Introduction

In responding to almost any type of stimulus, cells and organisms typically can detect the same percent change in a signal over a very wide range of stimulus intensities. At the cellular level this requires that target cells undergo a process of adaptation or desensitization, whereby, when they are exposed to a stimulus for a prolonged period, their response to it decreases. In this way a cell reversibly adjusts its sensitivity to the stimulus. In the case of chemical signaling, adaptation enables cells to respond to changes in the concentration of a signaling ligand (rather than to the absolute concentration of the ligand) over a very wide range of absolute concentrations. The general principle is simple: adaptation is achieved through a negative feedback that operates with a delay. The negative feedback means that a strong response modifies the machinery for making that response and so turns itself off; but thanks to the delay, a sudden change in the stimulus is able to make itself felt strongly for a short period before the negative feedback has time to act.

Adaptation to chemical signals can occur in various ways. In some cases it results from a gradual decrease in the number of specific cell-surface receptor proteins, which generally takes hours. In other cases it results from a rapid inactivation of such receptors, which can occur in minutes. In still other cases it is due to change in the proteins involved in transducing the signal following receptor activation, which usually occurs with an intermediate time course.

Slow Adaptation Depends on Receptor Down-Regulation ⁴²

After a protein hormone or growth factor binds to its receptor on the surface of a target cell, it is usually ingested by receptor-mediated endocytosis and delivered to endosomes (discussed in Chapter 13). Most receptors discharge their ligand in the acidic environment of endosomes and recycle back to the plasma membrane for reuse, while the ligand is delivered to lysosomes and is degraded. This process, therefore, represents a major pathway for the breakdown of many signaling proteins. Although many receptor molecules are retrieved from the endosome and recycled, a proportion of them fail to release their ligand and end up in lysosomes, where they are degraded along with the ligand. Thus, with continuous exposure to high concentrations of ligand, the number of cell-surface receptors gradually decreases, with a concomitant decrease in the sensitivity of the target cell to the ligand. By this type of mechanism, known as receptor down-regulation, a cell can slowly (over hours) adjust its sensitivity to the concentration of a stimulating ligand.

Rapid Adaptation Often Involves Receptor Phosphorylation ⁴³

Target-cell adaptation frequently involves a rapid ligand-induced phosphorylation of receptors, in addition to the slower down-regulation of the number of receptor molecules on the target cell. The best-understood example is the β₂-adrenergic receptor, which activates adenylyl cyclase via the stimulatory G protein G_s. When cells are exposed to a high concentration of adrenaline, they can desensitize within minutes by two pathways that depend on β₂-adrenergic receptor phosphorylation. In one, the rise in cyclic AMP caused by adrenaline binding activates A-kinase, which phosphorylates the β₂ receptor on a serine residue, thereby interfering with the receptor's ability to activate G_s. In the other, the activated β₂ receptor becomes a substrate for another, more specific protein kinase (called β-adrenergic kinase) that phosphorylates the carboxyl-terminal cytoplasmic tail of the activated receptor on multiple serine and threonine residues; this phosphorylated tail binds an inhibitory protein called β arrestin, which blocks the receptor's ability to activate G_s ( Figure 15-58). In vertebrate photoreceptor cells, rhodopsin, which, as we have seen, is structurally related to β-adrenergic receptors, is inactivated by a closely similar arrestin-based mechanism after it has been activated by the switching on of light. These cells have exceptionally rapid and sophisticated powers of adaptation, involving several mechanisms in addition to that based on arrestin; one of these was discussed earlier, on page 754.

Figure 15-58

Two mechanisms for the rapid desensitization of the β₂ -adrenergic receptor. Both depend on receptor phosphorylation. Both the phosphorylation in (A) and the binding of arrestin in (B) inhibit the ability of the activated receptor to interact (more...)

The A-kinase-dependent mechanism that desensitizes the β₂-adrenergic receptor operates whenever cyclic AMP levels rise in the cell. Hence, the activation of any type of receptor in the target cell that activates adenylyl cyclase can desensitize the β₂ receptor - an example of heterologous desensitization, where one ligand desensitizes target cells to another. The β-arrestin-dependent mechanism, by contrast, operates only when the β₂ receptor itself is activated by ligand binding - an example of homologous desensitization, where a ligand desensitizes target cells only to itself.

Some Forms of Adaptation Are Due to Downstream Changes ⁴⁴

Although most known mechanisms of adaptation involve changes in receptor proteins, adaptation can, in principle, result from a change in any of the components in the signaling pathway. There are several cases in which target-cell adaptation has been shown to involve a change in a trimeric G protein. This occurs, for example, in the response of yeast cells to mating pheromones.

Changes downstream from G proteins can also contribute to target-cell adaptation, as in the photoreceptor (see p. 754). In morphine addicts, for example, opiate-sensitive neurons in the brain become desensitized to morphine so that the addicts require much higher doses than normal individuals to relieve pain or to feel euphoric ( Figure 15-59). The adapted cells, however, usually have normal levels of functional cell-surface morphine (opiate) receptors. The mechanism of adaptation has been studied both in rats and in morphine-sensitive neural cell lines in culture. Morphine receptors activate the inhibitory G protein G_i, which inhibits adenylyl cyclase and thereby causes a decrease in intracellular cyclic AMP levels. This in turn decreases the activity of A-kinase and thereby the phosphorylation of several types of ion channels, which decreases the electrical firing of the neurons. Cells maintained for a long time in the presence of a high concentration of morphine adapt by a compensatory increase in their expression of the A-kinase and adenylyl cyclase genes, with the net effect that both adenylyl cyclase activity and intracellular cyclic AMP levels return to normal even though morphine is still bound to cell-surface receptors. Because the adapted cells have increased levels of adenylyl cyclase and A-kinase, however, when morphine is withdrawn, there is a marked increase in adenylyl cyclase and A-kinase activity, which causes cyclic AMP concentrations to rise to abnormally high levels. This increases the firing of the neurons and gives rise to the extremely unpleasant withdrawal symptoms (anxiety, sweating, tremors, hallucinations, etc.) experienced by morphine addicts who go "cold turkey."

Figure 15-59

The structure of morphine. Why do some of our cells have receptors for a drug that comes from poppy seeds? Pharmacologists long suspected that morphine may mimic some endogenous signaling molecule that regulates pain perception and mood. In 1975 two pentapeptides (more...)

Adaptation Plays a Crucial Role in Bacterial Chemotaxis ⁴⁵

Many of the mechanisms involved in chemical signaling between cells in multi-cellular animals have evolved from mechanisms used by unicellular organisms to respond to chemical changes in their environment. In fact, some of the same intracellular mediators, such as cyclic nucleotides, are used by both types of organisms. Among the best-studied reactions of unicellular organisms to extracellular signals are chemotactic responses, in which cell movement is oriented toward or away from a source of some chemical in the environment. We conclude this section with an account of bacterial chemotaxis, which, largely through the power of genetic analysis, provides a particularly clear and elegant illustration of the crucial role of adaptation in the response to chemical signals. The chemotaxis of eucaryotic cells is discussed in Chapter 16.

Motile bacteria will swim toward higher concentrations of nutrients ( attractants), such as sugars, amino acids, and small peptides, and away from higher concentrations of various noxious chemicals ( repellents) ( Figure 15-60). This relatively simple but highly adaptive chemotactic behavior has been most studied in E. coli and Salmonella typhimurium. We concentrate here chiefly on chemotaxis toward attractants; chemotaxis away from repellents depends on essentially the same mechanisms operating in reverse.

Figure 15-60

Bacterial chemotaxis. The photographs show Salmonella typhimurium bacteria being attracted to a small glass capillary tube containing the amino acid serine (A) and repelled from a capillary tube containing phenol (B). The pictures were taken 5 minutes (more...)

Bacteria swim by means of flagella that are completely different from the flagella of eucaryotic cells. The bacterial flagellum consists of a helical tube formed from a single type of protein subunit, called flagellin. Each flagellum is attached by a short flexible hook at its base to a small protein disc embedded in the bacterial membrane. Incredible though it may seem, this disc is part of a tiny "motor" that uses the energy stored in the transmembrane H⁺ gradient to rotate rapidly and turn the helical flagellum ( Figure 15-61).

Figure 15-61

Schematic drawing of the bacterial flagellar motor. The flagellum is linked to a flexible hook. The hook is attached to a series of protein rings (shown in red), which are embedded in the outer and inner (plasma) membranes and rotate with the flagellum (more...)

Because the flagella on the bacterial surface have an intrinsic "handedness," different directions of rotation have different effects on movement. Counterclockwise rotation allows all the flagella to draw together into a coherent bundle so that the bacterium swims uniformly in one direction. Clockwise rotation causes them to fly apart, so that the bacterium tumbles chaotically without moving forward ( Figure 15-62). In the absence of any environmental stimulus, the direction of rotation of the disc reverses every few seconds, producing a characteristic pattern of movement in which smooth swimming in a straight line is interrupted by abrupt, random changes in direction caused by tumbling ( Figure 15-63A).

Figure 15-62

Positions of the flagella on E. coli during swimming. When the flagella rotate counterclockwise (A), they are drawn together into a single bundle, which acts as a propeller to produce smooth swimming. When the flagella rotate clockwise (B), they fly apart and (more...)

Figure 15-63

The tracks of a swimming bacterium. In the absence of a chemotactic signal (A), periods of smooth swimming (runs) are interrupted by brief tumbles that randomly change the direction of swimming. Thus runs and tumbles occur in alternating sequence, each (more...)

The normal swimming behavior of bacteria is modified by chemotactic attractants or repellents, which bind to specific receptor proteins and affect the frequency of tumbling by increasing or decreasing the time that elapses between successive changes in direction of flagellar rotation. When bacteria are swimming in a favorable direction (toward a higher concentration of an attractant or away from a higher concentration of a repellent), they tumble less frequently than when they are swimming in an unfavorable direction (or when no gradient is present). Since the periods of smooth swimming are longer when a bacterium is traveling in a favorable direction, it will gradually progress in that direction - toward an attractant ( Figure 15-63B) or away from a repellent.

In its natural environment a bacterium detects a spatial gradient of attractants or repellents in the medium by swimming at a constant velocity and comparing the concentration of chemicals over time. (It does not monitor changes in concentration by using a spatial separation of receptors over its length; this would be extremely difficult given the very small size of a bacterium.) Changes over time can be produced artificially in the laboratory by the sudden addition or removal of a chemical to the culture medium. When an attractant is added in this way, tumbling is suppressed within a few tenths of a second, as expected. But after some time, even in the continuing presence of the attractant, tumbling frequency returns to normal. The bacteria remain in this adapted state as long as there is no increase or decrease in the concentration of the attractant; addition of more attractant will briefly suppress tumbling, whereas removal of the attractant will briefly enhance tumbling until the bacteria again adapt to the new level. Adaptation is a crucial part of the chemotactic response in that it enables bacteria to respond to changes in concentration rather than to steady-state levels of an attractant and to respond to these changes over an astonishingly wide range of attractant concentrations (from less than 10^-10 M to over 10^-3 M for some attractants).

Bacterial Chemotaxis Is Mediated by a Family of Four Homologous Transmembrane Receptors and a Phosphorylation Relay System ⁴⁶

The unraveling of the molecular mechanisms responsible for bacterial chemotaxis has depended largely on the isolation and analysis of mutants with defective chemotactic behavior. In this way it has been shown that chemotaxis to a number of chemicals depends on a small family of closely related transmembrane receptor proteins that are responsible for transmitting chemotactic signals across the plasma membrane. These chemotaxis receptors are methylated during adaptation (see below) and so are also called methyl-accepting chemotaxis proteins (MCPs). As we shall see, receptor activity is stimulated by an increase in repellent concentration and decreased by an increase in attractant concentration: a single receptor is affected by both sorts of molecules, with opposite consequences.

There are four types of plasma membrane chemotaxis receptors, each concerned with the response to a small group of chemicals. Type 1 and 2 receptors mediate responses to serine and aspartate, respectively, by directly binding these amino acids and transducing the binding event into an intracellular signal. A model of the structure of one of these receptors is shown in Figure 15-64. Type 3 and 4 receptors mediate responses to sugars and dipeptides, respectively, in a slightly less direct fashion ( Figure 15-65).

Figure 15-64

A model of the homodimeric structure of the aspartate chemotaxis receptor protein. The three-dimensional structure of the extracellular domain has been obtained by x-ray diffraction. The intracellular coiled-coil domains are predicted from amino acid sequence (more...)

Figure 15-65

The different types of chemotaxis receptors. Chemical attractants bind to type 1 or type 2 receptors in the plasma membrane or to binding proteins in the periplasmic space (between the inner and outer bacterial membranes) that then bind to type 3 or type (more...)

Genetic studies indicate that four cytoplasmic proteins - CheA, CheW, CheY, and CheZ - are involved in the intracellular signaling pathway that couples the chemotactic receptors to the flagellar motor. CheY acts at the effector end of the pathway to control the direction of flagellar rotation. When activated, it binds to the motor, causing it to rotate clockwise and thereby inducing tumbling; mutants that lack this protein swim constantly without tumbling. CheA is a histidine protein kinase. When bound to both an activated chemotactic receptor and CheW, it phosphorylates itself on a histidine residue and almost immediately transfers the phosphate to an aspartic acid residue on CheY. The phosphorylation of CheY activates the protein so that it binds to the flagellar motor and causes clockwise rotation and tumbling. CheZ rapidly inactivates phosphorylated CheY by stimulating its dephosphorylation ( Figure 15-66).

Figure 15-66

The phosphorylation relay system that enables the chemotaxis receptors to control the flagellar motor. The binding of a repellent increases the activity of the receptor, which binds CheW and CheA, thereby stimulating CheA to phosphorylate itself. CheA quickly (more...)

The binding of a repellent to a chemotactic receptor increases the activity of the receptor, which in turn increases the activity of CheA and thereby the phosphorylation of CheY, which causes tumbling. These phosphorylations occur rapidly: the time required for the tumbling response after adding a repellent is about 200 milliseconds. The binding of an attractant has the opposite effect. It decreases the activity of the receptor, which decreases the activity of CheA, so that CheY remains dephosphorylated, the motor continues to rotate counterclockwise, and the bacterium swims smoothly.

The function of CheY in bacterial chemotaxis is analogous to the function of Ras proteins in animal cell signaling. Like Ras, CheY functions as an on/off switch: it is on when phosphorylated and off when dephosphorylated, just as Ras is on with GTP bound and off with GDP bound. CheY is activated by CheA and inactivated by CheZ, just as Ras is activated by GNRPs and inactivated by GAPs (see Figure 15-50). Indeed, the three-dimensional structures of CheY and Ras are similar.

Receptor Methylation Is Responsible for Adaptation in Bacterial Chemotaxis ⁴⁶

Adaptation in bacterial chemotaxis results from the covalent methylation of the chemotaxis receptor proteins. When methylation is blocked by mutation, adaptation is markedly inhibited, and exposure of the mutant bacteria to an attractant results in the suppression of tumbling for days instead of for a minute or so. Binding of a chemoattractant to a chemotaxis receptor, therefore, has two separable consequences. (1) It rapidly decreases the activity of the receptor, thereby decreasing the activity of CheA and CheY and causing the flagellar motor to continue to rotate counterclockwise; this results in a suppression of tumbling. (2) It causes adaptation because, while the attractant is bound, the receptor is methylated by an enzyme in the cytoplasm, which increases the activity of the receptor over a period of a few minutes ( Figure 15-67).

Figure 15-67

The sequential activation and adaptation (via methylation) of a chemotaxis receptor. Note that the activity of the receptor, and there-fore the tumbling frequency of the bacterium, is the same in the resting and adapted states. The receptor is shown with (more...)

Receptor methylation is catalyzed by an enzyme ( methyl transferase) that acts on the receptor protein. As many as eight methyl groups can be transferred to a single receptor, the extent of methylation increasing at higher concentrations of attractant (where each receptor spends a larger proportion of its time with ligand bound). When the attractant is removed, the receptor is demethylated by a demethylating enzyme (methylesterase). Although the level of methylation changes during a chemotactic response, it remains constant once a bacterium is adapted because a balance is reached between the rates of methylation and demethylation. The methylesterase that removes methyl groups from the chemotactic receptors is also regulated by CheA-mediated phosphorylation, and this provides another form of negative feedback regulation that makes a further contribution to adaptation.

A variety of other regulatory interactions and feedback loops are probably still to be discovered in bacterial chemotaxis. Nonetheless, all of the genes and proteins involved in this highly adaptive behavior may now have been identified, and in most cases the protein sequences are known and the proteins are available in large quantities. It therefore seems likely that bacterial chemotaxis will be the first cell-signaling system to be understood completely in molecular terms. But even when all the molecules and their interactions have been defined, it may be difficult to comprehend how the signaling system operates as an integrated network, as we discuss next.

Summary

By adapting to high concentrations of a signaling ligand in a time-dependent, reversible manner, cells can adjust their sensitivity to the level of a stimulus and thereby respond to changes in a ligand's concentration over an enormously large range rather than to the absolute concentration of the ligand. Adaptation occurs in various ways: (1) ligand binding can induce the internalization of receptors, some of which are then degraded in lysosomes - a process called receptor down-regulation; (2) activated receptors can be reversibly inactivated by being phosphorylated or methylated; (3) G proteins can be reversibly inactivated; and (4) proteins downstream of G proteins in the signaling pathway can be up-regulated or down-regulated. At a molecular level the best-understood example of adaptation occurs in bacterial chemotaxis, in which the reversible methylation of key signal-transducing proteins in the plasma membrane helps the cell to swim toward an optimal environment.