Target-cell adaptation frequently involves a rapid ligand-induced phosphorylation of receptors, in

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 β 2 -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 β 2 -adrenergic receptor phosphorylation. In one, the rise in cyclic AMP caused by adrenaline binding activates A-kinase,

which phosphorylates the β 2 receptor on a serine residue, thereby interfering with the receptor's ability to activate G s . In the other, the activated β 2 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.

The A-kinase-dependent mechanism that desensitizes the β 2 -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 β 2 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 β 2 receptor itself is activated by ligand binding - an example of homologous desensitization, where a ligand desensitizes target

cells only to itself.

S o m e Fo rm s o f Ad a p t a t io n Are D u e t o D o w n s t re a m

Ch a n g e s 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."

Ad a p t a t io n P la y s a Cru c ia l Ro le in B a c t e ria l Ch e m o t a x is 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.

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).

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).

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).

B a c t e ria l Ch e m o t a x is I s Me d ia t e d b y a Fa m ily o f Fo u r Ho m o lo g o u s Tra n s m e m b ra n e Re c e p t o rs a n d a

P h o s p h o ry la t io n Re la y S y s t e m 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).

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).

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.

Re c e p t o r Me t h y la t io n I s Re s p o n s ib le fo r Ad a p t a t io n in