Molecular Biology of the Cell - Volume 2 - Alberts B., Bray D., Lewis J., Raff M., Roberts K., Watson J. 1993

Intercellular signaling
Adaptation of target cells

In responding to stimuli of almost any kind, Cells and whole organisms can generally perceive the same relative change in an external signal across a wide range of its absolute values. At THE CELLULAR LEVEL, this requires target cells that are continuously exposed to a certain stimulus to lose their ability to respond to it with their initial sensitivity. This phenomenon, known as adaptation or desensitization, allows cells to tune their sensitivity to a given stimulus. In the case of chemical signals, desensitization enables cells to detect specifically Changes in the concentration of the signaling Ligand (rather than its absolute concentration).

Desensitization to chemical signals is achieved through various mechanisms. Sometimes it results from a decrease in the number of surface receptors or their inactivation; in other cases, it is a consequence of modifications in the Proteins involved in signal Transduction downstream of receptor activation.

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12.5.1. Some forms of desensitization result from the endocytosis of surface receptors [31]

Protein Hormones and growth factors bound to the surface receptors of target cells are frequently taken up by The Cell via receptor-mediated endocytosis (Section 6.5.7). Endocytic vesicles (endosomes) subsequently transport their contents to Lysosomes, where the ligands and occasionally their receptors undergo degradation by hydrolytic Enzymes. This process not only represents an important pathway for the destruction of certain signaling ligands, but may also play a crucial role in regulating the concentration of specific receptor proteins On the surface of target cells. Although receptor degradation and replacement occur continuously, in the absence of ligand, the half-life of a receptor is approximately one day. Certain ligands markedly increase The rate of receptor degradation by inducing endocytosis; for example, in human fibroblasts grown in culture without EGF, the half-life of EGF receptors is about 10 hours, whereas upon The addition of excess EGF, the receptors are degraded with a half-life of 1 hour. At high concentrations of such ligands, the number of surface receptors progressively decreases, resulting in a diminished cellular sensitivity to the ligand.

Most types of internalized receptors, however, do not reach the lysosomes: they dissociate from their ligands within endosomes and are then recycled back to The Plasma Membrane for further use (Section 6.5.10). Yet even in this case, upon an increase in ligand concentration, a large proportion of such actively circulating receptors ends up inside the cell and becomes inaccessible to the extracellular ligand. This form of desensitization is termed receptor sequestration.

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12.5.2. Desensitization is frequently associated with receptor phosphorylation [32]

The desensitization of many cell-surface receptors relies on their reversible phosphorylation. As mentioned previously, the endocytosis of certain catalytic receptors (i.e., their removal from the surface or degradation) depends on their autophosphorylation at Tyrosine residues (Section 12.3.14). Another example is the desensitization of frog erythrocytes upon prolonged exposure to epinephrine. Their ß-adrenergic receptors are gradually modified such that within a few hours they become incapable of activating adenylate cyclase; these inactivated receptors are removed from the surface, apparently into endosomes. This modification consists in the phosphorylation of several Serine residues within the receptor protein (see Fig. 12-24) and is carried out by a specialized protein kinase that can phosphorylate only the activated form of the receptor. According to some evidence, phosphorylation indirectly switches off the function of ß-receptors by enabling them to bind to an inhibitory protein, thereby rendering them unable to activate C_s proteins. Rhodopsin, which is structurally related to ß-adrenergic receptors (Section 12.3.12), is inactivated following photoactivation through a similar mechanism: a specific rhodopsin kinase phosphorylates its activated molecules, allowing them to bind to the inhibitory protein arrestin, which prevents them from activating the G protein Transducin. This, however, is only one of the mechanisms employed by photoreceptors for light adaptation (Section 19.6.8).

12.5.3. Some forms of desensitization involve changes not in the receptors, but in G proteins [33]

Although most known desensitization mechanisms are driven by receptor modification, desensitization can in principle result from alterations in any component of the signaling pathway. It has been shown that in some cases it is linked to changes in the G protein. For example, if a fibroblast culture is incubated with prostaglandin PGE₁, which normally activates adenylate cyclase via a G_s protein, the cells soon become unresponsive not only to PGE₁, but also to other ligands whose receptors operate through the G_s-adenylate cyclase pathway (this is termed heterologous desensitization, as opposed to homologous desensitization, in which receptors are endocytosed or inactivated so that cells lose sensitivity only to a single ligand—the one that binds to those receptors). When G_s proteins from desensitized fibroblasts were added to the membranes of mutant cells lacking their own C_s protein, they proved to be ineffective activators of adenylate cyclase (compared to normal G_s proteins). The exact Nature of the alterations in the G_s protein in PGE₁-desensitized fibroblasts is not yet known, but it would come as no surprise if phosphorylation turns out to be the cause here as well.

Alterations in G proteins could also help explain certain aspects of drug addiction. In morphine addicts, target cells in the Brain are desensitized to morphine (Fig. 12-39), so a significantly higher dose of the drug is required to achieve the same analgesic or euphoric effect as in normal people. Desensitized cells, however, maintain a normal level of functional surface morphine receptors (opiate receptors). The Mechanism of desensitization has been studied using morphine-sensitive nerve cell lines. Morphine receptors on The surface of these cells trigger the activation of G_i proteins (Section 12.3.6), which inhibit adenylate cyclase and thereby reduce cAMP levels. Cells cultured in the presence of constant concentrations of morphine undergo desensitization, such that their adenylate cyclase activity and intracellular cAMP levels return to normal, even though morphine remains bound to surface receptors. If morphine is now washed out from the culture medium, a marked surge in adenylate cyclase activity occurs, and the intracellular cAMP concentration rises unusually high. This excess cAMP is likely responsible for the severely unpleasant withdrawal symptoms that appear upon abrupt cessation of drug use (anxiety, sweating, tremors, etc.). The actual mechanisms of morphine tolerance are not definitively known, but they are hypothesized to involve alterations in G_i proteins.

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Fig. 12-39. Structure of morphine, extracted from poppy seeds. Why do some of our cells possess receptors for drugs like morphine? Pharmacologists have long hypothesized that morphine mimics certain endogenous signaling molecules that regulate pain perception and mood. In 1975, two Peptides with morphine-like activity, named enkephalins, were isolated from pig brain.

Soon, longer peptides termed endorphins were isolated from the Pituitary Gland and other Tissues. All these so-called endogenous opiates share a common sequence of 4 Amino Acids and bind to the same surface receptors as morphine (and related drugs). Unlike morphine, however, they are rapidly degraded in the Organism and therefore do not accumulate in amounts sufficient to induce the tolerance observed in morphine addicts.

Fig. 12-40. Four possible modes of target cell desensitization upon prolonged exposure to a signaling molecule. A receptor is shown that normally activates or inhibits an effector enzyme (or ion channel) via a G protein. Although the inactivation mechanisms depicted here for the receptor and the G protein involve phosphorylation, Other types of modifications are also possible (these are described in the Discussion of bacterial chemotaxis, Section 12.5.4). Furthermore, receptor inactivation via phosphorylation does not always involve an inhibitory protein.

Fig. 12-41. Photographs of Salmonella typhimurium Bacteria accumulating near the tip of a Glass capillary containing The amino acid serine (A) and avoiding a capillary containing phenol (B). The photographs were taken 5 minutes after inserting the capillaries into dishes containing bacteria. Such an assay is a simple way to demonstrate chemotaxis. (V. A. Rubik, D. E. Koshland, Proc. Natl. Acad. Sci. USA 75: 2820–2824, 1978.)

The various mechanisms of target cell desensitization that we have examined are summarized in Fig. 12-40.

12.5.4. Adaptation plays a central role in bacterial chemotaxis [34]

Many types of chemical signaling between the cells of Multicellular animals may have evolved from the mechanisms by which unicellular organisms respond to chemical changes in their environment. Indeed, as already noted, certain intracellular messengers are utilized by organisms of both types. Among the best-studied responses of unicellular organisms to external signals are phenomena of chemotaxis, in which cell movement is directed toward or away from the source of a specific substance. Chemotaxis in Eukaryotic cells is discussed in Section 14.3.2 using the cellular slime mold Dictyostelium discoideum as an example, and in Section 11.6.4 using human neutrophils. We conclude this chapter with a description of bacterial chemotaxis, which—largely owing to the advances of genetic analysis—illustrates with exceptional clarity and elegance The Central Role of adaptation in responses to chemical signals.

Motile bacteria swim toward higher concentrations of nutrient substrates (attractants), such as sugars and amino acids, and "run away" from high concentrations of harmful substances (repellents) (Fig. 12-41). This relatively simple yet highly adaptive behavior—chemotaxis—has been most intensively studied in the bacteria E. coli and Salmonella typhimurium. Here we will focus primarily on chemotaxis toward attractants; movement away from repellents is governed by the same mechanisms operating in "reverse."

Fig. 12-42. Diagram of the "motor" that drives the E. coli flagellum. The "rotor" is a protein disk located within the plasma membrane. Driven by the transmembrane proton gradient, it rapidly rotates (at about 100 rev/s) within The Lipid Bilayer relative to another protein disk (the "stator"). The "rotor" shares a common axis with the hook and flagellum, causing them to rotate. A protein "bearing" serves to seal the outer membrane where the rotating rod passes through. In the drawing, stationary components are colored, while rotating components are shown in white. (Modified from M. L. de Pamphilis and J. Adler, J. Bacteriol. 105: 384-395; 396-407, 1971.)

Bacteria swim using flagella that are structurally much simpler than the flagella of eukaryotic cells (Section 11.3.2). A bacterial flagellum is a helical tube made of identical subunits of the flagellin protein. The base of each flagellum is attached via a short, flexible hook to a small protein disk embedded in the bacterial membrane. This disk forms part of a microscopic "motor" that drives the rapid Rotation of the helical flagellum by utilizing the energy of the transmembrane proton gradient (Fig. 12-42).

Because flagella on the bacterial surface are coiled into a helix in one specific direction, their direction of rotation is crucial for movement. Counterclockwise rotation allows all flagella to gather into a single bundle, enabling the bacterium to swim smoothly in one direction. However, during clockwise rotation, the bundle flies apart into individual flagella, and the bacterium begins to tumble haphazardly in place (Fig. 12-43). In the absence of external stimuli, the rotation of the disks reverses every few seconds, resulting in a characteristic pattern: straight-line motion is interrupted by sharp changes in direction during periods of tumbling (Fig. 12-44A).

Attractants and repellents modify standard bacterial movement. They bind to specific protein receptors and affect the frequency of tumbles by increasing or decreasing the interval between successive changes in the direction of flagellar rotation. When bacteria swim toward a higher concentration of an attractant, they tumble less frequently than when they swim in the opposite direction (or when no gradient exists). Because periods of straight-line motion are longer when moving "up" a concentration gradient, bacteria gradually approach the attractant source (Fig. 12-44A). Conversely, upon encountering an increasing concentration of a repellent, bacteria tumble more often than usual and consequently gradually move away from its source.

Fig. 12-43. Schematic representation of E. coli flagellar arrangement during cell movement. When the flagella rotate counterclockwise (A), they assemble into a single bundle that acts as a propeller and drives forward motion. When the flagellum rotates clockwise (B), the bundle disperses, and the bacterium begins to tumble.

Fig. 12-44. Trajectories of swimming bacteria. In the absence of a chemotactic signal (A), periods of straight-line motion are interrupted by brief tumbling episodes that randomly alter the direction of travel. In the presence of an attractant (B), tumbling is partially suppressed when the bacterium happens to move toward its higher concentration. This leads to the gradual approach of the bacterium toward the attractant source.

In natural environments, bacteria detect spatial gradients of various substances by sensing changes in their concentration over time as they move steadily from place to place (due to their small size, it would be extremely difficult for bacteria to detect spatial gradients by comparing concentrations at opposite ends of the cell). In laboratory settings, temporal concentration changes can be simulated by the rapid addition or removal of a chemical. If an attractant is added to the culture medium, tumbling is rapidly suppressed (within tenths of a second), as expected; however, after some time, despite the continued presence of the attractant, the tumbling frequency returns to normal. Bacteria remain in this adapted state until the attractant concentration changes again: adding an attractant rapidly suppresses tumbling, while removing it enhances it until the bacterium adjusts to the new level. Adaptation plays a crucial role in chemotaxis because it allows bacteria to respond not to a constant absolute value, but to changes in attractant concentration, enabling them to continue moving when travel is in the correct direction.

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12.5.5. Four Homologous Transmembrane Receptors Participate in Bacterial Chemotaxis [35]

The elucidation of the MOLECULAR MECHANISMS OF bacterial chemotaxis has been made possible largely through the isolation and analysis of mutants with various defects in this process. Such studies demonstrated that chemotactic responses to a range of substances depend on a small family of closely related transmembrane receptor proteins responsible for signal transduction across the plasma membrane. These chemotactic receptors become methylated during adaptation (see below) and are therefore frequently referred to as methyl-accepting chemotaxis proteins (Fig. 12-45).

The plasma membrane contains four types of chemotaxis receptors, each mediating a response to a narrow group of chemical compounds. Two of these mediate responses to serine and aspartate, respectively, by directly binding these amino acids and converting this event into an intracellular signal. The other two transmit responses to sugars and dipeptides, but they are activated indirectly via periplasmic substrate-binding proteins, which also participate in The transport of sugars and dipeptides across the plasma membrane (Section 6.4.13). These proteins, which "float" in the periplasmic space (between the outer bacterial membrane and the plasma membrane), specifically bind sugars and dipeptides and then form complexes with the appropriate chemotaxis receptors in the plasma membrane, thereby activating them (Fig. 12-46). Although the transport systems for these sugars and dipeptides and their corresponding chemotaxis systems utilize the same primary receptors (substrate-binding proteins), their remaining mechanisms differ. This is indicated by Mutations that abolish transport without affecting chemotaxis, and vice versa.

Fig. 12-45. Structure of a chemotactic receptor. The two segments of the polypeptide chain undergoing methylation contain a highly conserved sequence of 13 amino acids.

This diagram depicts The structure of the aspartate receptor, but chemotaxis receptors of all four types share a similar architecture. (From A. F. Russo and D. E. Koshland, Jr., Science 220: 1016-1020, 1983. Copyright 1983 by the AAAS.)

Fig. 12-46. Steps in signal transduction during bacterial chemotaxis. Chemical attractants bind to type 1 or type 2 chemotaxis receptors in the plasma membrane or to periplasmic substrate-binding proteins, which subsequently attach to type 3 or type 4 chemotaxis receptors. This results in receptor activation and the transmission of a signal into the cell that prompts the flagellar "motor" to rotate counterclockwise, thereby suppressing tumbling and prolonging periods of straight-line swimming. Attractants enter the periplasmic space from the outside through wide channels in the outer membrane (not shown).

12.5.6. Adaptation Results from Protein Methylation [35]

Compelling evidence indicates that adaptation in bacterial chemotaxis is mediated by the covalent attachment of a methyl group to the protein chemotaxis receptors. If methylation is blocked by a mutation, adaptation fails to occur, and the presence of an attractant suppresses tumbling for hours rather than minutes. Thus, activation of chemotaxis receptors has two distinct (and separate) consequences: (1) excitation rapidly develops as the activated receptor generates an intracellular signal prompting the flagellar motor to continue counterclockwise rotation, leading to forward cell movement without tumbling; and (2) slow adaptation occurs because the activated receptor becomes accessible to cytoplasmic enzymes for methylation, causing its activation to fade over a few minutes (Fig. 12-47).

Receptor methylation is catalyzed by a soluble enzyme (methyltransferase) that transfers a methyl group to a free carboxyl group of a glutamate residue within the activated receptor (Fig. 12-48). Up to four methyl groups can be transferred to a single receptor; the degree of methylation increases with rising attractant concentrations as each receptor spends a greater fraction of its time in a ligand-bound complex. Upon attractant removal, the receptor is demethylated by a soluble enzyme (Fig. 12-48). Although the methylation level fluctuates during chemotactic responses, it remains constant in adapted bacteria because a precise equilibrium is established between the rates of methylation and demethylation.

Fig. 12-47. Sequential processes of activation and adaptation (via methylation) of a chemotaxis receptor. Note that receptor activity (and thus the frequency of bacterial tumbling) is identical in the basal and adapted states. For simplicity, the receptor is shown with two methylation sites; in reality, each receptor has four. As ligand concentration increases, the fraction of time the receptor is occupied by the ligand rises. A higher ligand concentration induces a greater conformational change in the receptor than a low concentration does, bringing it closer to its maximally altered state. However, a slow increase in methylation restores the original conformation within a few minutes, with higher attractant concentrations corresponding to a greater number of methyl groups on the receptor. The receptor is now adapted. Although the diagram illustrates direct ligand binding to the receptor, in some cases the ligand first binds to a periplasmic substrate-binding protein, which subsequently interacts with the receptor.

12.5.7. Receptor Activation and Changes in Flagellar Rotation Are Coupled via a Protein Phosphorylation Cascade [36]

The activation of chemotaxis receptors by attractants and repellents must generate an intracellular signal that influences the direction of flagellar rotation. Genetic studies have shown that four cytoplasmic proteins—CheA, CheW, CheY, and CheZ—participate in this signal transduction pathway. CheY and CheZ act at the end of the effector pathway and control the direction of flagellar rotation, presumably by interacting with its "motor". CheY signals clockwise rotation, prompting the bacterium to tumble. Mutants lacking functional CheY swim entirely without tumbling. CheZ exerts the opposite effect of CheY, driving the "motor" counterclockwise, which leads to straight-line cell movement. The CheA and, likely, CheW proteins transmit the signal from the chemotaxis receptor to the CheY and CheZ proteins via a mechanism involving protein phosphorylation and dephosphorylation.

Fig. 12-48. Methylation and demethylation reactions of protein chemotactic receptors. Up to four methyl groups can be attached to each receptor via ester linkages.

Fig. 12-49. The phosphorylation system through which chemotactic receptors are believed to control the flagellar "motor." Binding of a repellent activates the receptor, leading to a brief phosphorylation of CheA. CheA rapidly transfers its covalently bound high-energy phosphate directly to CheY. CheY-phosphate binds to the flagellar motor and forces it to rotate clockwise, causing tumbling. Binding of an attractant has the opposite effect, decreasing the phosphorylation of CheA and CheY, and counterclockwise flagellar rotation results in smooth swimming. CheZ accelerates the dephosphorylation of CheY-phosphate, acting as a CheY antagonist. Each of these phosphorylated intermediates has a half-life of about 10 s, allowing the bacterium to respond rapidly to environmental changes (see Fig. 12-34). Exactly how the chemotactic receptor interacts with CheA and what role CheW plays in the process remain unknown.

In vitro experiments with purified proteins have demonstrated that CheA is a protein kinase that autophosphorylates in the presence of ATP and then rapidly transfers the phosphate group to CheY. Phosphorylated CheY is dephosphorylated with the participation of CheZ, which, as noted above, acts as a CheY antagonist in vivo. Apparently, CheY is activated (and triggers tumbling) upon being phosphorylated by the CheA kinase, whereas it is inactivated (allowing the bacterium to swim straight) when dephosphorylated by CheZ. It is thought that when attractants are bound by chemotactic receptors, the phosphorylation of CheA and CheY decreases, CheY is inactivated, and the bacterium tumbles less frequently and swims longer in a straight line; conversely, repellents activate CheA-dependent phosphorylation of CheY, leading to CheY activation and stimulating tumbling (Fig. 12-49).

This same set of proteins mediates adaptation. CheA phosphorylates the enzyme that demethylates the chemotaxis receptors (see Fig. 12-48), thereby increasing its activity and providing feedback Regulation of the chemotactic receptors.

Apparently, all the genes and proteins involved in bacterial chemotaxis have already been identified, and most of the proteins have been sequenced and isolated in large quantities. It appears that we are rapidly approaching a virtually complete molecular picture of this form of adaptive behavior.

Conclusion

By temporarily and reversibly adapting to high concentrations of a signaling ligand, cells can adjust their sensitivity to the magnitude of a stimulus, thereby responding to changes in ligand concentration rather than its absolute value. Adaptation is achieved in several ways: (1) the bound ligand may induce receptor internalization, where they remain sequestered for some time or are degraded in lysosomes; (2) activated receptors can be reversibly inactivated by phosphorylation or methylation; and (3) non-receptor Proteins of the signaling pathway (such as G proteins) can also be reversibly inactivated through as-yet-unclear mechanisms. The best-studied example of adaptation at THE MOLECULAR LEVEL is bacterial chemotaxis: the reversible methylation of a key membrane protein in the signal transduction chain enables cells to move in an optimal direction.

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