Signal Transduction

Cells can communicate with one another via chemical signals

Cells secrete chemical signals that can induce a physiological response in any (or all) of three ways ( Fig. 3-1 ): by entering the circulation and acting on distant tissues (endocrine), by acting on a neighboring cell in the same tissue (paracrine), or by stimulating the same cell that released the chemical (autocrine). Secreted factors that are produced by cells of one organ and enter the circulation to induce a response in a separate organ are called hormones, and the organs that secrete them—such as the pituitary, adrenal, and thyroid glands—are parts of the endocrine system. However, many other cells and tissues not classically thought of as endocrine in nature also produce hormones. For example, the kidney produces 1,25-dihydroxyvitamin D ₃ , and the salivary gland synthesizes nerve growth factor. Finally, physical contact between one cell and another, or between a cell and the matrix secreted by another cell, can transmit a signal (juxtacrine).

For paracrine and autocrine signals to be delivered to their proper targets, their diffusion must be limited. This restriction can be accomplished by rapid endocytosis of the chemical signal by neighboring cells, its destruction by extracellular enzymes, or its immobilization by the extracellular matrix. The events that take place at the neuromuscular junction are excellent examples of paracrine signaling. When an electrical impulse travels down an axon and reaches the nerve terminal ( Fig. 3-2 ), it stimulates release of the neurotransmitter acetylcholine (ACh). In turn, ACh transiently activates a ligand-gated cation channel on the muscle cell membrane. The resultant transient influx of Na ⁺ causes the membrane potential ( V _m ) to shift locally in a positive direction (i.e., depolarization), initiating events that result in propagation of an action potential along the muscle cell. The ACh signal is rapidly terminated by the action of acetylcholinesterase, which is present in the synaptic cleft. This enzyme degrades the ACh that is released by the neuron.

Soluble chemical signals interact with target cells via binding to surface or intracellular receptors

Four types of chemicals can serve as extracellular signaling molecules:

• 1

  Amines, such as epinephrine

• 2

  Peptides and proteins, such as angiotensin II and insulin

• 3

  Steroids, including aldosterone, estrogens, and retinoic acid

• 4

  Other small molecules, such as amino acids, nucleotides, ions (e.g., Ca ²⁺ ), and gases (e.g., nitric oxide)

For a molecule to act as a signal, it must bind to a receptor. Most receptors are proteins on the cell surface or within the cell that specifically bind a signaling molecule (the ligand) and induce a cellular response by interacting with an effector. In some cases, the receptor is itself the effector, as in ligand-gated ion channels that alter transmembrane ion conductance in response to an extracellular signal. In most cases, however, interaction of the ligand with its receptor results in association of the receptor with one or more intracellular effector molecules that in turn initiate the cellular response. Effectors include enzymes, channels, transport proteins, contractile elements, and transcription factors. The ability of a cell or tissue to respond to a specific signal is dictated by the complement of receptors it possesses and by the chain of intracellular reactions that are initiated by the binding of any one ligand to its receptor. Receptors can be divided into five categories on the basis of their mechanisms of signal transduction ( Table 3-1 ).

• 1

  Ligand-gated ion channels. Integral membrane proteins, these hybrid receptors/channels are involved in signaling between electrically excitable cells. The binding of a neurotransmitter such as ACh to its receptor—which in fact is merely part of the channel—results in transient opening of the channel and thus alters the ion permeability of the cell.

• 2

  G protein–coupled receptors. These integral plasma-membrane proteins work indirectly—through multiple intermediaries—to activate or inactivate downstream effectors, such as membrane-associated enzymes or channels. This group of receptors is named for the initial intermediary, which is a heterotrimeric GTP-binding complex called a G protein.

• 3

  Catalytic receptors. When activated by a ligand, these integral plasma-membrane proteins are either enzymes themselves or part of an enzymatic complex. In many cases, these receptors are either kinases that add phosphate groups to their substrates or phosphatases that remove substrate phosphate groups.

• 4

  Nuclear receptors. These proteins, located in the cytosol or nucleus, are ligand-activated transcription factors. These receptors link extracellular signals to gene transcription.

• 5

  Receptors that undergo cleavage. In response to ligand binding, some transmembrane proteins undergo regulated intramembranous proteolysis ( RIP; see pp. 87–88 ), N3-1 which liberates one or more fragments of their cytosolic domain that signal a cellular response by entering the nucleus to modulate gene expression.

TABLE 3-1

Classification of Receptors and Associated Signal-Transduction Pathways

CLASS OF RECEPTOR	SUBUNIT COMPOSITION OF RECEPTOR	LIGAND	SIGNAL-TRANSDUCTION PATHWAY DOWNSTREAM FROM RECEPTOR
Ligand-gated ion channels (ionotropic receptors)	Heteromeric or homomeric oligomers	Extracellular	Ion current
		GABA	Cl − >
		Glycine	Cl − >
		ACh: muscle	Na + , K + , Ca 2+
		ACh: nerve	Na + , K + , Ca 2+
		5-HT	Na + , K +
		Glutamate: non-NMDA	Na + , K + , Ca 2+
		Glutamate: NMDA	Na + , K + , Ca 2+
		ATP (opening)	Ca 2+ , Na + , Mg 2+
		Intracellular
		cGMP (vision)	Na + , K +
		cAMP (olfaction)	Na + , K +
		ATP (closes channel)	K +
		IP 3	Ca 2+
		Ca 2+ or ryanodine	Ca 2+
Receptors coupled to heterotrimeric (αβγ) G proteins	Single polypeptide that crosses the membrane seven times	Small transmitter molecules ACh Norepinephrine Peptides Oxytocin PTH NPY Gastrin CCK Odorants Certain cytokines, lipids, and related molecules	βγ directly activates downstream effector Muscarinic AChR activates atrial K + channel α activates an enzyme Cyclases that make cyclic nucleotides (cAMP, cGMP) Phospholipases that generate IP 3 and diacylglycerols Phospholipases that generate AA and its metabolites
Catalytic receptors	Single polypeptide that crosses the membrane once	ANP TGF-β	Receptor guanylyl cyclase Receptor serine/threonine kinases
	May be dimeric or may dimerize after activation	NGF, EGF, PDGF, FGF, insulin, IGF-1	Receptor tyrosine kinase
		IL-3, IL-5, IL-6, EPO, LIF, CNTF, GH, IFN-α, IFN-β, IFN-γ, GM-CSF	Tyrosine kinase–associated receptor
		CD45	Receptor tyrosine phosphatase
Intracellular (or nuclear) receptors	Homodimers of polypeptides, each with multiple functional domains	Steroid hormones	Bind to regulatory DNA sequences and directly or indirectly increase or decrease the transcription of specific genes
		Mineralocorticoids Glucocorticoids Androgens Estrogens Progestins
	Heterodimers of polypeptides, each with multiple functional domains	Others
		Thyroid hormones Retinoic acid Vitamin D Prostaglandin
Cleavage-activated receptors	Single polypeptide that crosses the membrane once	Jagged Delta	After receptor cleavage, cytosolic domain of receptor translocates to nucleus and regulates gene transcription

CCK, cholecystokinin; NMDA, N -methyl- d -aspartate; NPY, neuropeptide Y; PTH, parathyroid hormone.

Signaling events initiated by membrane-associated receptors can generally be divided into six steps:

• Step 1: Recognition of the signal by its receptor. The same signaling molecule can sometimes bind to more than one kind of receptor. For example, ACh can bind to both ligand-gated channels and G protein–coupled receptors. Binding of a ligand to its receptor involves the same three types of weak, noncovalent interactions that characterize substrate-enzyme interactions. Ionic bonds are formed between groups of opposite charge. In van der Waals interactions, a transient dipole in one atom generates the opposite dipole in an adjacent atom and thereby creates an electrostatic interaction. Hydrophobic interactions occur between nonpolar groups.

• Step 2: Transduction of the extracellular message into an intracellular signal or second messenger. Ligand binding causes a conformational change in the receptor that triggers the catalytic activities intrinsic to the receptor or causes the receptor to interact with membrane or cytoplasmic enzymes. The final consequence is the generation of a second messenger or the activation of a catalytic cascade.

• Step 3: Transmission of the second messenger's signal to the appropriate effector. The second messenger can be amplified and transmitted to distant regions within the cell by myriad events, such as activation of intra­cellular kinases and phosphatases that alter the activity of other enzymes and proteins, release or sequestration of intracellular ions, or regulation of metabolic pathways that generate ATP.

• Step 4: Modulation of the effector. These effectors represent a diverse array of molecules, such as enzymes, ion channels, cytoskeletal components, and transcription factors. The second message can modulate their expression or activity as well as alter their location or substrate availability.

• Step 5: Response of the cell to the initial stimulus. This collection of actions represents the summation and integration of input from multiple signaling pathways.

• Step 6: Termination of the response by feedback mechanisms at any or all levels of the signaling pathway.

Cells can also communicate by direct interactions—juxtacrine signaling

Second-messenger systems amplify signals and integrate responses among cell types

Once a signal has been received at the cell surface, it is typically amplified and transmitted to specific sites within the cells via second messengers. For a molecule to function as a second messenger, its concentration, activation, and location must be finely regulated. The cell achieves this control by rapidly producing or activating the second messenger and then inactivating or degrading it. To ensure that the system returns to a resting state when the stimulus is removed, counterbalancing activities function at each step of the cascade.

The involvement of second messengers in catalytic cascades provides numerous opportunities to amplify a signal. For example, the binding of a ligand to its receptor can generate hundreds of second-messenger molecules, which can in turn alter the activity of thousands of downstream effectors. This modulation usually involves the conversion of an inactive species into an active molecule or vice versa. An example of creating an active species would be G proteins that elicit the formation of the second messenger cAMP (see pp. 56–57 ), whereas an example of destroying an active species would be G proteins that elicit the breakdown of a related second messenger, cGMP; (see p. 58 ).

Second-messenger systems also allow both specificity and diversity. Distinct ligand-receptor combinations that activate the same intracellular signaling pathways often produce the same cellular response. For example, epinephrine, adrenocorticotropic hormone (ACTH), glucagon, and thyroid-stimulating hormone (TSH) all signal via cAMP to induce triacylglycerol breakdown. In contrast, a single signaling molecule can produce distinct responses in different cells, depending on the complement of receptors and signal-transduction pathways that are available in the cell as well as the specialized function that the cell carries out in the organism. For example, ACh stimulates contraction of skeletal muscle cells, inhibits contraction of heart muscle cells, and facilitates the exocytosis of secretory granules by pancreatic acinar cells. This signaling molecule achieves these different end points by interacting with distinct receptors on each cell. Finally, even within a single cell, a second messenger such as cAMP can act within microdomains to target a specific set of effector molecules, thereby conferring spatially based specificity. N3-4

The diversity and specialization of second-messenger systems are important to a multicellular organism, as can be seen in the coordinated response of an organism to a stressful situation. Under these conditions, the adrenal gland releases epinephrine. Different organ systems respond to epinephrine in a distinct manner, such as activation of glycogen breakdown in the liver, constriction of the blood vessels of the skin, dilation of the blood vessels in skeletal muscle, dilation of airways in the lung, and increased rate and force of heart contraction. The overall effect is an integrated response that readies the organism for attack, defense, or escape. In contrast, complex cell behaviors, such as proliferation and differentiation, are generally stimulated by combinations of signals rather than by a single signal. Integration of these stimuli requires crosstalk among the various signaling cascades.

As discussed below, most signal-transduction pathways use elaborate cascades of signaling proteins to relay information from the cell surface to effectors in the cell membrane, the cytoplasm, or the nucleus. In Chapter 4 , we discuss how signal-transduction pathways that lead to the nucleus can affect the cell by modulating gene transcription. These are genomic effects. Signal-transduction systems that project to the cell membrane or to the cytoplasm produce nongenomic effects, the focus of this chapter.

Ligand-gated ion channels transduce a chemical signal into an electrical signal

The property that defines the ligand-gated ion channel class of multisubunit membrane-spanning receptors is that the signaling molecule itself controls the opening and closing of an ion channel by binding to a site on the receptor. Thus, these receptors are also called ionotropic receptors to distinguish them from the metabotropic receptors, which act via “metabolic” pathways. One superfamily of ligand-gated channels includes the ionotropic receptors for ACh, serotonin, gamma-aminobutyric acid (GABA), and glycine. Most structural and functional information for ionotropic receptors comes from the nicotinic ACh receptor (AChR) present in skeletal muscle (see Fig. 8-7 ). The nicotinic AChR is a cation channel that consists of four membrane-spanning subunits, α, β, γ or ε, and δ, in a stoichiometry of 2 : 1 : 1 : 1. This receptor is called nicotinic because the nicotine contained in tobacco can activate or open the channel and thereby alter V _m . Note that the nicotinic AChR is very different from the muscarinic AChR discussed below, which is not a ligand-gated channel. Additional examples of ligand-gated channels are the inositol 1,4,5-trisphosphate (IP ₃ ) receptor and the Ca ²⁺ -release channel (also known as the ryanodine receptor). Both receptors are tetrameric Ca ²⁺ channels located in the membranes of intracellular organelles.

G proteins are heterotrimers that exist in many combinations of different α, β, and γ subunits

G proteins are members of a superfamily of GTP-binding proteins. This superfamily includes the classic heterotrimeric G proteins that bind to GPCRs as well as the so-called small GTP-binding proteins, such as Ras. Both the heterotrimeric and small G proteins can hydrolyze GTP and switch between an active GTP-bound state and an inactive GDP-bound state.

Heterotrimeric G proteins are composed of three subunits, α, β, and γ. At least 16 different α subunits (~42 to 50 kDa), 5 β subunits (~33 to 35 kDa), and 11 γ subunits (~8 to 10 kDa) are present in mammalian tissue. The α subunit binds and hydrolyzes GTP and also interacts with “downstream” effector proteins such as adenylyl cyclase. Historically, the α subunits were thought to provide the principal specificity to each type of G protein, with the βγ complex functioning to anchor the trimeric complex to the membrane. However, it is now clear that the βγ complex also functions in signal transduction by interacting with effector molecules distinct from those regulated by the α subunits. Moreover, both the α and γ subunits are involved in anchoring the complex to the membrane. The α subunit is held to the membrane by either a myristyl or a palmitoyl group, whereas the γ subunit is held via a prenyl group.

The multiple α, β, and γ subunits demonstrate distinct tissue distributions and interact with different receptors and effectors ( Table 3-2 ). Because of the potential for several hundred combinations of the known α, β, and γ subunits, G proteins are ideally suited to link a diversity of receptors to a diversity of effectors. The many classes of G proteins, in conjunction with the presence of several receptor types for a single ligand, provide a mechanism whereby a common signal can elicit the appropriate physiological response in different tissues. For example, when epinephrine binds β ₁ adrenergic receptors in the heart, it stimulates adenylyl cyclase, which increases heart rate and the force of contraction. However, in the periphery, epinephrine acts on α ₂ adrenergic receptors coupled to a G protein that inhibits adenylyl cyclase, thereby increasing peripheral vascular resistance and consequently increasing venous return and blood pressure.

Among the first effectors found to be sensitive to G proteins was the enzyme adenylyl cyclase. The heterotrimeric G protein known as G _s was so named because it stimulates adenylyl cyclase. A separate class of G proteins was given the name G _i because it is responsible for the ligand-dependent inhibition of adenylyl cyclase. Identification of these classes of G proteins was greatly facilitated by the observation that the α subunits of individual G proteins are substrates for ADP ribosylation catalyzed by bacterial toxins. The toxin from Vibrio cholerae activates G _s , whereas the toxin from Bordetella pertussis inactivates the cyclase-inhibiting G _i ( Box 3-1 ).

For their work in identifying G proteins and elucidating the physiological role of these proteins, Alfred Gilman and Martin Rodbell received the 1994 Nobel Prize in Physiology or Medicine. N3-5

G-protein activation follows a cycle

In their inactive state, heterotrimeric G proteins are a complex of α, β, and γ subunits in which GDP occupies the guanine nucleotide–binding site of the α subunit. After ligand binding to the GPCR ( Fig. 3-4 , step 1), a conformational change in the receptor–G protein complex facilitates the release of bound GDP and simultaneous binding of GTP to the α subunit (see Fig. 3-4 , step 2). This GDP-GTP exchange stimulates dissociation of the complex from the receptor (see Fig. 3-4 , step 3) and causes disassembly of the trimer into a free GTP-bound α subunit and separate βγ complex (see Fig. 3-4 , step 4). The GTP-bound α subunit interacts in the plane of the membrane with downstream effectors such as adenylyl cyclase and phospholipases (see Fig. 3-4 , step 5), or cleavage of its myristoyl or palmitoyl group can release the α subunit from the membrane. Similarly, the βγ subunit can activate ion channels or other effectors.

The α subunit is itself an enzyme that catalyzes the hydrolysis of GTP to GDP and inorganic phosphate (P _i ). The result is an inactive α-GDP complex that dissociates from its downstream effector and reassociates with a βγ subunit (see Fig. 3-4 , step 6); this reassociation terminates signaling and brings the system back to resting state (see Fig. 3-4 , step 1). The βγ subunit stabilizes α-GDP and thereby substantially slows the rate of GDP-GTP exchange (see Fig. 3-4 , step 2) and dampens signal transmission in the resting state.

The RGS (for “regulation of G-protein signaling”) family of proteins appears to enhance the intrinsic GTPase activity of some but not all α subunits. Investigators have identified at least 19 mammalian RGS proteins and shown that they interact with specific α subunits. RGS proteins promote GTP hydrolysis and thus the termination of signaling.

Activated α subunits couple to a variety of downstream effectors, including enzymes and ion channels

Activated α subunits can couple to a variety of enzymes. A major enzyme that acts as an effector downstream of activated α subunits is adenylyl cyclase ( Fig. 3-5 A ), which catalyzes the conversion of ATP to cAMP. This enzyme can be either activated or inhibited by G-protein signaling, depending on whether it associates with the GTP-bound form of Gα _s (stimulatory) or Gα _i (inhibitory). Thus, different ligands—acting through different combinations of GPCRs and G proteins—can have opposing effects on the same intracellular signaling pathway. N3-4

G proteins can also activate enzymes that break down cyclic nucleotides. For example, the G protein called transducin contains an α _t subunit that activates the cGMP phosphodiesterase, which in turn catalyzes the breakdown of cGMP to GMP (see Fig. 3-5 B ). This pathway plays a key role in phototransduction in the retina (see p. 368 ).

G proteins can also couple to phospholipases. These enzymes catabolize phospholipids, as discussed in detail below in the section on G-protein second messengers. This superfamily of phospholipases can be grouped into phospholipases A ₂ , C, or D on the basis of the site at which the enzyme cleaves the phospholipid. G proteins that include the α _q subunit activate phospholipase C, which breaks phosphatidylinositol 4,5-bisphosphate into two intracellular messengers, membrane-associated diacylglycerol and cytosolic IP ₃ (see Fig. 3-5 C ). Diacylglycerol stimulates protein kinase C, whereas IP ₃ binds to a receptor on the endoplasmic reticulum (ER) membrane and triggers the release of Ca ²⁺ from intracellular stores.

Some G proteins interact with ion channels. Agonists that bind to the β adrenergic receptor activate the L-type Ca ²⁺ channel (see pp. 190–193 ) in the heart and skeletal muscle. The α subunit of the G protein G _s binds to and directly stimulates L-type Ca ²⁺ channels and also indirectly stimulates this channel via a signal-transduction cascade that involves cAMP-dependent phosphorylation of the channel.

βγ subunits can activate downstream effectors

Following activation and disassociation of the heterotrimeric G protein, βγ subunits can also interact with downstream effectors. The neurotransmitter ACh released from the vagus nerve reduces the rate and strength of heart contraction. This action in the atria of the heart is mediated by muscarinic M ₂ AChRs, members of the GPCR family (see p. 341 ). These receptors can be activated by muscarine, an alkaloid found in certain poisonous mushrooms. Muscarinic AChRs are very different from the nicotinic AChRs discussed above, which are ligand-gated ion channels. Binding of ACh to the muscarinic M ₂ receptor in the atria activates a heterotrimeric G protein, which results in the generation of both activated Gα _i as well as a free βγ subunit complex. The βγ complex then interacts with a particular class of K ⁺ channels, increasing their permeability. This increase in K ⁺ permeability keeps the membrane potential relatively negative and thus renders the cell more resistant to excitation. The βγ subunit complex also modulates the activity of adenylyl cyclase and phospholipase C and stimulates phospholipase A ₂ . Such effects of βγ can be independent of, synergize with, or antagonize the action of the α subunit. For example, studies using various isoforms of adenylyl cyclase have demonstrated that purified βγ stimulates some isoforms, inhibits others, and has no effect on still others. Different combinations of βγ isoforms may have different activities. For example, β ₁ γ ₁ is one tenth as efficient at stimulating type II adenylyl cyclase as is β ₁ γ ₂ .

Some βγ complexes can bind to a special protein kinase called the β adrenergic receptor kinase (βARK). As a result of this interaction, βARK translocates to the plasma membrane, where it phosphorylates the ligand-receptor complex (but not the unbound receptor). This phosphorylation results in the recruitment of β-arrestin to the GPCR, which in turn mediates disassociation of the receptor-ligand complex and thus attenuates the activity of the same β adrenergic receptors that gave rise to the βγ complex in the first place. This action is an example of receptor desensitization. These phosphorylated receptors eventually undergo endocytosis, which transiently reduces the number of receptors that are available on the cell surface. This endocytosis is an important step in resensitization of the receptor system.