hormones (solid triangles) encounter their cognate receptor in the cytoplasm, where it exists in a complex with heat shock protein 90 (hsp). Ligand binding causes dissociation of hsp and a conformational change of the receptor. The receptoMigand complex then traverses the nuclear membrane and binds to DNA with specificity and high affinity at a glucocorticoid response element (GRE). This event triggers the assembly of a number of transcription coregula-tors (A), and enhanced transcription ensues. By contrast, thyroid hormones and retinoic acid (•) directly enter the nucleus, where their cognate receptors are already bound to the appropriate response elements with an associated transcription repressor complex ((3). This complex, which consists of molecules such as N-CoR or SMRT (see Table 43-6) in the absence of ligand, actively inhibits transcription. Ligand binding results in dissociation of the repressor complex from the receptor, allowing an activator complex to assemble. The gene is then actively transcribed.

ence a variety of other biologic processes, as shown in Figure 43-1.

G Protein-Coupled Receptors (GPCR)

Many of the group II hormones bind to receptors that couple to effectors through a GTP-binding protein intermediary. These receptors typically have seven hy-drophobic plasma membrane-spanning domains. This is illustrated by the seven interconnected cylinders extending through the lipid bilayer in Figure 43-4. Receptors of this class, which signal through guanine nu-cleotide-bound protein intermediates, are known as G protein-coupled receptors, or GPCRs. To date, over 130 G protein-linked receptor genes have been cloned from various mammalian species. A wide variety of responses are mediated by the GPCRs.

cAMP Is the Intracellular Signal for Many Responses

Cyclic AMP was the first intracellular signal identified in mammalian cells. Several components comprise a system for the generation, degradation, and action of cAMP.

A. Adenylyl Cyclase

Different peptide hormones can either stimulate (s) or inhibit (i) the production of cAMP from adenylyl cy-

Table 43-1. The DNA sequences of several hormone response elements (HREs).1

Hormone or Effector

Glucocorticoids Progestins Mineralocorticoids Androgens

Estrogens

Thyroid hormone Retinoic acid Vitamin D

cAMP

DNA Sequence

GRE PRE MRE ARE

RARE

VDRE

CRE TGACGTCA

betters indicate nucleotide; n means any one of the four can be used in that position. The arrows pointing in opposite directions illustrate the slightly imperfect inverted palindromes present in many HREs; in some cases these are called "half binding sites" because each binds one monomer of the receptor. The GRE, PRE, MRE, and ARE consist of the same DNA sequence. Specificity may be conferred by the intracellular concentration of the ligand or hormone receptor, by flanking DNA sequences not included in the consensus, or by other accessory elements. A second group of HREs includes those for thyroid hormones, estrogens, retinoic acid, and vitamin D. These HREs are similar except for the orientation and spacing between the half palindromes. Spacing determines the hormone specificity. VDRE (n=3), TRE (n=4), and RARE (n=5) bind to direct repeats rather than to inverted repeats. Another member of the steroid receptor superfamily, the retinoid X receptor (RXR), forms heterodimers with VDR, TR, and RARE, and these constitute the fra/is-acting factors. cAMP affects gene transcription through the CRE.

clase, which is encoded by at least nine different genes (Table 43-2). Two parallel systems, a stimulatory (s) one and an inhibitory (i) one, converge upon a single catalytic molecule (C). Each consists of a receptor, R or R;, and a regulatory complex, Gs and G;. Gs and G; are each trimers composed of a, P, and Y subunits. Because the a subunit in Gs differs from that in G;, the proteins, which are distinct gene products, are designated as and ai. The a subunits bind guanine nucleotides. The P and Y subunits are always associated (Py) and appear to function as a heterodimer. The binding of a hormone to Rs or Ri results in a receptor-mediated activation of G, which entails the exchange of GDP by GTP on a and the concomitant dissociation of Py from a.

The as protein has intrinsic GTPase activity. The active form, as^GTP, is inactivated upon hydrolysis of the GTP to GDP; the trimeric Gs complex (aPY) is then re-formed and is ready for another cycle of activation. Cholera and pertussis toxins catalyze the ADP-ribosylation of as and a;_2 (see Table 43-3), respec

NUCLEUS

CYTOPLASM

CYTOPLASM

Figure 43-3. The "information pathway." Information flows from the gene to the primary transcript to mRNA to protein. Hormones can affect any of the steps involved and can affect the rates of processing, degradation, or modification of the various products.

tively. In the case of as, this modification disrupts the intrinsic GTP-ase activity; thus, as cannot reassociate with PY and is therefore irreversibly activated. ADP-ribosylation of ai-2 prevents the dissociation of ai-2 from Py, and free a;_2 thus cannot be formed. as 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 43-3. There are 21 a, 5 P, and 8 Y subunit genes. Various combinations of these subunits provide a large number of possible aPY and cyclase complexes.

The a subunits and the PY complex have actions independent of those on adenylyl cyclase (see Figure 43-4 and Table 43-3). Some forms of a; stimulate K+ channels and inhibit Ca2+ channels, and some as molecules have the opposite effects. Members of the Gq family activate the phospholipase C group of enzymes. The PY 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 (aOLF) and vision (at). Some examples are listed in Table 43-3. GPCRs are implicated in a number of diseases and are major targets for pharmaceutical agents.

Figure 43-4. Components of the hormone receptor-G protein effector system. Receptors that couple to effectors through G proteins (GPCR) typically have seven membrane-spanning domains. In the absence of hormone (left), the heterotrimeric G-protein complex (a, P, y) is in an inactive guano-sine 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 Py subunits (wavy lines) and perhaps by myristoylated groups on a subunits (not shown). On binding of hormone to the receptor, there is a presumed conformational change of 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 a subunit, after which a and Py dissociate. The a subunit binds to and activates the effector (E). E can be adenylyl cyclase, Ca2+, Na+, or Cl- channels (as), or it could be a K+ channel (a) phospholipase CP (aq), or cGMP phosphodiesterase (at). The Py subunit can also have direct actions on E. (Modified and reproduced, with permission, from Granner DK in: Principles and Practice of Endocrinology and Metabolism, 3rd ed. Becker KL [editor]. Lippincott, 2000.)

Figure 43-4. Components of the hormone receptor-G protein effector system. Receptors that couple to effectors through G proteins (GPCR) typically have seven membrane-spanning domains. In the absence of hormone (left), the heterotrimeric G-protein complex (a, P, y) is in an inactive guano-sine 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 Py subunits (wavy lines) and perhaps by myristoylated groups on a subunits (not shown). On binding of hormone to the receptor, there is a presumed conformational change of 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 a subunit, after which a and Py dissociate. The a subunit binds to and activates the effector (E). E can be adenylyl cyclase, Ca2+, Na+, or Cl- channels (as), or it could be a K+ channel (a) phospholipase CP (aq), or cGMP phosphodiesterase (at). The Py subunit can also have direct actions on E. (Modified and reproduced, with permission, from Granner DK in: Principles and Practice of Endocrinology and Metabolism, 3rd ed. Becker KL [editor]. Lippincott, 2000.)

Table 4S-2. Subclassification of group II.A hormones.

Hormones That Stimulate

Hormones That Inhibit

Adenylyl Cyclase

Adenylyl Cyclase

(Hs)

(H)

ACTH

Acetylcholine

ADH

a2-Adrenergics

P-Adrenergics

Angiotensin II

Calcitonin

Somatostatin

CRH

FSH

Glucagon

hCG

LH

LPH

MSH

PTH

TSH

In prokaryotic cells, cAMP binds to a specific protein called catabolite regulatory protein (CRP) that binds directly to DNA and influences gene expression. In eu-karyotic cells, cAMP binds to a protein kinase called protein kinase A (PKA) that is a heterotetrameric molecule consisting of two regulatory subunits (R) and two catalytic subunits (C). cAMP binding results in the following reaction:

The R2C2 complex has no enzymatic activity, but the binding of cAMP by R dissociates R from C, thereby activating the latter (Figure 43-5). The active C subunit catalyzes the transfer of the Y phosphate of ATP to a serine or threonine residue in a variety of proteins. The consensus phosphorylation sites are -Arg-Arg/Lys-X-Ser/Thr- and -Arg-Lys-X-X-Ser-, where X can be any amino acid.

Protein kinase activities were originally described as being "cAMP-dependent" or "cAMP-independent." This

Table 43-3. Classes and functions of selected G proteins.

Table 43-3. Classes and functions of selected G proteins.

Class or Type

Stimulus

Effector

Effect

Gs as

Glucagon, ß-adrenergics

T Adenylyl cyclase

T Cardiac Ca2+, Cl-, and Na+ channels

Gluconeogenesis, lipolysis, glycogenolysis

aolf

Odorant

T Adenylyl cyclase

Olfaction

(^-adrenergics M2 cholinergics

i Adenylyl cyclase T Potassium channels i Calcium channels

Slowed heart rate

a0

Opioids, endorphins

T Potassium channels

Neuronal electrical activity

at

Light

T cGMP phosphodiesterase

Vision

Gq aq an

M, cholinergics ^-Adrenergics a,-Adrenergics

T Phospholipase C-pi T Phospholipase c-p2

T Muscle contraction and

T Blood pressure

Gl2 ai2

?

Cl- channel

?

'Modified and reproduced, with permission, from Granner DK in: Principles and Practice of Endocrinology and Metabolism, 3rd ed. Becker KL (editor). Lippincott, 2000.

2The four major classes or families of mammalian G proteins (Gs, G|, Gq, and G12) are based on protein sequence homology. Representative members of each are shown, along with known stimuli, effectors, and well-defined biologic effects. Nine iso-forms of adenylyl cyclase have been identified (isoforms I-IX). All isoforms are stimulated by as; ai isoforms inhibit types V and VI, and a0 inhibits types I and V. At least 16 different a subunits have been identified.

classification has changed, as protein phosphorylation is now recognized as being a major regulatory mechanism. Several hundred protein kinases have now been described. The 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, au-tophosphorylation, Km for ATP, and substrate specificity.

C. Phosphoproteins

The effects of cAMP in eukaryotic cells are all thought to be mediated by protein phosphorylation-dephosphor-ylation, 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. These substrates help 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 the protein cyclic AMP response ele ment binding protein (CREB). CREB binds to a cAMP responsive element (CRE) (see Table 43-1) 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.

D. Phosphodiesterases

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 43-5). 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 phospho-diesterase 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.

Active adenylyl cyclase

Cell membrane

Phosphatase

Physiologic effects

Figure 43-5. 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 and two catalytic subunits. The cAMP generated by the action of adenylyl cyclase (activated as shown in Figure 43-4) binds to the regulatory (R) 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.

heat-stable protein inhibitors regulate type I phosphatase activity. Inhibitor-1 is phosphorylated and activated by cAMP-dependent protein kinases; and in-hibitor-2, which may be a subunit of the inactive phosphatase, is also phosphorylated, possibly by glycogen synthase kinase-3.

cGMP Is Also an Intracellular Signal

Cyclic GMP is made from GTP by the enzyme gua-nylyl cyclase, which exists in soluble and membrane-bound forms. Each of these isozymes 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 re

Active adenylyl cyclase

+ R2

Mg2+ • ATP Protein Phosphoprotein

E. Phosphoprotein Phosphatases

Given the importance of protein phosphorylation, it is not surprising that regulation of the protein dephos-phorylation reaction is another important control mechanism (see Figure 43-5). 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. The best-studied role of regulation by the dephosphorylation of proteins is that of glycogen metabolism in muscle. Two major types of phosphoserine-phosphothreonine phosphatases have been described. Type I preferentially dephosphorylates the P subunit of phosphorylase kinase, whereas type II dephosphorylates the a subunit. Type I phosphatase is implicated in the regulation of glycogen synthase, phos-phorylase, 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 laxation 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 is an important regulator of a variety of cellular processes, including muscle contraction, stimulus-secretion coupling, the blood clotting cascade, enzyme activity, and membrane excitability. It is also an intracellular messenger of hormone action.

A. Calcium Metabolism

The extracellular calcium (Ca2+) concentration is about 5 mmol/L and is very rigidly controlled. Although substantial amounts of calcium are associated with intracel-lular organelles such as mitochondria and the endoplas-mic reticulum, the intracellular concentration of free or ionized calcium (Ca2+) is very low: 0.05-10 |mol/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-depen-dent 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 cy-tosol to the lumen of the endoplasmic reticulum. There are three ways of changing cytosolic Ca2+: (1) Certain hormones (class II.C, Table 42-3) 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 reticu-lum, 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.

B. Calmodulin

The calcium-dependent regulatory protein is calmod-ulin, 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 calmod-ulin (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 43-4.

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 P-adrenergic control, and various microfilament-medi-ated processes in noncontractile cells, including cell motility, cell conformation changes, mitosis, granule release, and endocytosis.

C. 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. The regulation of glycogen metabolism in liver by vaso-pressin and a-adrenergic catecholamines provides a good example. This is shown schematically in Figures 18-6 and 18-7.

Table43-4. Enzymes and proteins regulated by calcium or calmodulin.

Adenylyl cyclase Ca2+-dependent protein kinases Ca2+-Mg2+ ATPase

Ca2+-phospholipid-dependent protein kinase

Cyclic nucleotide phosphodiesterase

Some cytoskeletal proteins

Some ion channels (eg, l-type calcium channels)

Nitric oxide synthase

Phosphorylase kinase

Phosphoprotein phosphatase 2B

Some receptors (eg, NMDA-type glutamate receptor)

A number of critical metabolic enzymes are regulated by Ca2+, phosphorylation, or both, including glycogen synthase, pyruvate kinase, pyruvate carboxylase, glycerol-3-phosphate dehydrogenase, and pyruvate dehydrogenase.

D. Phosphatidylinositide Metabolism Affects Ca2+-Dependent Hormone Action

Some signal must provide communication between the hormone receptor on the plasma membrane and the in-tracellular Ca2+ reservoirs. This is accomplished by products of phosphatidylinositol metabolism. Cell sur face receptors such as those for acetylcholine, antidiuretic hormone, and artype 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 43-3 and Figure 43-6). Phospholipase C catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate to inositol trisphosphate (IP3) and 1,2-diacylglycerol (Figure 43-7). Diacylglycerol is itself capable of activating protein kinase C (PKC), the activity of which also depends upon Ca2+. IP3, by interacting with a specific intracellular receptor, is an effective releaser of Ca2+ from

Ca2+

Ca2+

Figure 43-6. Certain hormone-receptor interactions result in the activation of phospholipase C. This appears to involve a specific G protein, which also may activate a calcium channel. Phospholipase C results in generation of 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. (Courtesy of JH Exton.)

Figure 43-6. Certain hormone-receptor interactions result in the activation of phospholipase C. This appears to involve a specific G protein, which also may activate a calcium channel. Phospholipase C results in generation of 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. (Courtesy of JH Exton.)

Phosphatidylinositol 4,5-bisphosphate

1,2-Diacylglycerol

Figure 43-7. Phospholipase C cleaves PIP2 into diacylglycerol and inositol trisphosphate. R, generally is stearate, and R2 is usually arachido-nate. IP3 can be dephosphorylated (to the inactive I-1,4-P2) or phosphorylated (to the potentially active I-1,3,4,5-P4).

Phosphatidylinositol 4,5-bisphosphate

1,2-Diacylglycerol

Inositol 1,4,5-trisphosphate

Inositol 1,4,5-trisphosphate

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 43-4, the activation of G proteins can also have a direct action on Ca2+ channels. The resulting elevations of cy-tosolic 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, phosphatidyl-inositol, and polyphosphoinositides (see Chapter 14) 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 43-6. 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 phos-phorylation 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 recep-tors—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.

A. 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 the receptor binds ligand. The receptors are then autophosphorylated on tyrosine residues, and this initiates a complex series of events (summarized in simplified fashion in Figure 43-8). 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 (phospho-inositide-dependent kinase-1). This enzyme propagates the signal through several other kinases, including PKB (akt), SKG, and aPKC (see legend to Figure 43-8 for definitions and expanded abbreviations). An alternative

RECOGNITION (HYPERGLYCEMIA)

SIGNAL GENERATION

SIGNAL GENERATION

Protein translocation Enzyme activity

Gene transcription

EFFECTS

Glucose transporter Insulin receptor IGF-II receptor

+

p21Ras

Raf-1

MEK

MAP

kinase

I

Cell growth DNA synthesis Early response genes

Gene transcription

Glucose transporter Insulin receptor IGF-II receptor

Insulin receptor

PEPCK

HKII

Protein phosphatases

Glucagon

Glucokinase

Phosphodiesterases*

IGFBP1

> 100 others

Others

Cell growth DNA synthesis Early response genes

Figure 43-8. Insulin signaling pathways. The insulin signaling pathways provide an excellent example of the "recognition ^ hormone release ^ signal generation ^ effects" paradigm outlined in Figure 43-1. Insulin is released in response to hyperglycemia. Binding of insulin to a target cell-specific plasma membrane receptor results in a cascade of intracellular events. Stimulation of the intrinsic tyrosine kinase activity of the insulin receptor marks the initial event, resulting in increased tyrosine (Y) phosphorylation (Y ^ Y-P) of the receptor and then one or more of the insulin receptor substrate molecules (IRS 1-4). This increase in phosphotyrosine stimulates the activity of many intracellular molecules such as GTPases, protein kinases, and lipid kinases, all of which play a role in certain metabolic actions of insulin. The two best-described pathways are shown. First, phosphorylation of an IRS molecule (probably IRS-2) results in docking and activation of the lipid kinase, PI-3 kinase, which generates novel inositol lipids that may act as "second messenger" molecules. These, in turn, activate PDK1 and then a variety of downstream signaling molecules, including protein kinase B (PKB or akt), SGK, and aPKC. An alternative pathway involves the activation of p70S6K and perhaps other as yet unidentified kinases. Second, 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 several 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 physiologic processes, as shown. Each of the phosphorylation events is reversible through the action of specific phosphatases. For 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. (IGFBP, insulin-like growth factor binding protein; IRS 1-4, insulin receptor substrate isoforms 1-4); PI-3 kinase, phos-phatidylinositol 3-kinase; PTEN, phosphatase and tensin homolog deleted on chromosome 10; PKD1, phosphoinosi-tide-dependent kinase; PKB, protein kinase B; SGK, serum and glucocorticoid-regulated kinase; aPKC, atypical protein kinase C; p70S6K, p70 ribosomal protein S6 kinase; mTOR, mammalian target of rapamycin; GRB2, growth factor receptor binding protein 2; mSOS, mammalian son of sevenless; MEK, MAP kinase kinase and ERK kinase; MAP kinase, mitogen-activated protein kinase.)

pathway downstream from PKD1 involves p70S6K and perhaps other as yet unidentified kinases. A second major pathway involves mTOR. This enzyme is directly regulated by amino acids 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 43-8). 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 (MAP) kinase pathway and the anabolic effects of insulin is illustrated in Figure 43-8. The exact roles of many of these docking proteins, kinases, and phosphatases remain to be established.

B. The Jak/STAT Pathway Is Used by Hormones and Cytokines

Tyrosine kinase activation can also initiate a phosphor-ylation and dephosphorylation cascade that involves the action of several other protein kinases and the counter balancing 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 tyro-sine 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 cyto-plasmic 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 signal transducers and activators of transcription (STATs). The phosphorylated STAT protein dimerizes and translocates into the nucleus, binds to a specific DNA element such as the interferon response element, and activates transcription. This is illustrated in Figure 43-9. 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 (PLCy) 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.

x = SHC Dimerization

GRB2 and

PLCy nuclear

PI-3K translocation

x = SHC Dimerization

GRB2 and

PLCy nuclear

PI-3K translocation

Figure 43-9. 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 (Jakl, 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. 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. This results in activation of the MAP kinase pathway (through SHC or GRB2), PLCy, or PI-3 kinase.

C. The NF-kB Pathway Is Regulated by Glucocorticoids

The transcription factor NF-kB is a heterodimeric complex typically composed of two subunits termed p50 and p65 (Figure 43-10). Normally, NF-kB is kept sequestered in the cytoplasm in a transcriptionally inactive form by members of the inhibitor of NF-kB (IkB) family. Extracellular stimuli such as proinflammatory cytokines, reactive oxygen species, and mitogens lead to activation of the IkB kinase complex, IKK, which is a heterohexameric structure consisting of a, P, and Y sub-units. IKK phosphorylates IkB on two serine residues, and this targets IkB for ubiquitination and subsequent degradation by the proteasome. Following IkB degradation, free NF-kB can now translocate 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-kB is mediated by a variety of coactivators such as CREB binding protein (CBP), as described below (Figure 43-13).

Glucocorticoid hormones are therapeutically useful agents for the treatment of a variety of inflammatory and immune diseases. Their anti-inflammatory and im-munomodulatory actions are explained in part by the inhibition of NF-kB and its subsequent actions. Evidence for three mechanisms for the inhibition of NF-kB by glucocorticoids has been presented: (1) Glucocorticoids increase IkB mRNA, which leads to an increase of IkB protein and more efficient sequestration of NF-kB in the cytoplasm. (2) The glucocorticoid receptor competes with NF-kB for binding to coactivators. (3) The glucocorticoid receptor directly binds to the p65 subunit of NF-kB and inhibits its activation (Figure 43-10).

Diabetes 2

Diabetes 2

Diabetes is a disease that affects the way your body uses food. Normally, your body converts sugars, starches and other foods into a form of sugar called glucose. Your body uses glucose for fuel. The cells receive the glucose through the bloodstream. They then use insulin a hormone made by the pancreas to absorb the glucose, convert it into energy, and either use it or store it for later use. Learn more...

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