Signal Transduction by G-Protein-Coupled, Seven-Helix Transmembrane Receptors

The first three signaling pathways use seven-helix receptors coupled to trimeric G-proteins. Two highly specialized sensory systems—olfactory and visual reception—are particularly well characterized because their outputs are electrophysiological events that can be monitored at the level of single cells and single membrane ion channels on a rapid time scale. On a millisecond time scale, these two sensory transducers amplify minute stimuli to produce changes in the membrane potential that initiate a signal to the central nervous system. The response of cells to the hormone epinephrine through the β-adrenergic receptor provides an interesting contrast to these rapidly responding sensory systems. Although many of the protein components are similar, the response is slower and much more global, affecting cellular metabolism and responsiveness as a whole.

Detection of Odors by the Olfactory System

Metazoan sensory systems detect external stimuli with extremely high sensitivity and specificity. The chemosensory processes of smell and taste have evolved into highly specialized biochemical and electrophysiological pathways that connect individuals with their environment. Of these two sensory modalities, olfaction is more sensitive, allowing mammals to detect odorants at concentrations of a few parts per trillion in air and to distinguish among more than 10,000 different odorants. Most volatile chemicals with molecular weights of less than 1000 are perceived to have some odor. The olfactory system shares features with many systems that eukaryotes use for communication, including external pheromone signals of yeast and insects as well as internal hormonal signals, such as adrenaline, that circulate in the blood between organs of higher organisms.

Volatile odorant compounds first dissolve in the mucus that bathes the sensory tissue in the nasal cavity. Insects use a family of odorant-binding proteins to solubilize odorants in mucus. The role of such proteins is less well established in mammals. Odorant-binding proteins typically have low affinities for their ligands, so odorants exchange rapidly on and off their binding proteins in the mucus. When odorants dissociate, they can interact with receptor proteins located on specialized cilia of the olfactory neurons.

Sensory Neurons

Olfactory sensory neurons located in the nasal epithelium detect specific odorants and respond by sending action potentials to the brain (Fig. 27-1). These neurons have three specialized zones. An apical dendrite extends to the surface of the epithelium and sprouts approximately 12 sensory cilia specialized for responding to particular extracellular odorants. The response depends on high concentrations of four proteins in the ciliary membrane: a single type of odorant receptor, the trimeric G-protein G_olf, type III adenylyl cyclase, and cyclic nucleotide–gated ion channels. The cell body contains the nucleus, protein-synthesizing machinery, and plasma membrane pumps and channels that set the resting electrical potential of the plasma membrane. An axon projects from the base of each neuron to secondary neurons in the olfactory bulb at the front of the brain.

Figure 27-1 olfaction. A, Light micrograph of a section of sensory epithelium from the nasal passage of a mouse after staining (purple) for one olfactory receptor mRNA. Note that only a few cells express this gene. B, The sensory epithelium, highlighting one olfactory sensory neuron and the signal-transducing proteins concentrated in three parts of the cell. (BM, basement membrane.) C, Signal transduction mechanism. An odorant molecule binds a specific seven-helix plasma membrane receptor and changes its conformation. The activated receptor catalyzes the exchange of GDP for GTP on multiple trimeric G-proteins, causing dissociation of G_olfα from Gβγ. G_olfα-GTP activates adenylyl cyclase to produce multiple cAMPs. cAMP binds to and opens cyclic nucleotide–gated ion channels in the plasma membrane, depolarizing the plasma membrane. Ca²⁺ admitted by the cAMP-gated channel opens chloride channels (ClC), which augment membrane depolarization. Membrane depolarization triggers an action potential at the base of the axon that travels along the axon to secondary neurons in the brain. Multiple negative feedback loops (red) terminate stimulation. cAMP activates PKA, and Gβγ activates odorant receptor kinase (ORK [green]), both of which phosphorylate and inhibit the receptor. Ca²⁺ binds calmodulin, which inhibits the cAMP-gated channel and activates phosphodiesterase (PDE) to break down cAMP. D, Light micrograph of the olfactory bulb of a mouse expressing a marker enzyme (beta galactosidase) driven by the promoter for one odorant receptor. A histochemical reaction marks the axons of these cells blue. Axons from many olfactory sensory neurons expressing the same receptor converge on the same glomerulus in the olfactory bulb.

(A, From Ressler KJ, Sullivan SL, Buck LB: A zonal organization of odorant receptor gene expression in the olfactory epithelium. Cell 73:597–609, 1993. D, Courtesy of Charles Greer, Yale University, New Haven, Connecticut. Reference: Zou D-J, Feinstein P, Rivers AL, et al: Postnatal refinement of peripheral olfactory projections. Science 304:1976–1979, 2004.)

Olfactory sensory neurons are unique among adult neurons in their ability to replace themselves from precursor cells in the epithelium within 30 days after they are destroyed. If protected from viruses and environmental toxins, they turn over much more slowly, so the ability of self-renewal appears to be an adaptation to the hazards associated with exposure to the environment. Loss of olfactory function with age results, in part, from a decreased ability to maintain this neuronal replacement process. The presence of neurons in an epithelium might seem odd, but recall that the entire central nervous system derives from the embryonic ectoderm.

Photon Detection by the Vertebrate Retina

Overview of Visual Signal Processing

Photons are energetic but unconventional agonists. They are tiny, move very fast, and penetrate most biochemical materials. These properties create a formidable challenge for detecting photons and transducing their properties (intensity and wavelength) into a signal that can be transmitted to the brain. Nevertheless, vertebrate photoreceptor cells capture single photons and convert this energy into a highly amplified electrical response (Fig. 27-2). Phototransduction is the best-understood eukaryotic sensory process because the system is amenable to sophisticated biophysical, biochemical, and physiological analysis. Single-cell organisms use similar mechanisms to respond to light (see Fig. 38-19).

Figure 27-2 vertebrate visual transduction. A, Drawing of a rod cell. Disks in the outer segment are rich in rhodopsin. B–D, Drawings of small portions of an outer segment (upper panels) and the synaptic terminal of a rod cell (lower panels) in three physiological states. Active components are highlighted by bright colors. B, Resting cell in the dark. Constitutive production of cGMP keeps a subset of the plasma membrane cGMP-gated channels open most of the time, allowing an influx of Na⁺ and Ca²⁺. At the resting membrane potential, the synaptic terminal constitutively secretes the neurotransmitter glutamate. Ca²⁺ leaves the outer segment via a sodium/calcium exchange carrier in the outer segment, whereas Na⁺ leaves the cell via a sodium pump in the plasma membrane of the inner segment. C, Absorption of a photon activates one rhodopsin, allowing it to catalyze the exchange of GTP for GDP bound on many molecules of transducin (G_T). This dissociates G_Tα from Gβγ. Each G_Tα-GTP binds and activates one molecule of phosphodiesterase (attached to the disk membrane by N-terminal isoprenyl groups), which rapidly converts cGMP to GMP. As the concentration of free cGMP declines, the cGMP-gated channels close, leading to hyperpolarization of the plasma membrane and inhibition of glutamate secretion at the synaptic body. D, Recovery is initiated when rhodopsin kinase phosphorylates activated rhodopsin. Binding of arrestin to phosphorylated rhodopsin prevents further activation of G_T. Phosphodiesterase and an RGS protein cooperate to stimulate hydrolysis of GTP bound to G_T, returning G_T to the inactive G_Tα-GDP state. Synthesis of cGMP by guanylyl cyclase returns the cytoplasmic concentration of cGMP to resting levels and opens the cGMP-gated channels. Constitutive secretion of glutamate resumes.

Vertebrate photoreceptor cells are neurons located in a two-dimensional array in the retina, an epithelium inside the eye. The cornea and lens of the eye form an inverted real image of the outside world on the retina, so the intensity of the light across the field of view is encoded by the array of geographically separate photoreceptor cells. Photoreceptor cells lie at the base of a complex neural processing system. Having detected the rate of photon stimulation at a particular place in the visual field, photoreceptor neurons communicate this information to higher levels of the visual system. Initial processing of the information takes place in the retina, where secondary and tertiary neurons take input from multiple photoreceptors to derive local information regarding image contrast, as well as color and intensity. Neuroscience texts present more detailed information on higher levels of visual processing in the retina and brain.

The response of photoreceptor cells depends on the intensity of the light, that is, the flux of photons. Vertebrate retinas detect light with intensities that range over 10 orders of magnitude. Rod photoreceptors (Fig. 27-2A) detect low levels of light from about 0.01 photon per μm²per second (dim stars) to 10 photons per μm²per second but do not discriminate light of different colors. Cone photoreceptors (cones) respond to more intense light, up to about 10⁹photons per μm²per second (full sunlight). Three classes of cones with chromophores that are sensitive to different wavelengths of light allow humans to encode wavelength and color vision to operate (Box 27-2).

BOX 27-2 Color Vision

Three types of cones are responsible for our ability to discriminate colors. Cones are organized much like rods but express one of three different seven-helix photoreceptors, each with a distinct visual pigment. The absorption spectra of the photoreceptors overlap, so the central nervous system can perceive colors from deep purple to deep red by comparing the relative activation of the three types of cones at each point in the visual field. As in rods, signal transduction in cones depends on degradation of cGMP and closure of cGMP-gated channels. Activation of cones requires much more intense light, but cones respond faster than rods.

Rods and cones have three specialized regions with different molecular components and functions. The nucleus and the organelles in the inner segment maintain the cell’s structure and metabolism. A vestigial cilium connects the inner segment to the outer segment, which consists of a stack of internal membrane disks containing the photoreceptor protein, rhodopsin in the case of rods, surrounded by the plasma membrane. Disks form by invagination and pinching off of flattened sacks of plasma membrane. Rhodopsin synthesized in the cell body is transported to the plasma membrane along the secretory pathway and segregated into disk membranes. The lumen of the disks corresponds topologically to the lumen of the endoplasmic reticulum or the extracellular space.

Absorption of a photon activates rhodopsin and initiates a signaling cascade (Fig. 27-2) involving a trimeric G-protein and a cGMP phosphodiesterase, both attached to the cytoplasmic face of the disk membrane by covalent lipid groups. Active phosphodiesterase lowers the cytoplasmic concentration of cGMP and closes cGMP-gated channels in the plasma membrane. Closing these channels reduces the release of glutamate at the synapse with the next neuron in the visual circuit. Signals flow through the system as follows:

1. A chromophore bound to a seven-helix receptor absorbs light and changes the conformation of the receptor.

2. Active receptor catalyzes the exchange of GDP for GTP on a trimeric G-protein.

3. G-proteins activate an enzyme, cGMP phos-phodiesterase.

4. Phosphodiesterase rapidly reduces the cytoplasmic concentration of cGMP.

5. The reduction in cytoplasmic cGMP closes cGMP-gated cation channels, hyperpolarizing the plasma membrane.

6. The change in membrane potential reduces the rate of transmitter release at the synapse between the photoreceptor cell and the next neuron in the visual circuit.

Feedback loops operate at every level in this signal transduction pathway, turning off the response to a flash of light. The following sections explain how these reactions achieve their spectacular sensitivity in rods.

Regulation of Metabolism through the β-adrenergic Receptor

Epinephrine, a catecholamine that is also called adrenaline (Fig. 27-3), is secreted by the neuroendocrine cells of the adrenal gland and other tissues when an animal is startled, is stressed, or otherwise needs to respond vigorously. Norepinephrine, a closely related catecholamine, is secreted by sympathetic neurons, including those that regulate the contractility of the heart. These hormones flow through the blood and stimulate cells of many types throughout the body to heighten their metabolic activity. Skeletal muscle and liver cells respond by breaking down glycogen to glucose to provide energy. Smooth muscle cells of arteries relax to facilitate blood flow. Norepinephrine stimulates heart cells to contract more frequently and with greater force (see Fig. 11-12) and stimulates brown fat cells to dissipate energy as heat (see Fig. 28-6). The variety of physiological responses depends on selective expression of a family of nine adrenergic receptors and their associated signaling hardware in particular differentiated cells (Table 27-1).

Figure 27-3 β-adrenergic signaling mechanism. Active components are shown in bright colors. Upper right, Pathway of epinephrine synthesis. Lower left, Time course of the amplification of the signal by the catalytic cascade of signal-transducing enzymes.A, Epinephrine binds the seven-helix β-adrenergic receptor, shifting its equilibrium to the active conformation. B, Active receptor catalyzes the exchange of GDP for GTP on G_sα, dissociating G_sα-GTP from Gβγ.C, G_sα-GTP activates adenylyl cyclase, which produces multiple cAMPs.D, cAMP activates protein kinase A (PKA) by dissociating the regulatory subunit, RII (not shown to scale).E, PKA phosphorylates and partially activates multiple molecules of phosphorylase kinase (PK).F, Ca²⁺ binds to calmodulin (CM) associated with phosphorylase kinase, completing activation.G, Phosphorylase kinase phosphorylates and activates phosphorylase.H, Phosphorylase catalyzes the conversion of glycogen to glucose-6 phosphate. Negative feedback loops (red) terminate stimulation. cAMP activates PKA and Gβγ activates β-adrenergic receptor kinase, both of which phosphorylate and inhibit the receptor from catalyzing nucleotide exchange. PP1, protein phosphatase 1.

Epinephrine binding to the β-adrenergic receptor is the classic example of a pathway utilizing a seven-helix receptor (see Fig. 24-2), a trimeric G-protein (see Fig. 25-9), and adenylyl cyclase (see Fig. 26-2) to produce cAMP. This second messenger mediates a wide variety of cellular responses by activating protein kinase A (PKA; see Fig. 25-3), which changes the activity of many different cellular proteins by phosphorylation. Differentiated cells vary in their responses to epinephrine and norepinephrine, because they express different targets for PKA. In the heart, PKA phosphorylates voltage-gated Ca-channels, increasing Ca²⁺ release, and phospholamban, a small membrane protein that stimulates the Ca²⁺ pump of the smooth endoplasmic reticulum to clear Ca²⁺ from the cytoplasm (see Fig. 39-15). These changes strengthen contraction. Smooth muscle PKA phosphorylates and inhibits myosin light chain kinase, preventing it from initiating contraction and increasing blood flow (see Fig. 39-21). Liver PKA activates enzymes that break down glycogen, releasing glucose into the circulation.

This section explains how β-adrenergic receptors regulate the production of glucose-6 phosphate from glycogen (Fig. 27-3). As with vision and olfaction, the response to epinephrine is sensitive, highly amplified, and subject to negative feedback control. Five stages of amplification along the seven-step pathway allow binding of a single molecule of epinephrine to a re-ceptor to activate millions of enzyme molecules that produce many million molecules of glucose-6 phosphate:

The positive response to epinephrine is transient, owing to reactions that counterbalance the positive arm of the pathway, even in the continued presence of epinephrine. First, each activating reaction is reversible, either spontaneously or after being catalyzed by specific enzymes. Second, the system has several negative feedback loops.

Activating steps are reversed in several different ways. Epinephrine dissociates rapidly from receptors, so if the plasma concentration of epinephrine declines, the b-receptor equilibrium shifts promptly toward the inactive state. Activated GTP-G_sα hydrolyzes its bound nucleotide slowly, at a rate of about 0.05s⁻¹. GDP-G_sα then rebinds Gβγ, returning the complex to its inactive state. cAMP is degraded to 5′AMP by a phosphodiesterase, an enzyme activated by Ca²⁺-calmodulin. This convergence allows signaling pathways that release Ca²⁺ (see Fig. 26-12) to modulate the β-adrenergic pathway.

The negative feedback loops operate on a range of time scales. Gγ is anchored to the membrane lipid bilayer by a C-terminal C-20 geranylgeranyl group, so Gβγ subunits released from G_sα provide a membrane-binding site for cytoplasmic β-adrenergic receptor kinase (now called GRK2). Over seconds to minutes, membrane-associated GRK2 phosphorylates serines in the C-terminal cytoplasmic tail of active receptors. β-arrestin binding to phosphorylated receptors has three negative effects: It blocks interactions of the active receptor with G-proteins, attracts cAMP phosphodiesterase to the membrane, and on a time scale of many minutes, its interactions with β-arrestin, clathrin, and adapter proteins (see Fig. 22-10) mediates removal of receptors from the cell surface by endocytosis. Prolonged stimulation results in receptor ubiquitination (see Fig. 23-2), endocytosis, and degradation. Active PKA produced by the pathway independently phosphorylates the receptor with the same negative consequences as phosphorylation by GRK2.

In addition to these effects on glucose metabolism, active β-adrenergic receptors produce at least two other signals. Gβγ subunits activate calcium channels in some cells. This Ca²⁺ can augment glycogen breakdown at the phosphorylase kinase step. In addition to its negative effects, β-arrestin binding to phosphorylated receptors can initiate a positive signal: activation of the mitogen-activated protein (MAP) kinase pathway (see Fig. 27-6). β-arrestin serves as a membrane-anchoring site for the cytoplasmic tyrosine kinase, c-Src, which initiates signaling to the MAP kinase cascade.

Figure 27-6 egf receptor tyrosine kinase signaling pathway through map kinase. A, Ligand binding changes the conformation of the extracellular domains of the receptor.B, Extracellular domains dimerize, bringing together the tyrosine kinase domains of two receptor subunits in the cytoplasm. Direct interactions and transphosphorylation activate the kinases and create specific docking sites for effector proteins with SH2 domains.C, Phospholipase Cγ (PLCg) binds one phosphotyrosine and is activated by phosphorylation to break down phosphatidyl 4,5-bisphosphate-(PIP₂) into diacylglycerol and IP₃.D, A complex of the adapter protein Grb2 and the nucleotide exchange factor SOS binds another phosphotyrosine. (The gene for SOS protein got its name—“son of sevenless”—as a downstream component of the sevenless growth factor receptor gene required for the development of photoreceptor cell number seven in the fly eye.) SOS catalyzes the exchange of GDP for GTP on the membrane-associated small GTPase Ras. Ras-GTP attracts the cytoplasmic serine/threonine kinase Raf to the plasma membrane.E, Raf phosphorylates and activates the dual-function kinase MEK.F, MEK phosphorylates and activates MAP kinase.G, MAP kinase enters the nucleus and activates latent transcription factors.

(Receptor drawings based on originals by Daniel J. Leahy, Johns Hopkins University, Baltimore, Maryland. Unliganded receptor PDB file: 2AHX. Reference: Bouyain S, Longo PA, Li S, et al: The extracellular region of ErbB4 adopts a tethered conformation in the absence of ligand. Proc Natl Acad Sci U S A 102:15024–15029, 2005.)

Signaling Pathways Influencing Gene Expression

Many extracellular ligands influence gene expression, all through just three kinds of generic pathways (Fig. 27-4). The ligands for the first generic pathway are small and hydrophobic, such as steroids, vitamin A, and thyroid hormone. These ligands penetrate the plasma membrane and bind nuclear receptors in the cytoplasm. The ligands for the other two generic pathways include small charged molecules, peptides, and proteins that cannot penetrate the plasma membrane. Therefore, they must bind receptors on the cell surface to initiate pathways that activate transcription factors. In all cases, activated transcription factors cooperate with other nuclear proteins to regulate the expression of specific genes:

Figure 27-4 the three signaling pathways by which extracellular ligands influence gene expression. A, Nuclear receptor pathway for small hydrophobic ligands that penetrate the plasma membrane (see Fig. 15-22A for an example).B, Pathways employing a plasma membrane receptor and a cytoplasmic protein kinase that enters the nucleus to activate a latent transcription factor. (See Fig. 15-22 for the PKA pathway, Fig. 27-6 for a receptor tyrosine kinase pathway, and Fig. 27-8 for a cytoplasmic tyrosine kinase pathway.)C, Pathways employing a plasma membrane receptor and activating a latent transcription factor in the cytoplasm. The list includes six known pathways of this type. (See Fig. 27-8 for NF-AT, Fig. 27-9 for a STAT pathway, Fig. 27-10 for a Smad pathway, Fig. 15-22 for the NF-kB pathway, Chapter 24 for the Notch and Hedgehog pathways, and Fig. 30-8 for the β-catenin pathway.) CREB, cAMP response element–binding protein; ECM, extracellular matrix; TCR, T-cell receptor; TNF, tumor necrosis factor.

Figure 27-5 A, Four MAP kinase pathways. The pathways are arranged vertically. The levels of the pathways are arranged horizontally. Dual-specificity protein kinases phosphorylate S/T and Y residues.B, Positive and negative feedback loops along a generic MAP kinase pathway. K, kinase.

Figure 27-7 insulin signaling pathways in an adipose cell. Active components are shown in bright colors.A, Insulin binds the preformed dimeric receptor, bringing together the tyrosine kinase domains in the cytoplasm.B, The tyrosine kinase domains activate each other by transphosphorylation of activation loops (Fig. 27-3F). Receptor kinases then phosphorylate a variety of downstream targets: the adapter protein Cbl, which activates a nucleotide exchange protein (GEF), which activates the small GTPase TC10 (C); the adapter protein SHC, which binds Grb2-SOS and slowly initiates the MAP kinase pathway (D); the adapter protein IRS, which binds Grb2-SOS and rapidly initiates the MAP kinase pathway (E); and another IRS phosphotyrosine, which binds phosphatidylinositol 3-kinase (PI3K)(F). G, PI3K phosphorylates PIP₂ to make PIP₃.H, PIP₃ binds and activates several protein kinases: Akt (PKB), PKCl, and PKCz.I, These kinases, together with activated TC10, stimulate fusion (J) of vesicles carrying the glucose transporter GLUT4 with the plasma membrane.K, GLUT4 transports glucose into the cell. CAP binds Cbl to the plasma membrane protein flotillin. The PTB domain of IRS binds phosphotyrosine and the PH domain binds PIP₃.

Figure 27-8 t-lymphocyte activation. A, Resting T cell with inactive nonreceptor tyrosine kinase Lck and the T-cell receptor complex (TCR) with unphosphorylated cytoplasmic phosphorylation sites (ITAMs).B, An encounter with an antigen-presenting cell with an MHC-antigenic peptide complex complementary to the particular TCR initiates signaling. Active Lck phosphorylates various ITAMs.C, The nonreceptor tyrosine kinase ZAP-70 is activated by binding via its two SH2 domains to phosphorylated ITAMs on the zeta chains.D, Ribbon diagrams of MHC II (green) with bound peptide from moth cytochrome c (orange). The main model is reduced in size and tilted 90 degrees forward in the view in the upper right corner, the same orientation as in the panels B, C, E, and H. E, Active ZAP-70 phosphorylates various targets, including the transmembrane protein LAT and the adapter protein SLP76, which then propagate the signal. Phospholipase Cg binds a LAT phosphotyrosine and produces IP₃ and DAG. IP₃ releases Ca²⁺ from vesicular stores. Ca²⁺ activates calcineurin (protein phosphatase 2B), which activates the latent transcription factor NF-AT. Vav, the nucleotide exchange factor of the small GTPase Rac, is activated by binding to SLP76. Grb2-SOS binds another phosphorylated ITAM and initiates the MAP kinase cascade.F, Micrographs of the time course of the interaction of a T cell with an artificial membrane mimicking a specific antigen-presenting cell. Each image comprises a superimposition interference reflection micrograph, showing the closeness of contact as shades of gray (with white being closest apposition), and a fluorescence micrograph, showing TCRs (green) and ICAM1 (red). The stable arrangement of ICAM1 around concentrated TCRs is called an immunologic synapse. G–H, Immunologic synapse with a central zone of TCRs bound to MHC complexes and peripheral ICAM1 bound to the integrin LFA. Gads is an adapter protein; RAFT is a lipid raft.

(D, PDB file: 1KT2. Reference: Fremont DH, Dai S, Chiang H, et al: Structural basis of cytochrome c presentation by IE^k. J Exp Med 195:1043–1052, 2002. F, Courtesy of M. Dustin, New York University, New York.)

Figure 27-9 cytokine jak/stat signaling pathway. A, Cytokine binds a preformed receptor dimer (Fig. 24-7), bringing together the cytoplasmic domains with a bound tyrosine kinase, JAK.B, JAKs activate each other by transphosphorylation and then phosphorylate other tyrosines on the receptor.C, The SH2 domain of the latent transcription factor STAT binds a receptor phosphotyrosine.D, JAK phosphorylates the STATs, which then dissociate from the receptor.E, Growth factor receptor tyrosine kinases can also activate STATs.F, The STATs form an active dimer by reciprocal SH2-phosphotyrosine interactions.G, The STAT dimer enters the nucleus.H, The STAT dimer activates the expression of various genes. One of these genes encodes SOCS1, which creates negative feedback by inhibiting further STAT activation.

Figure 27-10 TGF-β/Smad signaling pathway. A, TGF-β binding assembles a complex consisting of two RII receptors and two RI receptors.B, RII phosphorylates and activates RI.C, An autoinhibited R-Smad binds RI in a complex with the adapter SARA.D, RI kinase phosphorylates R-Smad, which promotes dissociation.E, Co-Smad binds to R-Smad.F, The Smad heterodimer enters the nucleus.G, The Smad heterodimer associates with other DNA-binding proteins to activate or inhibit transcription of specific genes.

MAP Kinase Pathways to the Nucleus

Cascades of three protein kinases terminating in a MAP kinase (mitogen-activated protein kinase) relay signals from diverse stimuli and receptors to the nucleus (Fig. 27-5). The first kinase activates the second kinase by phosphorylating serine residues. The second kinase activates MAP kinase by phosphorylating both a tyrosine and a serine residue in the activation loop (see Fig. 25-3E). Active MAP kinase enters the nucleus and phosphorylates transcription factors, which regulate gene expression. Key targets include genes that advance or restrain the cell cycle, depending on the system. MAP kinases also regulate the synthesis of nucleotides required for making RNA and DNA.

A variety of cell surface receptors initiate pathways that activate MAP kinase cascades. Many of these pathways pass through the small guanosine triphosphatase (GTPase) Ras, allowing cells to integrate diverse growth-promoting signals to control the cell cycle (see Fig. 41-8). Receptor tyrosine kinases for growth factors (Fig. 27-6) and insulin (Fig. 27-7) send signals through Ras. Other receptors use nonreceptor tyrosine kinases coupled to Ras and MAP kinase, such as T-lymphocyte receptors via zeta-associated protein kinase (ZAP-kinase) (Fig. 27-8). Seven-helix receptors can also activate MAP kinase pathways. For example, β-arrestin not only inactivates β-adrenergic receptors but also couples them to a MAP kinase pathway. Budding yeast activates MAP kinase pathways in two ways. Mating pheromones bind seven-helix receptors that release Gβγ subunits of trimeric G-proteins, which activate the first kinase in the cascade. On the other hand, osmotic shock activates a two-component receptor (Fig. 27-11) upstream of another MAP kinase pathway that regulates the synthesis of glycerol, which is used to adjust cytoplasmic osmolarity.

Figure 27-11 two-component bacterial signaling systems. A, Atomic model of the aspartate receptor Tar. The atomic structures of the extracellular and cytoplasmic domains were determined by X-ray crystallography. The transmembrane α-helices are models based on the primary structure. The two polypeptides are shown inred and blue. Each polypeptide starts in the cytoplasm and passes twice through the lipid bilayer.B, Bacterial chemotaxis signaling proteins. Scale models of the molecular components and pathway of information transfer. The domains shown on the right are color coded in the molecular models on the left. An accessory protein, CheW, facilitates binding of the histidine kinase CheA to the aspartate receptor Tar. CheY and CheB are response regulators. CheR is a methyl transferase.C, Bacterial osmoregulation. The histidine kinase forms the cytoplasmic domain of the receptor. OmpR is the response regulator with a DNA-binding domain. Scale models of the molecules(left) and pathway of information transfer(right).

(A, Modified from material provided courtesy of S. H. Kim, University of California, Berkeley. Reference: Kim KK, Yokata H, Kim SH: Four helical-bundle structure of the cytoplasmic domain of a serine chemotaxis receptor. Nature 400:787–792, 1999. PDB file: 1QU7. B–C, Based on material provided courtesy of A. M. Stock, Howard Hughes Medical Institute, and Robert Wood Johnson Medical School. Reference: Stock AM, Robinson WL, Goudreau PN: Two-component signal transduction. Annu Rev Biochem 69:183–215, 2000.)

Animal cells have multiple MAP kinase cascades with particular isoforms of the three kinases linked in series and leading to different effectors (Fig. 27-5). The kinases that make up these pathways are expressed selectively in various cells and tissues. However, deletion of single MAP kinases in mice is generally not lethal, so cross talk between pathways is likely to be extensive.

A cascade of kinases provides opportunities to integrate inputs from converging pathways and to amplify signals. Amplification can be so strong that a MAP kinase cascade acts like an all-or-nothing switch. For example, frog oocytes that are arrested in the G2 stage of the cell cycle react to the hormone progesterone by either remaining arrested or entering the cell cycle. Progesterone activates a MAP kinase cascade consisting of Mos, MEK1, and the p42 MAP kinase. In individual cells, the MAP kinase is either unphosphorylated and inactive or doubly phosphorylated and fully active. This switch-like response depends in part on the fact that both MEK1 and MAP kinase require two independent phosphorylation events for activation. In addition, active MAP kinase provides two types of positive feedback (Fig. 27-5B). MAP ki-nase not only activates Mos by phosphorylation but also drives Mos expression. Consequently, a marginal stimulus turns some cells on strongly and others not at all rather than producing a graded response in all of the cells.

On the other hand, both yeast and mammals anchor two or three of the kinases in certain MAP kinase pathways to a common scaffold protein. Physical association of the enzymes insulates these pathways from parallel pathways but precludes amplification.

Growth Factor Receptor Tyrosine Kinase Pathway through Ras to Map Kinase

Protein and polypeptide growth factors control the expression of genes required for growth and development. For example, the protein epidermal growth factor (EGF) controls proliferation and differentiation of epithelial cells in vertebrates. Platelet-derived growth factor (PDGF) stimulates the proliferation of connective tissue cells required to heal wounds (see Fig. 32-11). Similar proteins specify the differentiation of cells in fly eyes and the reproductive tract of nematode worms.

Growth factor signaling pathways transfer information from the cell surface through at least eight different protein molecules to the nucleus (Fig. 27-6). Conservation of the main features of the mechanism in vertebrates, worms, and flies made it possible to piece together this complex pathway by pooling information from different systems. Genetic tests identified the components and established the order of their interactions. Many components were identified independently as oncogenes and by biochemical isolation and reconstitution of individual steps.

Information flows from growth factors to the nucleus as follows:

The routes from the cell surface through Ras and MAP kinase to nuclear transcription factors are not simple linear pathways. The signal is amplified at some steps and influenced by both positive and negative feedback loops at multiple levels. For example, pathways through phospholipase Cγ1 and phosphatidylinositol-3 kinase produce Ca²⁺ and lipid second messengers that activate PKC isoforms (see Fig. 26-6), which provide negative feedback by phosphorylating growth factor receptors. Active receptors are also modified by addition of a single ubiquitin, a signal for inactivation by endocytosis (see Fig. 23-2).

Growth factor pathways are double-edged swords. They are essential for normal growth and development, but malfunctions cause disease by inappropriate cellular proliferation. One example is the release of PDGF at the sites of blood vessel injury. Normally, PDGF stimulates wound repair (see Fig. 32-11), but excess stimulation of the proliferation of smooth muscle cells in the walls of injured blood vessels is an early event in the development of arteriosclerosis.

Many components of growth factor signaling pathways were discovered during the search for genes that cause cancer. As Jean Marx put it, “growth pathways are liberally paved with oncogene products.”^*Several of the genes were identified in cancer-causing viruses as oncogenes that are capable of transforming cells in tissue culture. Oncogenes include sis, a retroviral homolog of PDGF; erbB, a homolog of the EGF receptor; and raf kinase. Subsequently, the normal homologs of these genes were found to have mutations in human can-cers. Cancer-causing mutations typically make the protein constitutively active, producing a positive sig-nal for growth in the absence of external stimuli (see Fig. 41-10).

For example, two types of mutations can increase the concentration of active Ras. Point mutations (such as substitution of valine for glycine-12) can constitutively activate Ras by reducing its GTPase activity. Alternatively, mutations that inactivate GAPs, such as NF1 (the gene causing neurofibromatosis, the so-called elephant man disease), reduce the rate that Ras hydrolyzes GTP. In both cases, the high concentration of active Ras-GTP transmits positive signals for growth in the absence of external stimuli, predisposing individuals to malignant disease.

Insulin Pathways to GLUT4 and MAP Kinase

The insulin receptor tyrosine kinase not only stimulates the MAP kinase cascade but also triggers the acute response of muscle and adipose cells to the elevation of blood glucose following a meal (Fig. 27-7). High blood glucose levels stimulate β cells in the islets of Langerhans in the pancreas to secrete insulin, a small protein hormone. Insulin receptors are found on many cells, particularly muscle and fat cells. The insulin receptor is a stable dimer of two identical subunits, each consisting of two polypeptides covalently linked by a disulfide bond. One polypeptide forms the insulin-binding extracellular domain. The other has a single transmembrane helix connected to a cytoplasmic tyrosine kinase domain. Insulin binding changes the conformation of the extracellular domains in a way that brings together the tyrosine kinase domains on the other side of the membrane. Transphosphorylation of the juxtaposed kinase domains (see Fig. 25-3E) stimulates their kinase activities (Fig. 27-7D). The kinases propagate the signal by phosphorylating adapter proteins including IRS (insulin receptor substrates, isoforms 1 to 4), SHC (for SH2 and collagen-like), and Cbl. Each plays a distinct role in the ensuing response. This strategy differs from growth factor receptors, which use phosphotyrosine on the receptor itself to dock SH2-domain effector enzymes.

The best-known effects of insulin are to stimulate glucose uptake from blood (particularly into skeletal muscle and white fat) and the synthesis of glycogen, protein, and lipid. Glucose uptake is accomplished by the glucose carrier, GLUT4 (see Fig. 9-5). Resting cells store the GLUT4 uniporter in the membranes of cytoplasmic vesicles. Insulin stimulates fusion of these vesicles with the plasma membrane, making GLUT4 avail-able to transport glucose into the cell. This membrane fusion event requires two separate signals, both of which are downstream from the insulin receptor. Binding of PI-3 kinase to a particular phosphotyrosine on IRS initiates one signal. PI-3 kinase synthesizes PIP₃, which activates the protein kinases PKB/Akt, PKCg, and PKCγ. Phosphorylation of the adapter protein Cbl initiates the second signal. Cbl activates a nucleotide exchange protein (guanine nucleotide exchange factor [GEF]), which activates the small GTPase TC10. Within minutes of insulin stimulation, the three kinases and TC10-GTP cooperate to release GLUT4 vesicles from intracellular tethers and promote their fusion with the plasma membrane. PKB also stimulates the conversion of the newly acquired glucose to its storage form, glycogen, by releasing glycogen synthase (the enzyme that makes glycogen) from inhibition by glycogen synthase kinase (GSK) 3. The importance of PKB in the response to insulin was verified by the discovery that an inactivating mutation of PKB causes a rare form of diabetes.

Insulin is also a growth factor for some cells, acting through the Ras/MAP kinase pathway to nuclear transcription factors. The signaling circuit to Ras has two arms that operate on different time scales. The fast pathway, acting within seconds, is through tyrosine phosphorylation of IRS, which binds Grb2-SOS and initiates the MAP kinase pathway. The slow arm, acting over a period of minutes, is through phosphorylation of SHC, which binds larger quantities of Grb2-SOS and slowly initiates a sustained response of the MAP kinase pathway. Normal growth and tissue differentiation of many animals depend on insulin-like growth factors, which act on receptors similar to insulin receptor and use IRS1 to channel growth-promoting signals to the nucleus.

T-Lymphocyte Pathways through Nonreceptor Tyrosine Kinases

Some signaling pathways that control cellular growth and differentiation operate through cytoplasmic protein tyrosine kinases separate from the plasma membrane receptors. The best-characterized path-ways control the development and activation of lymphocytes in the immune system. T lymphocytes are the example in this section. T lymphocytes defend against intracellular pathogens, such as viruses, and assist B lymphocytes in producing antibodies (see Fig. 28-9).

T cells are activated during interactions of recep-tors (called T-cell receptors or TCRs) and accessory proteins on their surface with peptide antigens bound to histocompatibility proteins on the surface of an antigen-presenting cell (Fig. 27-8). Some interactions of T cells with the antigen-presenting cells are generic; others are specific. These interactions on the surface of the T lymphocyte trigger a network of interactions among protein tyrosine kinases, adapter proteins, and effector proteins on the inner surface of the plasma membrane. Tyrosine phosphorylation of multiple membrane and cytoplasmic proteins activates three separate pathways to the nucleus. Two activate cytoplasmic transcription factors; the third uses the Ras/MAP kinase pathway to activate transcription factors in the nucleus.

The T-cell antigen receptor is a complex of eight transmembrane polypeptides (Fig. 27-8A). The α and β chains, each with two extracellular immunoglobulin-like domains, provide antigen-binding specificity. Similar to antibodies, one of these immunoglobulin domains is constant and one is variable in sequence. The genes for T-cell receptors are assembled from separate parts, similar to the rearrangement of antibody genes (see Fig. 28-10). Genomic sequences for variable domains are spliced together randomly in developing lymphocytes from a panel of sequences, each encoding a small part of the protein. This combinatorial strategy creates a diversity of T-cell antigen receptors, with one type expressed on any given T cell. Variable sequences of α and β chains provide binding sites for a wide range of different peptide antigens bound to proteins, collectively termed the major histocompatibility complex (MHC) antigens, and presented on the surface of cells (Fig. 27-8D). These peptides are fragments of viral proteins or other foreign matter that have been degraded inside the cell, inserted into compatible MHC molecules during their assembly in the endoplasmic reticulum, and transported to the cell surface. Assembly of T-cell receptors in the endoplasmic reticulum requires six additional transmembrane polypeptides, each with one or more short sequence motifs, called immunoreceptor tyrosine activation motifs (ITAMs), in their cytoplasmic domains.

The expression of single types of α and β chains provides individual T cells with specificity for a particular peptide. Although T-cell antigen receptors bind specifically, their affinity for the complex of peptide and MHC is low (K_d in the range of 10μM). Given the small number (hundreds) of unique MHC-peptide complexes found on the target cell surface, this low affinity would not be sufficient for a lymphocyte to form a stable complex with an antigen-presenting cell. Accessory proteins called coreceptors, such as CD4 (also the receptor for human immunodeficiency virus [HIV]) and CD8 (see Fig. 30-3), bind directly to any MHC protein and reinforce interaction of the two cells.

Two classes of protein tyrosine kinases are required to transmit a signal from the engaged TCR to effector systems. The first class of kinases, including Lck and Fyn, are relatives of the product of the Src gene (see Fig. 25-3C), the first oncogene to be characterized (Box 27-5). These tyrosine kinases are anchored to the plasma membrane by myristolated N-terminal glycines and inhibited by a phosphotyrosine near the C-terminus (see Fig. 25-3C). This tyrosine is phosphorylated by a kinase, Csk, and dephosphorylated by the transmembrane protein tyrosine phosphatase, CD45 (see Fig. 25-6B). Apparently, CD45 keeps Lck partially dephosphorylated and therefore partially active in resting lymphocytes. Zeta-associated protein–70 kD (ZAP-70) is the most important of the second class of protein tyrosine kinases. Two SH2 domains allow ZAP-70 to bind tyrosine-phosphorylated ITAMs on ζ chains.

Physical contact of a T lymphocyte with an antigen-presenting cell carrying an MHC-peptide specific for its T-cell receptor generates multiple signals as follows:

When a T cell recognizes an antigen-presenting cell with an appropriate peptide bound to MHC on its surface, the TCRs and adhesion proteins in the interface between the cells rearrange to form an “immunologic synapse” (Fig. 27-8F). TCRs initially gather around a region of contact between integrins (LFA) on the T cell and immunoglobulin-cellular adhesion molecules (ICAMs) on the antigen-presenting cell. With time, these zones reverse positions, yielding a stable immuno-logic synapse, with a ring of adhesion molecules (Fig. 27-8G-H) around a central region with concentrated MHCs and TCRs. In this crowded central region, even a few specific MHC-peptides can activate multiple TCRs in serial fashion. Each active TCR generates a signal to the nucleus and is then internalized and degraded.

The best available immunosuppressive drugs used in human organ transplantation block lymphocyte proliferation by inhibiting calcineurin, the phosphatase that activates NF-AT. When given within 1 hour of the stimulus, these drugs completely block T-cell activation, but they have little effect after several hours once the genetic program has been initiated. Cyclosporin and FK506 bind to separate cytoplasmic proteins, cyclophi-lin, and FK-binding protein. Both of these drug-protein complexes bind calcineurin and inhibit phosphatase activity. Considering that many cells express calcineurin, the effects of these drugs on lymphocytes is amazingly specific, with relatively few side effects. Specificity arises from the low concentration of calcineurin in lymphocytes: only 10,000 molecules in T cells compared with 300,000 in other cells. Hence, low concentrations of inhibitor can selectively block calcineurin in T lymphocytes. Cyclosporin made human heart and liver transplantation feasible.

The response to T cell receptor activation depends on the particular state of differentiation of the T cell that encounters its partner antigenic peptide. Stimulation causes some T cells to secrete toxic peptides that kill the antigen-presenting cell, others to synthesize and secrete lymphokines (immune system hormones), others to proliferate and differentiate, and yet others to commit to apoptosis (see Fig. 46-8).

Cytokine Receptor, JAK/STAT Pathways

Many polypeptide hormones and growth factors (collectively called cytokines; see Fig. 24-6) regulate gene expression through a three-protein relay without a second messenger—the most direct signal transduction pathway from extracellular ligands to the nucleus (Fig. 27-9). Growth hormone uses this mechanism to drive overall growth of the body, erythropoietin directs the proliferation and maturation of red blood cell precursors, and several interferons and interleukins mediate antiviral and immune responses. Slime molds and animals use these pathways, but these proteins are not present in fungi or plants.

The three components in these pathways are a plas-ma membrane receptor that lacks intrinsic enzymatic activity, an associated tyrosine kinase (JAK), and a latent, cytoplasmic transcription factor called a STAT (signal transducer and activator of transcription). Crystal structures of the extracellular domain of the human erythropoietin receptor (see Fig. 24-6) suggest that the inactive receptor is preformed dimer in the membrane. Most of these receptors use two fibronectin III domains to bind their ligands.

JAKs were originally given the lighthearted name “just another kinase.” In view of their role between diverse receptors and transcription factors, the revisionist name “Janus kinase” (for the Greek god who opens doors) has been suggested. The tyrosine kinase domain is located near the C-terminus next to an inactive kinase-like domain. The N-terminal half of these proteins and kinase-like domain mediate the association of JAKs with receptors. Some cytokine receptors bind and activate a single type of JAK; others are promiscuous (see Fig. 24-6).

JAKs activate STATs by tyrosine phosphorylation, which promotes the formation of active dimers. These dimers enter the nucleus and bind to specific promoter sequences. The signal moves from the cytokine receptor to JAK to STAT to the nucleus as follows:

Three different mechanisms turn down the response to cytokine activation. Phosphatases inactivate the receptor, kinase, and intranuclear STATs. Endocytosis also turns off active receptors. A slowly acting, negative feedback loop limits the duration of the response. One of the genes expressed in response to STAT encodes SOCS1. Once synthesized, the SOCS1 protein inhibits further STAT activation by interaction with the cytokine receptor.

Selective expression of specific cytokine receptors, four JAKs, and seven STATs prepares differentiated mammalian cells to respond specifically to various cytokines. Active STAT dimers are either homodimers or heterodimers of two different STATs. A variety of STATs, with some unique and some common subunits, bind regulatory sites for the family of genes required to activate the target cells. The products of genes controlled by STATs not only contribute to differentiated cellular functions; some also drive proliferation. Accordingly, loss of JAK function causes certain immune deficiencies, while patients with mutations in STAT5b are resistance to growth hormone and fail to grow. The opposite effect follows from a mutation that renders JAK2 constitutively active: Red blood cells proliferate out of control, independent of stimulation by erythropoietin.

The three-protein pathway from a cytokine receptor to JAK to activated STAT is appealing in its simplicity, but in reality, these pathways do not operate in isolation. On one hand, converging signals from EGF- and PDGF-receptors can phosphorylate and activate STATs, a second input to STAT-responsive genes. On the other hand, some cytokine receptors can regulated gene expression through Shc and Grb2-SOS to Ras and MAP kinases and other pathways.

Serine/Threonine Kinase Receptor Pathways through Smads

Metazoans use a family of dimeric polypeptide growth factors related to transforming growth factor-β(TGF-β [see Fig. 24-8]) to specify developmental fates during embryogenesis and to control cellular differentiation in adults. More than 40 genes in this family are divided into two classes: (i) those related to TGF-β and activins and (ii) a large family of bone morphogenetic proteins. All activate a short pathway consisting of receptor serine/threonine kinases and a family of eight mobile transcription factors called Smads. The receptors consist of two types of subunits called RI and RII.

Ligand binding brings together two RI and two RII receptors, allowing the RII receptors to activate the RI receptors by transphosphorylation. Active RI receptors phosphorylate “regulated Smads” (R-Smads), such as Smad2 and Smad3. Phosphorylated R-Smads form active heterodimers with Smad4, called a co-Smad, because it is not subjected to phosphorylation itself. Other Smads regulate these pathways by inhibiting phosphorylation of R-Smads. After activation of a receptor, information is transmitted to the nucleus as follows (Fig. 27-10):

The Smad pathway activated by TGF-β regulates cellular proliferation and differentiation of many cell types, including epithelial and hematopoietic cells. Although its name implies that it should drive transformation, TGF-β actually stops the cell cycle in G1 by promoting expression of negative regulators of cyclin-dependent kinases (see Fig. 41-3).

In accord with the ability of TGF-β pathways to inhibit cellular growth, many human tumors have loss of function mutations in the genes for TGF-β receptors or Smads. Mutations in an accessory receptor for TGF-β cause malformed blood vessels in the human disease hereditary hemorrhagic telangiectasia. Mice with homozygous loss of function mutations in genes for the components of the TGF pathway die during embryonic development.

Like other signaling pathways, these receptors and Smads do not operate in isolation. MAP kinases and cyclin dependent kinases can phosphorylate Smads, and active RI receptors can activate MAP kinase pathways.

Two-Component Phosphotransfer Systems

Prokaryotes, fungi, and plants transduce stimuli ranging from nutrients to osmotic pressure using signaling systems consisting of as few as two proteins, a receptor-linked histidine kinase, and a “response regulator” activated by phosphorylation of an aspartic acid (Box 27-6 and Fig. 27-11). Such “two-component” systems from bacteria are one of the few signaling pathways in which the dynamics of information transfer are well understood. Extensive collections of mutants in these pathways and sensitive single-cell assays for responses, such as flagellar rotation, provide tools for rigorous tests of concepts and mathematical models derived from biochemical experiments on isolated components.

For years, two-component systems appeared to be restricted to prokaryotes, but response regulators eventually were discovered in yeast and plants. Bacteria have genes for up to 70 response regulators, with 32 response regulators and 30 histidine kinases in Escherichia coli. Archaea have genes for up to 24 response regulators. The slime mold Dictyostelium has more than 10 histidine kinases, whereas fungi have just one or two of these systems. Plants use a two-component system to regulate fruit ripening in response to the gas ethylene.

Bacterial Chemotaxis

The two-component system regulating bacterial chemotaxis (Fig. 27-12) is the best-understood signaling pathway of any kind. E. coli cells use five types of plasma membrane receptors to sense a variety of different chemicals. These receptors are also called methyl-accepting chemotaxis proteins, as they are regulated by methylation. The most abundant, with about 2000 copies per cell, is Tar (Fig. 27-11A-B), the receptor for the nutrients aspartic acid (Tar-D) and maltose, protons (as part of pH sensing), temperature, and the repellent nickel. A few thousand Tar molecules are concentrated at one end of the cell (Fig. 27-12A). Clustering facilitates interactions between receptor molecules, but this polarized distribution has nothing to do with sensing the direction of chemical gradients.

Figure 27-12 Motility of bacteria is determined by the direction of rotation of their flagella. A, Arrangement of signal transduction components and flagella inEscherichia coli. B, Flagella that are rotating counterclockwise (viewed from the tip of the flagella) form a bundle that pushes the cell smoothly forward.C, When flagella rotate clockwise, the bundle flies apart, and the cell tumbles in place. D, An attractive chemical biases movement toward its source by modulating the frequency of runs and tumbles.

(A, Reference: Parkinson JS, Blair DF: DoesE. coli have a nose? Science 259:1701–1702, 1993.)

The chemotactic signaling system guides swimming bacteria toward attractive chemicals and away from repellents in a biased random walk. Environmental chemicals influence the behavior of the cell by biasing the direction that the rotary flagellar motor turns (see Figs. 38-23 and 38-24 for details on the motor itself). In its default mode, the motor turns counterclockwise, and the bacterium swims smoothly in a more or less linear path. When the flagella turn the other way, the bacterium tumbles about in one place. A tumble allows a bacterium to reorient its direction randomly, so when it resumes smooth swimming, it usually heads off in a new direction. In the absence of chemoattractants, bacteria swim for about 0.9s and then tumble for about 0.1s, allowing for random reorientations every second.

A gradient of chemical attractant promotes the length of runs up the gradient by suppressing tumbling if the concentration of attractant increases over time (Fig. 27-12D). A two-component signaling pathway senses the attractant and controls the frequency of tumbling. The degree of saturation of the flagellar motor with the response regulator CheY determines which way it rotates.

Ligand-free Tar stimulates the phosphorylation of the associated histidine kinase CheA. (“Che” refers to a gene required for chemotaxis, as most of these components were discovered by mutagenesis. A lowercase “p” represents the shorthand for phosphorylation in these bacterial systems.) CheAp activates the response regulator, CheY, by transferring phosphate from histidine to D57 of CheY. CheYp has a higher affinity for the flagellar motor than CheY, and ligand-free receptor maintains an equilibrium with the rotors partially saturated with CheYp. With several bound CheYps, the motor reverses from its free-running, counterclockwise state about 10% of the time, inducing a brief tumble about once per second.

Information about aspartate in the environment flows rapidly through the pathway as changes in the concentrations of the phosphorylated species CheAp and CheYp. A key point is that Tar with bound aspartate, Tar-D, does not activate histidine phosphorylation of CheA. Hence aspartate binding to Tar reduces the saturation of the flagellar motors with CheYp and the frequency of tumbles.

For the cell to respond to aspartate on a subsecond time scale, an accessory protein, CheZ, is required to increase the rate of CheYp dephosphorylation more than 100-fold from its slow spontaneous rate of 0.03s⁻¹. Given this fast dissipation of CheYp, maintenance of a tumbling rate of about 1s⁻¹, in the absence of an attractant, requires constant flow of phosphate from ATP to CheAp to CheYp (Fig. 27-13A). (In most other two-component pathways, dephosphorylation of the re-sponse regulator is much slower, allowing responses over a period of minutes rather than milliseconds.) The following sections examine chemotaxis on the system level, starting with the response to a rapid change in concentration of aspartate.

Figure 27-13 signaling during bacterial chemotaxis. A, Absence of aspartate. Ligand-free Tar (1) allows CheA to phosphorylate CheY and CheB (3). Constant dephosphorylation of CheYp drives a cycle of phosphorylation (2). The steady-state concentration of CheYp keeps the motor partially saturated (4). The partially saturated motor turns counterclockwise 90% of the time (runs [thick arrow]) and clockwise 10% of the time (tumbles [thin arrow]) (5).B, Rapid response to the presence of aspartate. Aspartate binding turns off Tar (1). Constant dephosphorylation depletes CheYp on a time scale of tens of milliseconds (2). CheYp dissociates from the motor (4). The motor, without CheYp, rotates counterclockwise, so the bacterium runs continuously (5).C, Slower, adaptive response to the presence of aspartate. Inactive CheA stops phosphorylating CheB, allowing dephosphorylation of CheBp on a time scale of seconds; this inactivation of CheB allows net methylation of Tar by CheR (1). Even with bound aspartate, methylated Tar is partially active, allowing phosphorylation of CheA (2). CheY is phosphorylated (3). CheYp rebinds the motor (4). The flagella turn clockwise part of the time, causing occasional tumbles (5).

Temporal Sensing of Gradients

Bacteria are too small and move too fast to detect a spatial gradient directly. Instead, they sense the gradient as a change in concentration of attractant or repellent as a function of time. When a bacterium swims up a gradient of chemoattractant, the concentration of attractant increases with time, and the signaling mechanism suppresses tumbling. Fewer tumbles bias movement toward the attractant. When a cell swims down the gradient, tumbling is more frequent, allowing for reorientation.

A sudden increase in the concentration of aspartate yields a smooth swimming response within 200μs due to rapid reequilibration of the concentrations of all of the cytoplasmic signaling components (Fig. 27-13B). Aspartate binds Tar and inhibits autophosphorylation of CheA. Because CheYp has a half-life less than 100μs, the concentrations of CheAp and CheYp decrease rapidly. CheYp dissociates from the flagellar motor and the motor persists in the counterclockwise, smooth swimming direction. If the concentration change with time is due to a gradient of aspartate, the bacterium tends to swim steadily up the gradient toward the source.

The opposite sequence of events takes place if a bacterium swims down a gradient of aspartate. The fraction of Tar with bound aspartate declines, CheAp and CheYp concentrations rise, and tumbling is more frequent, providing opportunities to reorient and swim back up the gradient.

Adaptation

After a step change in aspartate, bacteria respond quickly with smooth swimming, but within tens of seconds to minutes, they return to their normal pattern of intermittent tumbling. In fact, the steady-state tumbling frequency is independent of the concentration of aspartate. This remarkable capacity to adapt is accomplished by a negative feedback loop provided by reversible methylation of the receptor (Fig. 27-13C). Methylated Tar has a somewhat lower affinity for aspartate than unmethylated Tar, but Me-Tar with bound aspartate is more effective at stimulating CheA phosphorylation than Tar with bound aspartate.

Two relatively slow enzymes determine the level of Tar methylation (Fig. 27-11B). CheR adds methyls to four glutamic acid residues on each receptor polypeptide, whereas CheB removes them. CheR is constitutively active but sensitive to the overall metabolic state of the cell, as it depends on the concentration of S-adenosyl methionine, a methyl donor that is used in many metabolic reactions.

A variable rate of demethylation determines the level of Tar methylation. CheB methylesterase is a response regulator that is activated by phosphorylation by CheAp. CheB is autoinhibited by its response regulator domain blocking the active site. Phosphorylation displaces the response regulator domain from the active site, making CheBp much more active than CheB.

Adaptation occurs because aspartate binding to Tar activates two different pathways on different time scales. On a millisecond time scale, the concentrations of both CheAp and CheYp decline, CheYp dissociates from the motor, and the cell swims smoothly. The rapid reduction in CheAp also reduces the concentration of CheBp, but on a second time scale, as the rate of CheBp dephosphorylation is only 0.1s⁻¹. The slow decline in CheBp gradually reduces methylesterase activity and results in a higher level of Tar methylation. This, in turn, allows the receptor, still saturated with aspartate, to reactivate CheA phosphorylation. Remarkably, the cell returns exactly to its prestimulus frequencies of runs and tumbles. This robust adaptation mechanism is an integral feedback system, just like a thermostat on a heater. Both methylation and demethylation are sensitive to the conformation of the receptor, so another level of complexity contributes to the capacity of the system to adapt.

Extended Range of Response

An amazing feature of this system is its ability to respond with fast changes in flagellar rotation and slow adaptation to changes of just a few percentage points in aspartate concentration over a range of five orders of magnitude. Clearly, a simple bimolecular reaction of aspartate with Tar cannot change the fractional saturation of Tar over such an extended range of concentrations. This extended range of sensitivity is valuable for the survival of the bacterium and must depend on some sort of amplification at the level of the receptor. A likely mechanism is that aspartate binding to one Tar activates many surrounding Tars in the receptor clusters at the end of the cell.

Bacterial chemotaxis illustrates some of the classical features of signaling pathways, including high sensitivity due to amplification at the level of CheA phosphorylation, negative feedback through methylation of Tar, and branching networks that respond on different time scales to the same stimulus. The mechanism has been tested thoroughly by mutating all of the signaling components and observing the consequences. For example, loss of either CheA or CheY results in smooth-running bacteria. Cells without CheZ respond slowly to a change in aspartate. Loss of CheR and CheB results in bacteria that respond to signals over a limited range of concentrations but are unable to adapt to them.