Rl

Receptor-ligand complex

This binding, like that of an enzyme to its substrate, depends on the concentrations of the interacting components and can be described by an equilibrium constant:

Receptor Ligand k _ 1 Receptor-ligand complex

1/Kd where Ka is the association constant and Kd is the dissociation constant.

Like enzyme-substrate binding, receptor-ligand binding is saturable. As more ligand is added to a fixed amount of receptor, an increasing fraction of receptor molecules is occupied by ligand (Fig. 1a). A rough measure of receptor-ligand affinity is given by the concentration of ligand needed to give half-saturation of the receptor. Using Scatchard analysis of receptor-ligand binding, we can estimate both the dissociation constant Kd and the number of receptor-binding sites in a given preparation. When binding has reached equilibrium, the total number of possible binding sites, Bmax, equals the number of unoccupied sites, represented by [R], plus the number of occupied or ligand-bound sites, [RL]; that is, Bmax = [R] + [RL]. The number of unbound sites can be expressed in terms of total sites minus occupied sites: [R] = Bmax — [RL]. The equilibrium expression can now be written

Rearranging to obtain the ratio of receptor-bound lig-and to free (unbound) ligand, we get

From this slope-intercept form of the equation, we can see that a plot of [bound ligand]/[free ligand] versus [bound ligand] should give a straight line with a slope of _Ka (_1/Kd) and an intercept on the abscissa of 5max, the total number of binding sites (Fig. 1b). Hormone-ligand interactions typically have Kd values of 10_9 to 10_11 m, corresponding to very tight binding.

Scatchard analysis is reliable for the simplest cases, but as with Lineweaver-Burk plots for enzymes, when the receptor is an allosteric protein, the plots deviate from linearity.

Bound hormone, [RL]

Bound hormone, [RL]

FIGURE 1 Scatchard analysis of a receptor-ligand interaction. A radiolabeled ligand (L)—a hormone, for example—is added at several concentrations to a fixed amount of receptor (R), and the fraction of the hormone bound to receptor is determined by separating the receptor-hormone complex (RL) from free hormone. (a) A plot of [RL] versus [L] + [RL] (total hormone added) is hyperbolic, rising toward a maximum for [RL] as the receptor sites become saturated. To control for nonsaturable, nonspecific binding sites (eicosanoid hormones bind nonspecifically to the lipid bilayer, for example), a separate series of binding experiments is also necessary. A large excess of unlabeled hormone is added along with the dilute solution of labeled hormone. The unlabeled molecules compete with the labeled molecules for specific binding to the saturable site on the receptor, but not for the nonspecific binding. The true value for specific binding is obtained by subtracting nonspecific binding from total binding. (b) A linear plot of [RL]/[L] versus [RL] gives Kd and Bmax for the receptor-hormone complex. Compare these plots with those of V0 versus [S] and 1/V0 versus 1/[S] for an enzyme-substrate complex (see Fig. 6-12, Box 6-1).

each other at several levels, generating a wealth of interactions that maintain homeostasis in the cell and the organism.

We consider here the molecular details of several representative signal-transduction systems. The trigger for each system is different, but the general features of signal transduction are common to all: a signal interacts with a receptor; the activated receptor interacts with cellular machinery, producing a second signal or a change in the activity of a cellular protein; the metabolic activity (broadly defined to include metabolism of RNA, DNA, and protein) of the target cell undergoes a change; and finally, the transduction event ends and the cell returns to its prestimulus state. To illustrate these general features of signaling systems, we provide examples of each of six basic signaling mechanisms (Fig. 12-2).

1. Gated ion channels of the plasma membrane that open and close (hence the term "gating") in response to the binding of chemical ligands or changes in transmembrane potential. These are the simplest signal transducers. The acetylcholine receptor ion channel is an example of this mechanism (Section 12.2).

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

3. Receptor proteins (serpentine receptors) that indirectly activate (through GTP-binding proteins, or G proteins) enzymes that generate intracellular second messengers. This is illustrated by the ^-adrenergic receptor system that detects epinephrine (adrenaline) (Section 12.4).

4. Nuclear receptors (steroid receptors) that, when bound to their specific ligand (such as the hormone estrogen), alter the rate at which specific genes are transcribed and translated into cellular proteins. Because steroid hormones function through mechanisms intimately related to the regulation of gene expression, we consider them here only briefly (Section 12.8) and defer a detailed discussion of their action until Chapter 28.

5. Receptors that lack enzymatic activity but attract and activate cytoplasmic enzymes that act on downstream proteins, either by directly converting them to gene-regulating proteins or by activating a cascade of enzymes that finally activates a gene regulator. The JAK-STAT system exemplifies the first mechanism (Section 12.3); and the TLR4 (Toll) signaling system in humans, the second (Section 12.6).

Gated ion channel

Opens or closes in response to concentration of signal ligand (S) or membrane potential.

Serpentine receptor

External ligand binding to receptor (R) activates an intracellular GTP-binding protein (G), which regulates an enzyme (Enz) that generates an intracellular second messenger, X.

Receptor with no intrinsic enzyme activity

Interacts with cytosolic protein kinase, which activates a gene-regulating protein (directly or through a cascade of protein kinases), changing gene expression.

Gated ion channel

Opens or closes in response to concentration of signal ligand (S) or membrane potential.

Olfactory Processing Coupled Protein
FIGURE 12-2 Six general types of signal transducers.

6. Receptors (adhesion receptors) that interact with macromolecular components of the extracellular matrix (such as collagen) and convey to the cytoskeletal system instructions on cell migration or adherence to the matrix. Integrins (discussed in Chapter 10) illustrate this general type of transduction mechanism.

As we shall see, transductions of all six types commonly require the activation of protein kinases, enzymes that transfer a phosphoryl group from ATP to a protein side chain.

SUMMARY 12.1 Molecular Mechanisms of Signal Transduction

■ All cells have specific and highly sensitive signal-transducing mechanisms, which have been conserved during evolution.

■ A wide variety of stimuli, including hormones, neurotransmitters, and growth factors, act through specific protein receptors in the plasma membrane.

■ The receptors bind the signal molecule, amplify the signal, integrate it with input from other receptors, and transmit it into the cell. If the signal persists, receptor desensitization reduces or ends the response.

■ Eukaryotic cells have six general types of signaling mechanisms: gated ion channels; receptor enzymes; membrane proteins that act through G proteins; nuclear proteins that bind steroids and act as transcription factors; membrane proteins that attract and activate soluble protein kinases; and adhesion receptors that carry information between the extracellular matrix and the cytoskeleton.

12.2 Gated Ion Channels

Ion Channels Underlie Electrical Signaling in Excitable Cells

The excitability of sensory cells, neurons, and myocytes depends on ion channels, signal transducers that provide a regulated path for the movement of inorganic ions such as Na+, K+, Ca2 + , and Cl— across the plasma membrane in response to various stimuli. Recall from Chapter 11 that these ion channels are "gated"; they may be open or closed, depending on whether the associated receptor has been activated by the binding of its specific ligand (a neurotransmitter, for example) or by a change in the transmembrane electrical potential, Vm. The Na+K+ ATPase creates a charge imbalance across the plasma membrane by carrying 3 Na+ out of the cell for every 2 K+ carried in (Fig. 12-3a), making the inside negative relative to the outside. The membrane is said to be polarized. By convention, Vm is negative when the inside of the cell is negative relative to the outside. For a typical animal cell, Vm = —60 to —70 mV.

Because ion channels generally allow passage of either anions or cations but not both, ion flux through a channel causes a redistribution of charge on the two sides of the membrane, changing Vm. Influx of a positively charged ion such as Na+, or efflux of a negatively charged ion such as Cl—, depolarizes the membrane and brings Vm closer to zero. Conversely, efflux of K+ hy-perpolarizes the membrane and Vm becomes more negative. These ion fluxes through channels are passive, in contrast to active transport by the Na+K+ ATPase.

The direction of spontaneous ion flow across a polarized membrane is dictated by the electrochemical

The electrogenic Na+K+ ATPase establishes the membrane potential.

Plasma membrane

[Na+]

High

Low

[K+]

Low

High

[Ca2+

High

Low

[Cl"]

High n- "

The electrogenic Na+K+ ATPase establishes the membrane potential.

Ions tend to move down their electrochemical gradient across the polarized membrane.

Ions tend to move down their electrochemical gradient across the polarized membrane.

FIGURE 12-3 Transmembrane electrical potential. (a) The electrogenic Na+K+ ATPase produces a transmembrane electrical potential of —60 mV (inside negative). (b) Blue arrows show the direction in which ions tend to move spontaneously across the plasma membrane in an animal cell, driven by the combination of chemical and electrical gradients. The chemical gradient drives Na+ and Ca2+ inward (producing depolarization) and K+ outward (producing hyperpolar-ization). The electrical gradient drives Cl— outward, against its concentration gradient (producing depolarization).

TABLE 12-2 Ion Concentrations in Cells and Extracellular Fluids (mM)

TABLE 12-2 Ion Concentrations in Cells and Extracellular Fluids (mM)

Cell type

In

Out

In

Out

In

Out

In

Out

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