CELL SIGNALING AND SIGNAL TRANSDUCTION::

CELL SIGNALING AND SIGNAL TRANSDUCTION::
• Conversion of information into a chemical change, signal transduction, is a universal property of living cells.


MOLECULAR MECHANISMS OF SIGNAL TRANSDUCTION::
• Signal transductions are remarkably specific and exquisitely sensitive. Specificity is achieved by precise molecular complementarity between the signal and receptor molecules, mediated by the same kinds of weak (noncovalent) forces that mediate enzyme-substrate and antigen-antibody interactions.

 Thyrotropin-releasing hormone, for example, triggers responses in the cells of the anterior pituitary but not in hepatocytes, which lack receptors for this hormone.
 Epinephrine alters glycogen metabolism in hepatocytes but not in erythrocytes; in this case, both cell types have receptors for the hormone, but whereas hepatocytes contain glycogen and the glycogen-metabolizing enzyme that is stimulated by epinephrine, erythrocytes contain neither.

• Three factors account for the extraordinary sensitivity of signal transducers: the high affinity of receptors for signal molecules, cooperativity (often but not always) in the ligand-receptor interaction, and amplification of the signal by enzyme cascades.

o The affinity between signal (ligand) and receptor can be expressed as the dissociation constant Kd, usually 10-10 M or less—meaning that the receptor detects picomolar concentrations of a signal molecule.
 Receptor-ligand interactions are quantified by Scatchard analysis, which yields a quantitative measure of affinity (Kd) and the number of ligand-binding sites in a receptor sample
o Cooperativity in receptor-ligand interactions results in large changes in receptor activation with small changes in ligand concentration (recall the effect of cooperativity on oxygen binding to hemoglobin).
o Amplification by enzyme cascades results when an enzyme associated with a signal receptor is activated and, in turn, catalyzes the activation of many molecules of a second enzyme.

 When a signal is present continuously, desensitization of the receptor system results (Fig. 12–1c); when the stimulus falls below a certain threshold, the system again becomes sensitive. Think of what happens to your visual transduction system when you walk from bright sunlight into a darkened room or from darkness into the light.
o A final noteworthy feature of signal-transducing systems is integration, the ability of the system to receive multiple signals and produce a unified response appropriate to the needs of the cell or organism.


EXAMPLES OF EACH OF SIX BASIC SIGNALING MECHANISMS
1. Gated ion channels of the plasma membrane that open and close (hence the term “gating”) in response to the binding of chemical ligands or changes in transmembrane potential. These are the simplest signal transducers. The acetylcholine receptor ion channel is an example of this mechanism.

2. Receptor enzymes, plasma membrane receptors that are also enzymes. When one of these receptors is activated by its extracellular ligand, it catalyzes the production of an intracellular second messenger. An example is the insulin receptor (Section 12.3).

3. Receptor proteins (serpentine receptors) that indirectly activate (through GTP-binding proteins or G proteins) enzymes that generate intracellular second messengers. This is illustrated by the β-adrenergic receptor system that detects epinephrine (adrenaline) (Section 12.4).
4. Nuclear receptors (steroid receptors) that, when bound to their specific ligand (such as the hormone estrogen), alter the rate at which specific genes are transcribed and translated into cellular proteins. Because steroid hormones function through mechanisms intimately related to the regulation of gene expression.

5. Receptors that lack enzymatic activity but attract and activate cytoplasmic enzymes that act on downstream proteins, either by directly converting them to gene-regulating proteins or by activating a cascade of enzymes that finally activates a gene regulator. The JAK-STAT system exemplifies the first mechanism (Section 12.3); and the TLR4 (Toll) signaling system in humans, the second (Section 12.6).
6. Receptors (adhesion receptors) that interact with macromolecular components of the extracellular matrix (such as collagen) and convey to the cytoskeletal system instructions on cell migration or adherence to the matrix. Integrins illustrate this general type of transduction mechanism.












12.2 Gated Ion Channels
1.Ion Channels Underlie Electrical Signaling in Excitable Cells::
The excitability of sensory cells, neurons, and myocytes depends on ion channels, signal transducers that provide a regulated path for the movement of inorganic ions such as Na+, K+, Ca2+, and Cl-across the plasma membrane in response to various stimuli.

The Na+K+ ATPase creates a charge imbalance across the plasma membrane by carrying 3 Na+out of the cell for every 2 K+ carried in (Fig. 12–3a), making the inside negative relative to the outside. The membrane is said to be polarized. By convention, Vm is negative when the inside of the cell is negative relative to the outside. For a typical animal cell, Vm = -60 to -70 mV.

Because ion channels generally allow passage of either anions or cations but not both, ion flux through a channel causes a redistribution of charge on the two sides of the membrane, changing Vm.

Influx of a positively charged ion such as Na+, or efflux of a negatively charged ion such as Cl- ,depolarizes the membrane and brings Vm closer to zero. Conversely, efflux of K+ hyperpolarizes the membrane and Vm becomes more negative. These ion fluxes through channels are passive, in contrast to active transport by the Na+K+ ATPase.

The force (∆G) that causes a cation (say, Na+) to pass spontaneously inward through an ion channel is a function of the ratio of its concentrations on the two sides of the membrane (Cin/Cout) and of the difference in electrical potential (∆Ψor Vm):



At this equilibrium potential (E), the driving force (∆G) tending to move an ion is zero.
The equilibrium potential is different for each ionic species because the concentration gradients differ for each ion.

In most cells at rest, more K+ channels than Na+, Cl-, or Ca2+ channels are open and thus the resting potential is closer to the E for K+ (-98 mV) than that for any other ion.


The precisely timed opening and closing of ion channels and the resulting transient changes in membrane potential underlie the electrical signaling by which the nervous system stimulates the skeletal muscles to contract, the heart to beat, or secretory cells to release their contents. Moreover, many hormones exert their effects by altering the membrane potentials of their target cells.

2.THE NICOTINIC ACETYLCHOLINE RECEPTOR IS A LIGAND-GATED ION CHANNEL::
One of the best-understood examples of a ligand-gated receptor channel is the nicotinic acetylcholine receptor.
The receptor channel opens in response to the neurotransmitter acetylcholine (and to nicotine, hence the name). This receptor is found in the postsynaptic membrane of neurons at certain synapses and in muscle fibers (myocytes) at neuromuscular junctions.


Acetylcholine released by an excited neuron diffuses a few micrometers across the synaptic cleft or neuromuscular junction to the postsynaptic neuron or myocyte, where it interacts with the acetylcholine receptor and triggers electrical excitation (depolarization) of the receiving cell. The acetylcholine receptor is an allosteric protein with two high-affinity binding sites for acetylcholine, about 3.0 nm from the ion gate, on the two α subunits.

Either Na+or Ca2+ can now pass, and the inward flux of these ions depolarizes the plasma membrane, initiating subsequent events that vary with the type of tissue.
In a postsynaptic neuron, depolarization initiates an action potential(electrical impulse) ; at a neuromuscular junction, depolarization of the muscle fiber triggers muscle contraction

Normally, the acetylcholine concentration in the synaptic cleft is quickly lowered by the enzyme acetylcholinesterase, present in the cleft.





3. VOLTAGE-GATED ION CHANNELS PRODUCE NEURONAL ACTION POTENTIALS::

Signaling in the nervous system is accomplished by networks of neurons, specialized cells that carry an electrical impulse (action potential) from one end of the cell (the cell body) through an elongated cytoplasmic extension (the axon).

The electrical signal triggers release of neurotransmitter molecules at the synapse, carrying the signal to the next cell in the circuit.
o Three types of voltage-gated ion channels are essential to this signaling mechanism.

Three types of voltage-gated ion channels are essential to this signaling mechanism.
Along the entire length of the axon are voltage-gated Na+channels (Fig. 12–5; see also Fig. 11–50), which are closed when the membrane is at rest (Vm = -60 mV) but open briefly when the membrane is depolarized locally in response to acetylcholine (or some other neurotransmitter).

The depolarization induced by the opening of Na+ channels causes voltage-gated K+ channels to open, and the resulting efflux of K+ repolarizes the membrane locally.

A brief pulse of depolarization traverses the axon as local depolarization triggers the brief opening of neighboring Na+channels, then K+ channels. After each opening of a Na+ channel, a short refractory period follows during which that channel cannot open again, and thus a unidirectional wave of depolarization sweeps from the nerve cell body toward the end of the axon.

The voltage sensitivity of ion channels is due to the presence at critical positions in the channel protein of charged amino acid side chains that interact with the electric field across the membrane. Changes in transmembrane potential produce subtle conformational changes in the channel protein.

At the distal tip of the axon are voltage-gated Ca2+ channels. When the wave of depolarization reaches these channels, they open, and Ca2+ enters from the extracellular space. The rise in cytoplasmic [Ca2+] then triggers release of acetylcholine by exocytosis into the synaptic cleft.
Acetylcholine diffuses to the postsynaptic cell where it binds to acetylcholine receptors and triggers depolarization. Thus the message is passed to the next cell in the circuit

 We see, then, that gated ion channels convey signals in either of two ways: by changing the cytosolic concentration of an ion (such as Ca2+), which then serves as an intracellular second messenger (the hormone or neurotransmitter is the first messenger), or by changing Vm and affecting other membrane proteins that are sensitive to Vm. The passage of an electrical signal through one neuron and on to the next illustrates both types of mechanism.




FIGURE 12–5 Role of voltage-gated and ligand-gated ion channels in neural transmission. Initially, the plasma membrane of the presynaptic neuron is polarized (inside negative) through the action of the electrogenic Na+K+ ATPase, which pumps 3 Na+ out for every 2 K+pumped into the neuron . 1 A stimulus to this neuron causes an action potential to move along the axon (white arrow), away from the cell body. The opening of one voltage-gated Na+ channel allows Na+entry, and the resulting local depolarization causes the adjacent Na+ channel to open, and so on. The directionality of movement of the action potential is ensured by the brief refractory period that follows the opening of each voltage-gated Na+ channel. 2 When the wave of depolarization reaches the axon tip, voltagegated Ca2+ channels open, allowing Ca2+ entry into the presynaptic neuron. 3 The resulting increase in internal [Ca2+] triggers exocytic release of the neurotransmitter acetylcholine into the synaptic cleft. 4 Acetylcholine binds to a receptor on the postsynaptic neuron, causing its ligand-gated ion channel to open. 5 Extracellular Na+ and Ca2+ enter through this channel, depolarizing the postsynaptic cell. The electrical signal has thus passed to the cell body of the postsynaptic neuron and will move along its axon to a third neuron by this same sequence of events.

4.Neurons Have Receptor Channels That Respond to Different Neurotransmitters::
Animal cells, especially those of the nervous system, contain a variety of ion channels gated by ligands, voltage, or both.
The neurotransmitters 5-hydroxytryptamine (serotonin), glutamate, and glycine can all act through receptor channels that are structurally related to the acetylcholine receptor.
 Serotonin and glutamate trigger the opening of cation (K+, Na+, and Ca2+) channels, whereas glycine opens Cl- specific channels.

Cation and anion channels are distinguished by subtle differences in the amino acid residues that line the hydrophilic channel.

Cation channels have negatively charged Glu and Asp side chains at crucial positions. When a few of these acidic residues are experimentally replaced with basic residues, the cation channel is converted to an anion channel.

The receptor channels for acetylcholine, glycine, glutamate, and γ-aminobutyric acid (GABA) are gated by extracellular ligands.

Intracellular second messengers— such as cAMP, cGMP (3', 5’-cyclic GMP, a close analog of cAMP), IP3 (inositol 1, 4, 5-trisphosphate), Ca2+, and ATP—regulate ion channels of another class.


12.3 RECEPTOR ENZYMES::
These proteins have a ligand-binding domain on the extracellular surface of the plasma membrane and an enzyme active site on the cytosolic side, with the two domains connected by a single transmembrane segment.
 Commonly, the receptor enzyme is a protein kinase that phosphorylates Tyr residues in specific target proteins; the insulin receptor is the prototype for this group.
 In plants, the protein kinase of receptors is specific for Ser or Thr residues.

1. THE INSULIN RECEPTOR IS A TYROSINE-SPECIFIC PROTEIN KINASE::
Insulin regulates both metabolism and gene expression:

The insulin signal passes from the plasma membrane receptor to insulin-sensitive metabolic enzymes and to the nucleus, where it stimulates the transcription of specific genes.

The active insulin receptor consists of two identical α chains protruding from the outer face of the plasma membrane and two transmembrane β subunits with their carboxyl termini protruding into the cytosol.

FIGURE 12–6 Regulation of gene expression by insulin. The insulin receptor consists of two α chains on the outer face of the plasma membrane and two β chains that traverse the membrane and protrude from the cytoplasmic face. Binding of insulin to the α chains triggers a conformational change that allows the autophosphorylation of Tyr residues in the carboxyl-terminal domain of the β subunits. Autophosphorylation further activates the Tyr kinase domain, which then catalyzes phosphorylation of other target proteins. The signaling pathway by which insulin regulates the expression of specific genes consists of a cascade of protein kinases, each of which activates the next. The insulin receptor is a Tyr-specific kinase; the other kinases (all shown in blue) phosphorylate Ser or Thr residues. MEK is a dual-specificity kinase, which phosphorylates both a Thr and a Tyr residue in ERK (extracellular regulated kinase); MEK is mitogen-activated, ERK-activating kinase; SRF is serum response factor. Abbreviations for other components are explained in the text.

• step 1 ). The α chains contain the insulinbinding domain, and the intracellular domains of the β chains contain the protein kinase activity that transfers a phosphoryl group from ATP to the hydroxyl group of Tyr residues in specific target proteins One of these target proteins (Fig. 12–6, step 2 ) is insulin receptor substrate-1 (IRS-1). Once phosphorylated on its Tyr residues, IRS-1 becomes the point of nucleation for a complex of proteins

• (step 3 ) that carry the message from the insulin receptor to end targets in the cytosol and nucleus, through a long series of intermediate proteins. First, a P –Tyr residue in IRS-1 is bound by the SH2 domain of the protein Grb2. (SH2 is an abbreviation of Src homology 2; the sequences of SH2 domains are similar to a domain in another protein Tyr kinase, Src (pronounced sark).) Grb2 also contains a second protein-binding domain, SH3, that binds to regions rich in Pro residues. Grb2 binds to a proline-rich region of Sos, recruiting Sos to the growing receptor complex.

• When bound to Grb2, Sos catalyzes the replacement of bound GDP by GTP on Ras, one of a family of guanosine nucleotide–binding proteins (G proteins) that mediate a wide variety of signal transductions (Section 12.4).

• When GTP is bound, Ras can activate a protein kinase, Raf-1 (step 4 ), the first of three protein kinases—Raf-1, MEK, and ERK—that form a cascade in which each kinase activates the next by phosphorylation.

• (step 5 ). The protein kinase ERK is activated by phosphorylation of both a Thr and a Tyr residue. When activated, it mediates some of the biological effects of insulin by entering the nucleus and phosphorylating proteins such as Elk1, which modulates the transcription of about 100 insulin-regulated genes (step 6 ).

• The proteins Raf-1, MEK, and ERK are members of three larger families, for which several nomenclatures are employed. ERK is a member of the MAPK family (mitogen-activated protein kinases; mitogens are signals that act from outside the cell to induce mitosis and cell division).

ACTIVATION OF GLYCOGEN SYNTHASE BY INSULIN::


 The enzyme phosphoinositide 3- kinase (PI-3K) binds IRS-1 through the former’s SH2 domain.
 Thus activated, PI-3K converts the membrane lipid phosphatidylinositol 4,5-bisphosphate (see Fig. ), also called PIP2, to phosphatidylinositol 3,4,5-trisphosphate (PIP3).
 When bound to PIP3, protein kinase B (PKB) is phosphorylated and activated by yet another protein kinase, PDK1.
 The activated PKB then phosphorylates Ser or Thr residues on its target proteins, one of which is glycogen synthase kinase 3 (GSK3).

 In its active, nonphosphorylated form, GSK3 phosphorylates glycogen synthase, inactivating it and thereby contributing to the slowing of glycogen synthesis.
 When phosphorylated by PKB, GSK3 is inactivated. By thus preventing inactivation of glycogen synthase in liver and muscle, the cascade of protein phosphorylations initiated by insulin stimulates glycogen synthesis.
 In muscle, PKB triggers the movement of glucose transporters (GLUT4) from internal vesicles to the plasma membrane, stimulating glucose uptake from the blood.

 PKB also functions in several other signaling pathways, including that triggered by ∆9-tetrahydrocannabinol (THC), the active ingredient of marijuana and hashish. THC activates the CB1 receptor in the plasma membrane of neurons in the brain, triggering a signaling cascade that involves MAPKs.
 One consequence of CB1 activation is the stimulation of appetite, one of the well-established effects of marijuana use.

 The insulin receptor is the prototype for a number of receptor enzymes with a similar structure and receptor Tyr kinase activity.

 The receptors for epidermal growth factor and platelet-derived growth factor, for example, have structural and sequence similarities to the insulin receptor, and both have a protein Tyr kinase activity that phosphorylates IRS-1.



The JAK-STAT transduction mechanism for the erythropoietin receptor

FIGURE 12–9 The JAK-STAT transduction mechanism for the erythropoietin receptor. Binding of erythropoietin (EPO) causes dimerization of the EPO receptor, which allows the soluble Tyr kinase JAK to bind to the internal domain of the receptor and phosphorylate it on several Tyr residues. The STAT protein STAT5 contains an SH2 domain and binds to the P –Tyr residues on the receptor, bringing the receptor into proximity with JAK. Phosphorylation of STAT5 by JAK allows two STAT molecules to dimerize, each binding the other’s P –Tyr residue. Dimerization of STAT5 exposes a nuclear localization sequence (NLS) that targets STAT5 for transport into the nucleus. In the nucleus, STAT causes the expression of genes controlled by EPO. A second signaling pathway is also triggered by autophosphorylation of JAK that is associated with EPO binding to its receptor. The adaptor protein Grb2 binds P –Tyr in JAK and triggers the MAPK cascade, as in the insulin system (see Fig. 12–6).

 One example is the system that regulates the formation of erythrocytes in mammals.
 The cytokine (developmental signal) for this system is erythropoietin (EPO), a 165 amino acid protein produced in the kidneys.
 When EPO binds to its plasma membrane receptor (Fig. 12–9), the receptor dimerizes and can now bind the soluble protein kinase JAK (Janus kinase).

 This JAK-STAT system operates in a number of other signaling pathways, including that for the hormone leptin.
 Activated JAK can also trigger, through Grb2, the MAPK cascade, which leads to altered expression of specific genes.
 Src is another soluble protein Tyr kinase that associates with certain receptors when they bind their ligands.
 Src was the first protein found to have the characteristic P –Tyr-binding domain that was subsequently named the Src homology (SH2) domain.
 Yet another example of a receptor’s association with a soluble protein kinase is the Toll-like receptor (TLR4) system through which mammals detect the bacterial lipopolysaccharide (LPS), a potent toxin.

Receptor Guanylyl Cyclases Generate the Second Messenger cGMP::
Guanylyl cyclases are another type of receptor enzyme. When activated, a guanylyl cyclase produces guanosine 3',5'-cyclic monophosphate (cyclic GMP, cGMP) from GTP:


FIGURE 12–10 Two types (isozymes) of guanylyl cyclase that participate in signal transduction. (a) One isozyme exists in two similar membrane-spanning forms that are activated by their extracellular ligands: Atrial natriuretic factor, ANF (receptors in cells of the renal collecting ducts and the smooth muscle of blood vessels), and guanylin (receptors in intestinal epithelial cells). The guanylin receptor is also the target of a type of bacterial endotoxin that triggers severe diarrhea.
(b) The other isozyme is a soluble enzyme that is activated by intracellular nitric oxide (NO); this form is found in many tissues, including smooth muscle of the heart and blood vessels.

Cyclic GMP is a second messenger that carries different messages in different tissues. In the kidney and intestine it triggers changes in ion transport and water retention; in cardiac muscle (a type of smooth muscle) it signals relaxation; in the brain it may be involved both in development and in adult brain function.
Guanylyl cyclase in the kidney is activated by the hormone atrial natriuretic factor (ANF), which is released by cells in the atrium of the heart when the heart is stretched by increased blood volume. Carried in the blood to the kidney, ANF activates guanylyl cyclase in cells of the collecting ducts.

The resulting rise in [cGMP] triggers increased renal excretion of Na+ and, consequently, of water, driven by the change in osmotic pressure. Water loss reduces the blood volume, countering the stimulus that initially led to ANF secretion.

Vascular smooth muscle also has an ANF receptor— guanylyl cyclase; on binding to this receptor, ANF causes relaxation (vasodilation) of the blood vessel, which increases blood flow while decreasing blood pressure.
A similar receptor guanylyl cyclase in the plasma membrane of intestinal epithelial cells is activated by an intestinal peptide, guanylin, which regulates Cl- secretion in the intestine. This receptor is also the target of a heat-stable peptide endotoxin produced by Escherichia coli and other gram-negative bacteria.
The elevation in [cGMP] caused by the endotoxin increases Cl- secretion and consequently decreases reabsorption of water by the intestinal epithelium, producing diarrhea.

 A distinctly different type of guanylyl cyclase is a cytosolic protein with a tightly associated heme group , an enzyme activated by nitric oxide (NO).
 Nitric oxide is produced from arginine by Ca2+ dependent NO synthase, present in many mammalian tissues, and diffuses from its cell of origin into nearby cells.
 NO is sufficiently nonpolar to cross plasma membranes without a carrier. In the target cell, it binds to the heme group of guanylyl cyclase and activates Cgmp production.
 In the heart, cGMP reduces the forcefulness of contractions by stimulating the ion pump(s) that expel Ca2+ from the cytosol.
 Nitric oxide (NO) is a short-lived messenger that acts by stimulating a soluble guanylyl cyclase, raising [cGMP] and stimulating PKG.

Most of the actions of cGMP in animals are believed to be mediated by cGMP-dependent protein kinase, also called protein kinase G or PKG, which, when activated by cGMP, phosphorylates Ser and Thr residues in target proteins.



12.4 G Protein–Coupled Receptors and Second Messengers::
The β-adrenergic receptor, which mediates the effects of epinephrine on many tissues, is the prototype for this type of transducing system.

The β-Adrenergic Receptor System Acts through the Second Messenger cAMP
Adrenergic receptors are of four general types, α1, α2, β1, and β2, defined by subtle differences in their affinities and responses to a group of agonists and antagonists.

Agonists are structural analogs that bind to a receptor and mimic the effects of its natural ligand; antagonists are analogs that bind without triggering the normal effect and thereby block the effects of agonists.

 Isoproterenol and propranolol are synthetic analogs, one an agonist with an affinity for the receptor that is higher than that of epinephrine, and the other an antagonist with extremely high affinity.

The β-adrenergic receptor is an integral protein .This protein is a member of a very large family of receptors, all with seven transmembrane helices,that are commonly called serpentine receptors, G protein–coupled receptors (GPCR), or 7 transmembrane segment (7tm) receptors.

Transduction of the epinephrine signal: the β-adrenergic pathway.

The binding of epinephrine to a site on the receptor deep within the membrane (Fig. 12–12, step 1) promotes a conformational change in the receptor’s intracellular domain that affects its interaction with the second protein in the signal-transduction pathway, a heterotrimeric GTP-binding stimulatory G protein, or GS, on the cytosolic side of the plasma membrane.

When GTP is bound to Gs, Gs stimulates the production of cAMP by adenylyl cyclase (see below) in the plasma membrane.

The function of Gs as a molecular switch resembles that of another class of G proteins typified by Ras.Structurally, Gs and Ras are quite distinct G proteins of the Ras type are monomers whereas the G proteins that interact with serpentine receptors are trimers of three different subunits,α ,β ,γ.


When the nucleotide-binding site of Gs (on the α subunit) is occupied by GTP, Gs is active and can activate adenylyl cyclase with GDP bound to the site, Gs is inactive.

(step 2 ). As this occurs, the β and γsubunits of Gs dissociate from the α subunit, and Gsα, with its bound GTP, moves in the plane of the membrane from the receptor to a nearby molecule of adenylyl cyclase (step 3 ). The Gsα is held to the membrane by a covalently attached palmitoyl group.

Adenylyl cyclase is an integral protein of the plasma membrane, with its active site on the cytosolic face. It catalyzes the synthesis of cAMP from ATP:
The association of active Gsα with adenylyl cyclase stimulates the cyclase to catalyze cAMP synthesis , raising the cytosolic [cAMP].

This stimulation by Gsα is self-limiting; Gsα is a GTPase that turns itself off by converting its bound GTP to GDP.

Activation of cAMP-dependent protein kinase, PKA.

The inactive form of PKA contains two catalytic subunits (C) and two regulatory subunits (R) which are similar in sequence to the catalytic and regulatory domains of PKG (cGMP-dependent protein kinase).

The tetrameric R2C2 complex is catalytically inactive, because an autoinhibitory domain of each R subunit occupies the substrate-binding site of each C subunit.

Signal transduction by adenylyl cyclase entails several steps that amplify the original hormone signal.
First, the binding of one hormone molecule to one receptor catalytically activates several Gs molecules. Next, by activating a molecule of adenylyl cyclase, each active Gsα molecule stimulates the catalytic synthesis of many molecules of cAMP. The second messenger cAMP now activates PKA, each molecule of which catalyzes the phosphorylation of many molecules of the target protein—phosphorylase b kinase in Figure 12–16.
This kinase activates glycogen phosphorylase b, which leads to the rapid mobilization of glucose from glycogen. The net effect of the cascade is amplification of the hormonal signal by several orders of magnitude, which accounts for the very low concentration of epinephrine (or any other hormone) required for hormone activity.

Cyclic AMP, the intracellular second messenger in this system, is short-lived. It is quickly degraded by cyclic nucleotide phosphodiesterase to 5'-AMP (Fig. 12–12, step 7 ), which is not active as a second messenger:

The intracellular signal therefore persists only as long as the hormone receptor remains occupied by epinephrine. Methyl xanthines such as caffeine and theophylline (a component of tea) inhibit the phosphodiesterase, increasing the half-life of cAMP and thereby potentiating agents that act by stimulating adenylyl cyclase.



The β-Adrenergic Receptor Is Desensitized by Phosphorylation

When the receptor is occupied by epinephrine, β-adrenergic receptor kinase (βARK) phosphorylates Ser residues near the carboxyl terminus of the receptor. Normally located in the cytosol, βARK is drawn to the plasma membrane by its association with th Gsβγ subunits and is thus positioned to phosphorylate the receptor. The phosphorylation creates a binding site for the protein β-arrestin (βarr), also called arrestin 2, and binding of β-arrestin effectively prevents interaction between the receptor and the G protein.




Two Second Messengers Are Derived from Phosphatidylinositols::
A second class of serpentine receptors are coupled through a G protein to a plasma membrane phospholipase C (PLC) that is specific for the plasma membrane lipid phosphatidylinositol 4,5-bisphosphate . This hormone-sensitive enzyme catalyzes the formation of two potent second messengers: diacylglycerol and inositol 1,4,5-trisphosphate, or IP3 (not to be confused with PIP3, p. 431).

Calcium Is a Second Messenger in Many Signal Transductions::
In many cells that respond to extracellular signals, Ca2+ serves as a second messenger that triggers intracellular responses, such as exocytosis in neurons and endocrine cells, contraction in muscle, and cytoskeletal rearrangement during amoeboid movement.

Changes in intracellular [Ca2+] are detected by Ca2+-binding proteins that regulate a variety of Ca2+ dependent enzymes. Calmodulin (CaM) is an acidic protein with four high-affinity Ca2+ binding sites. When intracellular [Ca2+] rises to about 10-6 M (1 µM), the binding of Ca2_ to calmodulin drives a conformational change in the protein (Fig. 12–21).
Calmodulin associates with a variety of proteins and, in its Ca2+ bound state, modulates their activities. Calmodulin is a member of a family of Ca2+ binding proteins that also includes troponin ,which triggers skeletal muscle contraction in response to increased [Ca2+]. This family shares a characteristic Ca2+ binding structure, the EF hand.

Calmodulin is also an integral subunit of a family of enzymes, the Ca2+/calmodulin-dependent protein kinases (CaM kinases I–IV). When intracellular [Ca2+] increases in response to some stimulus, calmodulin binds Ca2+, undergoes a change in conformation, and activates the CaM kinase. The kinase then phosphorylates
a number of target enzymes, regulating their activities. Calmodulin is also a regulatory subunit of phosphorylase b kinase of muscle, which is activated by Ca2+.

Thus Ca2+ triggers ATP-requiring muscle contractions while also activating glycogen breakdown, providingfuel for ATP synthesis.


SUMMARY
A large family of plasma membrane receptors with seven transmembrane segments acts through heterotrimeric G proteins. On ligand binding, these receptors catalyze the exchange of GTP for GDP bound to an associated G protein, forcing dissociation of the α subunit of the G protein. This subunit stimulates or inhibits the activity of a nearby membrane-bound enzyme, changing the level of its second messenger product.
The β-adrenergic receptor binds epinephrine, then through a stimulatory G protein, Gs, activates adenylyl cyclase in the plasma membrane. The cAMP produced by adenylyl cyclase is an intracellular second messenger that stimulates cAMP-dependent protein kinase, which mediates the effects of epinephrine by phosphorylating key proteins, changing their enzymatic activities or structural features.

Cyclic AMP is eventually eliminated by cAMP phosphodiesterase, and Gs turns itself off by hydrolysis of its bound GTP to GDP. When the epinephrine signal persists, β-adrenergic receptor–specific protein kinase and arrestin 2 temporarily desensitize the receptor and cause it to move into intracellular vesicles. In some cases, arrestin also acts as a scaffold protein, bringing together protein components of a signaling pathway such as the MAPK cascade.

Some serpentine receptors are coupled to a plasma membrane phospholipase C that cleaves PIP2 to diacylglycerol and IP3. By opening Ca2+ channels in the endoplasmic reticulum, IP3 raises cytosolic [Ca2+]. Diacylglycerol and Ca2+ act together to activate protein kinase C, which phosphorylates and changes the activity of specific cellular proteins. Cellular [Ca2+] also regulates a number of other enzymes, often through calmodulin.

SUMMARY Multivalent Scaffold Proteins and Membrane Rafts

■ Many signaling proteins have domains that bind phosphorylated Tyr, Ser, or Thr residues in other proteins; the binding specificity for each domain is determined by sequences that adjoin the phosphorylated residue.
■ SH2 and PTB domains bind to proteins containing P –Tyr residues; other domains bind P –Ser and P –Thr residues in various contexts.
■ Plextrin homology domains bind the membrane phospholipid PIP3.
■ Many signaling proteins are multivalent, with several different binding modules. By combining the substrate specificities of various protein kinases with the specificities of domains that bind phosphorylated Ser, Thr, or Tyr residues, and with phosphatases that can rapidly inactivate a pathway, cells create a large number of multiprotein signaling complexes.
■ Membrane rafts and caveolae sequester groups of signaling proteins in small regions of the plasma membrane, enhancing their interactions and making signaling more efficient.

SUMMARY Signaling in Microorganisms and Plants

■ Bacteria and unicellular eukaryotes have a variety of sensory systems that allow them to sample and respond to their environment. In the two-component system, a receptor His kinase senses the signal and autophosphorylates a His residue, then phosphorylates the response regulator on an Asp residue.
■ Plants respond to many environmental stimuli, and employ hormones and growth factors to coordinate the development and metabolic activities of their tissues. Plant genomes encode hundreds of signaling proteins, including some very similar to those used in signal transductions in mammalian cells.
■ Two-component signaling mechanisms common in bacteria have been acquired in altered forms by plants. Cyanobacteria use typical two-component systems in the detection of chemical signals and light; plants use related proteins—which autophosphorylate on Ser/Thr, not His, residues—to detect ethylene.
■ Plant receptorlike kinases (RLKs), with an extracellular ligand-binding domain, a single transmembrane segment, and a cytosolic protein kinase domain, participate in detecting a wide variety of stimuli, including peptides that originate from pathogens, brassinosteroid hormones, self-incompatible pollen, and developmental signals. RLKs autophosphorylate Ser/Thr residues, then activate downstream proteins that in some cases are MAPK cascades. The end result of many such signals is increased transcription of specific genes.


SUMMARY Sensory Transduction in Vision, Olfaction, and Gustation
■ Vision, olfaction, and gustation in vertebrates employ serpentine receptors, which act through heterotrimeric G proteins to change the Vm of the sensory neuron.
■ In rod and cone cells of the retina, light activates rhodopsin, which stimulates replacement of GDP by GTP on the G protein transducin. The freed α subunit of transducin activates cGMP phosphodiesterase, which lowers [cGMP] and thus closes cGMP-dependent ion channels in the outer segment of the neuron. The resulting hyperpolarization of the rod or cone cell carries the signal to the next neuron in the pathway, and eventually to the brain.
■ In olfactory neurons, olfactory stimuli, acting through serpentine receptors and G proteins, trigger either an increase in [cAMP] (by activating adenylyl cyclase) or an increase in [Ca2+] (by activating PLC). These second messengers affect ion channels and thus the Vm.
■ Gustatory neurons have serpentine receptors that respond to tastants by altering [cAMP], which in turn changes Vm by gating ion channels.
■ There is a high degree of conservation of signaling proteins and transduction mechanisms across species.

SUMMARY Regulation of Transcription by Steroid Hormones
■ Steroid hormones enter cells and bind to specific receptor proteins.
■ The hormone-receptor complex binds specific regions of DNA, the hormone response elements, and regulates the expression of nearby genes by interacting with transcription factors.
■ Two other, faster-acting mechanisms produce some of the effects of steroids. Progesterone triggers a rapid drop in [cAMP], mediated by a plasma membrane receptor, and binding of progesterone to the classic soluble steroid receptor activates a MAPK cascade.

SUMMARY Regulation of the Cell Cycle by Protein Kinases
■ Progression through the cell cycle is regulated by the cyclin-dependent protein kinases (CDKs), which act at specific points in the cycle, phosphorylating key proteins and modulating their activities. The catalytic subunit of CDKs is inactive unless associated with the regulatory cyclin subunit.
■ The activity of a cyclin-CDK complex changes during the cell cycle through differential synthesis of CDKs, specific degradation of cyclin, phosphorylation and dephosphorylation of critical residues in CDKs, and binding of inhibitory proteins to specific cyclin-CDKs.

SUMMARY Oncogenes, Tumor Suppressor Genes, and Programmed Cell Death
■ Oncogenes encode defective signaling proteins. By continually giving the signal for cell division, they lead to tumor formation. Oncogenes are genetically dominant and may encode defective growth factors, receptors, G proteins, protein kinases, or nuclear regulators of transcription.
■ Tumor suppressor genes encode regulatory proteins that normally inhibit cell division; mutations in these genes are genetically recessive but can lead to tumor formation.
■ Cancer is generally the result of an accumulation of mutations in oncogenes and tumor suppressor genes.
■ Apoptosis can be triggered by extracellular signals such as TNF through plasma membrane receptors.