Second Messengers

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CHAPTER 26 Second Messengers

This chapter considers the remarkable variety of small molecules that carry signals inside living cells. These second messengers are chemically diverse, ranging from hydrophobic lipids confined to membrane bilayers, to an inorganic ion (Ca²⁺), to nucleotides (cyclic adenosine monophosphate [cAMP] and cyclic guanosine monophosphate [cGMP]), to a gas (nitric oxide). The messages that these molecules carry are encoded by their concentrations. In the simplest case, a rise or fall in the concentration of the second messenger conveys a signal from its source to its target. In other cases, the signal depends on the rate or frequency of the fluctuations in the concentration of the second messenger. The local concentration of a second messenger depends on the rate of production, the rate of diffusion from the site of production, and the rate of removal. Most second messengers are produced by enzymes that can switch on and off rapidly, allowing modulation of the concentration of second messengers on a millisecond time scale. In the case of Ca²⁺, the cytoplasmic concentration is determined by channels that release the ion from membrane-delimited stores and by pumps that remove it from cytoplasm.

The physical state of second messengers has important consequences. Lipid-derived second messengers reach different targets in the cell depending on whether they are more soluble in the lipid bilayer or in water. Similarly, Ca²⁺ acts only locally in cytoplasm, where a high concentration of binding sites impedes its free diffusion. Cyclic nucleotides diffuse rapidly through cytoplasm, but their concentrations may rise and fall locally, owing to restricted sites of synthesis combined with rapid degradation at particular sites in the cell.

The complexity of the signaling pathways is determined by the number of sources and the number of targets of each second messenger. Generally, multiple signal sources and multiple second messenger targets generate more complexity than one can fully appreciate. This chapter deals with this complexity only in passing. Chapter 27 considers a few model systems in which it is possible to understand how signals are integrated and transduced.

This chapter discusses second messengers in the following four sections: Cyclic Nucleotides, Lipid-Derived Second Messengers, Calcium, and Nitric Oxide. All of these topics are interrelated, as multiple second messengers participate in many signal-ing systems. For example, nitric oxide controls the production of cGMP, and ino-sitol triphosphate derived from a membrane lipid controls the release of Ca²⁺ into cytoplasm.

Cyclic Nucleotides

Two cyclic nucleoside monophosphates—adenosine 3′,5′-cyclic monophosphate (cAMP) and guanosine 3′,5′-cyclic monophosphate (cGMP)—are employed as second messengers (Fig. 26-1). Both act by binding reversibly to specific proteins. Enzymes that produce and degrade cyclic nucleotides determine the concentrations of these messengers available to bind targets. These enzymes turn over substrates rapidly, so they can amplify signals massively on a millisecond time scale, under the control of diverse signaling pathways (see Chapter 27). Cyclases make cyclic nucleotides in a single step from the corresponding nucleoside triphosphate, either adenosine triphosphate (ATP) or guano-sine triphosphate (GTP).

Figure 26-1 cyclic nucleotide metabolism. Synthesis and degradation of cAMP and cGMP, including regulatory inputs and targets. G_sα, G_iα, and G_tα are trimeric GTPase a subunits (see Fig. 25-9). C A, calcium; NO, nitric oxide.

Cyclic nucleotides diffuse in the cytoplasm at about the same rate as in free solution, activating a modest repertoire of downstream targets, including protein kinases (see Figs. 25-3 and 25-4), cyclic nucleotide–gated ion channels (see Fig. 10-10), and, in the case of cAMP, one class of nucleotide exchange factors (Epac) for small GTPases (Rap1 and Rap2). The components of this system are quite ancient, since the protein domains that bind cAMP or cGMP are homologous to the cAMP-binding domain of CAP, a bacterial transcription factor. Enzymes called cyclic nucleotide phosphodiesterases degrade cAMP and cGMP to inactive nucleoside 5′-monophosphates. Eleven genes encode more than 40 different phosphodiesterases, which vary in their specificities for the two cyclic nucleotides, expression in various tissues, and localization to cellular compartments.

A family (10 human genes) of enzymes called adenylyl cyclases synthesize cAMP from ATP. Multiple transmembrane segments anchor most of these enzymes to the plasma membrane. Two homologous catalytic domains reside in the cytoplasm (C₁ and C₂ in Fig. 26-2). These cytoplasmic domains can be produced experimentally as soluble proteins separately from the transmembrane domains and can then be recombined to make a fully active enzyme. Both domains are necessary because the active site lies at the interface of the two domains. One adenylyl cyclase is a soluble enzyme that can concentrate in the nucleus. Generally, the concentration of adenylyl cyclases is very low relative to the trimeric G-proteins that regulate their activity.

Figure 26-2 adenylyl cyclase. A, Topology of the polypeptide. The C_1a and C_2a regions fold together to form the active enzyme. B, Atomic structure of the catalytic domains of adenylyl cyclase associated with G_sα. ATP is bound to the active site. Breaks in the chain are due to disordered regions.

(PDB file: 1CJK. Reprinted with permission from Tesmer JJ, Sunahara RK, Gilman AG, Sprang R: Crystal structure of the catalytic domains of adenylyl cyclase in a complex with G_sαGTP_γS. Science 278:1907–1916, 1997.) Copyright 1997 AAAS.

Multiple regulatory mechanisms act synergistically to regulate adenylyl cyclases. GTP-G_sα the GTPase subunit of a trimeric G-protein, activates many membrane-associated adenylyl cyclases by binding far from the active site (Fig. 26-2) and inducing a conformational change. Ca²⁺-calmodulin or protein kinase C (PKC) activates some adenylyl cyclases. GTP-G_iα the GTPase subunit of another trimeric G-protein, or protein kinase A (PKA) inhibits some cyclases. Gβγ subunits of trimeric G-proteins activate some adenylyl cyclases but inhibit others. These diverse regulatory mechanisms allow adenylyl cyclases to integrate a variety of input signals. The diterpene forskolin from a Coleus plant binds to and activates adenylyl cyclases. Forskolin is useful experimentally for manipulating the cAMP concentration in cells and, on the basis of its ability to bind adenylyl cyclases, was essential in the initial purification of the enzyme by affinity chromatography. Regulation of a soluble form of adenylyl cyclase differs from that of all other isoforms. It is activated by bicarbonate, an essential step in sperm maturation.

In resting cells, the concentration of cAMP is so low, approximately 10⁻⁸M, that it does not bind its targets. Stimulation of appropriate receptors (such as the seven-helix β-adrenergic receptor, see Fig. 27-3) increases the cytoplasmic cAMP concentration more than 100-fold, enough to saturate the PKA regulatory subunits (Fig. 26-3). Dissociation of the regulatory (R) subunits frees the active PKA catalytic subunits to phosphorylate cytoplasmic and membrane substrates and to move into the nucleus to activate the transcription factor CREB (cyclic nucleotide regulatory element–binding protein) (see Fig. 15-22).

Figure 26-3 Images of cAMP transients in cultured aplysia sensory neurons. Neurons were injected with protein kinase A (PKA) labeled on the catalytic subunit with fluorescein and on the regulatory subunit with rhodamine. Fluorescence energy transfer between the dyes on the two subunits provides an assay for cAMP, which dissociates the subunits and reduces energy transfer. Fluorescent dyes also allow detection of the subunits inside the neuron. A, Free cAMP in the resting cell is <50nM (blue). B–C, Stimulation with serotonin activates adenylyl cyclase and increases cytoplasmic cAMP to the micromolar range (red), especially within fine processes with a high surface-to-volume ratio. Images 120μm wide were taken just inside the cell near the coverslip. D, Another resting neuron imaged at the level of the nucleus. At the low resting level of cAMP, <50nM (blue) labeled PKA is excluded from the nucleus. E, Stimulation with serotonin plus forskolin (to stimulate adenylyl cyclase) and isobutylmethylxanthine (IBMX) to inhibit phosphodiesterases that catalyze cAMP breakdown, raises the concentration of cAMP around the nucleus (yellow). F, Two hours later, the free catalytic subunit of PKA (pink) accumulates in the nucleus.

(Courtesy of R. Y. Tsien, University of California, San Diego. F, Reprinted with permission from Bacskai BJ, Hochner B, Mahaut-Smith M, et al: Spatially resolved dynamics of cAMP and protein kinase A in Aplysia neurons. Science 260:222–226, 1993. Copyright 1993 AAAS.)

Outside the animal kingdom, cAMP has many functions. In bacteria, cAMP controls gene expression in response to nutritional conditions. The cellular slime mold Dictyostelium uses cAMP as an extracellular signal, acting through a seven-helix receptor, for its social interactions (see Fig. 38-12).

Guanylyl cyclases are dimeric enzymes similar to adenylyl cyclases. In fact, mutation of just two amino acid residues can convert a guanylyl cyclase to an adenylyl cyclase. Vertebrates express two types of guanylyl cyclases (see Fig. 24-9). A family of transmembrane receptors with cytoplasmic cyclase domains respond to ligand binding by producing cGMP. Cytoplasmic guanylyl cyclases are activated by nitric oxide and carbon monoxide, which bind a heme group in a regulatory domain (see the section titled “Nitric Oxide” later in this chapter).

Lipid-Derived Second Messengers

Phosphoglycerides (see Fig. 7-2) and sphingolipids (see Fig. 7-3) not only form cellular membranes but also participate in a wide range of signaling mechanisms. The long list of intracellular and extracellular second messengers derived from lipids will undoubtedly expand in the future. Three membrane lipids are the primary sources of these signaling molecules (Fig. 26-4):

1. Phosphatidylinositol and its various phosphorylated derivatives, discussed later, are minor lipids of the cytoplasmic leaflet of the plasma membrane and some organelle membranes.

2. Phosphatidylcholine is a major membrane phosphoglyceride found in both leaflets of the plasma membrane and organelle membranes (see Fig. 7-2).

3. Sphingomyelin, the major membrane sphingolipid, concentrates in the outer leaflet of the plasma membrane (see Fig. 7-3).

Figure 26-4 production of lipid second messengers by enzymatic attacks on three parent lipids. A, PIP₂ (phosphatidylinositol 4,5-bisphosphate).B, PC (phosphatidylcholine).C, Sphingomyelin. Second messengers are named and are surrounded by colored boxes.

Enzyme Reactions That Produce Lipid Second Messengers

Just three kinds of enzymes—phospholipases, lipid kinases, and lipid phosphatases—produce most lipid-derived second messengers. Remarkably, most conceivable products produced by these enzymes from the three parent lipids participate in signaling reactions, either directly or indirectly. The second messengers produced by these reactions partition between the aqueous phase of the cytoplasm (products designated by italics in this section) and the hydrophobic phase of the membrane bilayer (products designated by bold italics in this section). In the following paragraphs, the details of the various enzymatic reactions and the structures of the lipid derivatives are less important than is the broader principle that cells use the full range of chemical diversity in their membrane lipids to create chemical signals to regulate cellular activities.

Three types of enzyme reactions generate lipid second messengers:

1. Different phospholipases cleave the four ester bonds of phosphoglycerides (Fig. 26-4). Corresponding enzymes attack the two ester bonds and single amide bond of sphingomyelin. Cells have three main intracellular phospholipases. Phospholipase A2 (PLA2) removes the C2 fatty acid, yielding a free fatty acid, which partitions into the cytoplasm with the aid of fatty acid–binding proteins, and a lysophosphoglyceride. The corresponding ceramidase removes the fatty acid from sphingomyelin. When phospholipase C (PLC) cleaves the phosphorylated head group (such as inositol 1,4,5-triphosphate [IP₃]) from a phosphoglyceride, it leaves behind diacylglycerol (DAG) in the membrane bilayer. The corresponding sphingomyelinase leaves behind ceramide in the membrane bilayer. Phospholipase D (PLD) cleaves the polar head group from phosphoglycerides, producing phosphatidic acid that re-mains in the bilayer. Production of phosphatidic acid from phosphatidylcholine generates choline, which has no known signaling activity. Note that the phospholipase A1 that cleaves the ester bond linking the fatty acid to the C1 position on the glycerol is not yet known to participate in signaling.

2. Lipid kinases add phosphate groups to diacylglycerol to make phosphatidic acid and to phosphatidylinositol to make a variety of polyphosphoinositides, including phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-bisphosphate (PIP₂), and phosphatidylinositol 3,4,5-trisphosphate (PIP₃). PIP₂ is a substrate for a family of phosphoinositide-specific phospholipase Cs that produce the important signaling molecules DAG and IP₃ (Fig. 26-4A).

3. Lipid phosphatases remove phosphate from phosphatidic acid (another way to make DAG) and phosphates from inositol head groups.

Cells also use transferases that add or exchange head groups or fatty acids on membrane phosphoglycerides (see Fig. 7-2). These enzymes are essential for lipid biosynthesis but have not been implicated in signaling. For example, transferases add choline (as the nucleotide conjugate cytidine diphosphate [CDP]–choline) to DAG to make phosphatidylcholine). In the case of phosphatidylinositol, the head group is provided by dephosphorylation of IP₃ to inositol, which is recombined enzymatically with a CDP conjugate of DAG to make phosphatidylinositol.

Sequential action of two or more enzymes produces some lipid second messengers. For example, cells can make DAG in two steps from phosphatidylcholine: PLD first makes phosphatidic acid, which is dephosphorylated by phosphatidic acid phosphatase. PLD followed by PLA2 makes lysophosphatidic acid from phosphatidylcholine. PLA2 also initiates the production of a huge family of fatty acid derivatives called eicosanoids by cleaving the fatty acid arachidonic acid from phosphatidylcholine. Cyclooxygenases and lipoxygenases then modify arachidonic acid to make prostaglandins, thromboxanes, and leukotrienes (Figs. 26-9 and 26-10), which leave the cell to interact with cell surface receptors.

Figure 26-9 pathway of prostaglandin synthesis. Cyclooxygenase-1 (ribbon diagram) is a homodimer that is bound to the surface of a lipid bilayer by hydrophobic membrane-binding helices. This enzyme has two active sites that convert arachidonic acid to prostaglandin H₂. A hydrophobic channel 2.5nm long leads from the bilayer to the active sites. Nonsteroidal anti-inflammatory drugs compete with arachidonic acid for binding to the cyclooxygenase active site, and aspirin covalently modifies serine 530 in the active site. Specific prostaglandin synthases convert prostaglandin H₂ into various products. PC, phosphatidylcholine; PE, phosphatidyl ethanolamine; PI, phosphatidylinositol; TXA₂, thromboxane A. (PDB file: 1CQE.)

Figure 26-10 PATHWAYS OF LEUKOTRIENE SYNTHESIS.

Agonists and Receptors

Most signaling pathways have the potential to produce lipid second messengers, depending on the expression of the appropriate enzymes. Some pathways activate enzymes directly; receptor tyrosine kinases bind and activate certain phosphatidylinositol phospholipase Cs (PI-PLCs). Seven-helix receptors signal through trimeric G-proteins to activate another PI-PLC. Other pathways are less direct; one leads from G-protein-coupled receptors or receptor tyrosine kinases to DAG and PKC, activating an isoform of phospholipase D.

Targets of Lipid Second Messengers

Downstream targets of the lipid second messengers are diverse but can be generalized to some extent into three categories (Fig. 26-5):

1. Most lipid second messengers derived from phosphoglycerides and retained in the membrane bilayer exert their physiological effects on PKC isozymes (Fig. 26-6). Ceramide is also retained in the bilayer, where it activates another protein kinase and a protein phosphatase (see Fig. 26-11). Phosphatidic acid activates a lipid kinase, PI5 kinase, which phosphorylates phosphatidylinositol (Fig. 26-7).

2. IP₃ and sphingosine-1-phosphate use different mechanisms to release Ca²⁺ from vesicular stores in the cytoplasm (Fig. 26-12).

3. All water-soluble lipid second messengers containing or derived from fatty acids (platelet-activating factor [PAF], lysophosphatidic acid [LPA], eicosanoids) leave the cell and bind to G-protein-coupled, seven-helix receptors on the surface of target cells.

Figure 26-5 generation of lipid second messengers and their cellular targets. LysoPtdCho, lysophosphatidyl choline; PKC, protein kinase C; PP2A, protein phosphatase 2A; PtdIns (3,4,5)-P₃, PIP₃; PtdOH, phosphatidic acid; SMase, sphingomyelinase.

(Adapted from Liscovitch M, Cantley LC: Lipid second messengers. Cell 77:329–334, 1994.)

Figure 26-6 protein kinase c (pkc) family. A, Domain organization of PKC isozymes with ligand-binding sites. PS indicates the pseudosubstrate sequence that binds to and inhibits the kinase catalytic site. B, Tissue distribution.C, Activators. DAG, diacylglycerol; FFA, free fatty acid, LysoPC, lysophosphatidylcholine; PIP₃ is phosphatidylinositol 3,4,5-trisphosphate; PS, phosphatidylserine.

Figure 26-11 sphingomyelin/ceramide signaling pathway. Stimulation of the tumor necrosis factor (TNF) receptor activates a neutral sphingomyelinase, which cleaves choline from sphingomyelin. Ceramide flips across the bilayer and activates a cytoplasmic kinase as well as PKC-ζ and a protein phosphatase.

Figure 26-7 synthesis and turnover of phosphoinositides. Enzymes regulated by receptor tyrosine kinases and seven-helix receptors are purple. Enzymes regulated by receptor tyrosine kinases are blue. Second messengers aregreen. PA, phosphatidic acid; PI-3k, phosphatidylinositol 3 kinase; PI-4k, phosphatidylinositol 4 kinase; PI-5k, phosphatidylinositol 5 kinase; PtdIns, phosphatidylinositol; PTEN, phosphatase and tensin homolog deleted on chromosome 10 (a tumor suppressor).

Figure 26-12 Pathways of Ca²⁺ release and uptake. Ca²⁺ pumps in the plasma membrane and endoplasmic reticulum membrane use ATP hydrolysis to pump Ca²⁺ out of the cytoplasm. A variety of receptors activate phospholipase C (PLC) isozymes to produce IP₃ from PIP₂. IP₃ diffuses to the endoplasmic reticulum, where it opens IP₃ receptors (IP₃R), releasing Ca²⁺ from the lumen into cytoplasm. Voltage-sensitive l-type Ca²⁺ channels respond to membrane depolarization by admitting extracellular Ca²⁺. Depletion of Ca²⁺ stored in the endoplasmic reticulum activates plasma membrane Ca²⁺ channels, likely members of the Trp channel family, which admit extracellular Ca²⁺ to replenish stores.

With so much potential for information transfer (multiple agonists, multiple membrane transduction mechanisms, multiple lipid second messengers, and multiple downstream targets), where is the specificity in these systems? Do all cells respond in all possible ways? The answer is no, and the explanation is that the protein hardware required for these reactions is selectively expressed in differentiated cells and carefully localized at particular sites in cells, such as the plasma membrane, nucleus, or cytoskeleton. In addition, targeting of PKC isozymes to particular cellular compartments ensures that only selected substrates are phosphorylated in response to lipid second messenger production. Thus, each cell takes a limited number of items from the lipid second messenger menu and uses them to produce a selective, customized response to each agonist.

Lipid second messengers participate in many processes that are considered elsewhere in the book. For example, regulated secretion (see Chapter 21) in response to an agonist binding a seven-helix or tyrosine kinase receptor requires a transient Ca²⁺ signal in the cytoplasm. The Ca²⁺ is released from the membrane stores by IP₃, which is produced by the action of a PI-PLC on PIP₂. IP₃-mediated Ca²⁺ release from the endoplasmic reticulum also controls smooth muscle contraction (see Fig. 39-21).

Protein Kinase C

Many lipid second messengers, including DAG, PIP₃, arachidonic acid, phosphatidic acid, and lysophosphatidylcholine activate one or more of the 10 protein kinase C (PKC) isozymes expressed by vertebrate cells (Fig. 26-6). These multiple PKC isozymes provide a selective response to various lipid second messengers. Some, but not all, PKC isozymes also require Ca²⁺ for activation. Sphingosine may inhibit some PKC isozymes.

PKCs have a protein kinase catalytic domain (see Fig. 25-3) and N-terminal regulatory domains. A pseudosubstrate sequence binds intramolecularly to the active site and inhibits activity by blocking substrate binding. Pseudosubstrates have alanine at the phosphorylation site instead of serine as substrates have. During apoptosis, caspases cleave off this regulatory domain (see Fig. 46-10) producing constitutively active PKC isoforms.

Lipid second messengers activate PKC by binding C1 regions and dissociating the pseudosubstrate from the active site. Phorbol esters, pharmacological activators of PKC that promote tumor formation, bind PKC in a similar fashion. Binding DAG or phorbol esters also requires phosphoglycerides, such as phosphatidylserine. Ca²⁺-dependent PKC isozymes have C2 regions that mediate binding to phospholipids in the presence of Ca²⁺.

Activated PKCs have many potential targets in cells and have been implicated in the regulation of cellular activities ranging from gene expression to cell motility to the generation of lipid second messengers. PKC isozymes differ in their activity toward protein substrates. Localization of various PKC isozymes to different parts of the cell might provide additional specificity. Receptors for activated C kinases bind C2 regions and target PKC isozymes to the plasma membrane, cytoskeleton, or nucleus.

PKCs can provide either positive or negative feedback to the signaling pathways that turn them on. PKC activates PLD and PLA2 and provides positive feedback, because those enzymes produce more DAG to sustain the activation of PKC. On the other hand, PKC provides negative feedback when it phosphorylates and inhibits both growth factor receptors and PI-PLCγ1. PKC also phosphorylates and inhibits PI-PLCβ, generating negative feedback after activation of seven-helix receptors. Negative feedback makes both of these signaling events transient.

Phosphoinositide Signaling Pathways

Although minor in terms of mass in biological membranes, phosphoinositides are major players in signal-ing (Fig. 26-7). The parent compound, phosphatidyl-inositol (PI), is a phosphoglyceride with a cyclohexanol head group called inositol. Specific lipid kinases phosphorylate the 4 and 5 hydroxyl groups of phosphatidylinositol to form PI(4-)P and PI(4,5-)P₂, usually referred to simply as PIP and PIP₂. A specific phosphatase can remove the D5 phosphate. Inactivation of the single copy of this gene on the X-chromosome causes Lowe’s syndrome with cataracts, renal failure, and mental retardation.

PIP₂ is a substrate for a family of receptor-controlled PI-PLCs that cleave off the phosphorylated head group, producing two potent second messengers: IP₃ and DAG. Water-soluble IP₃ activates Ca²⁺ release channels in the endoplasmic reticulum (Fig. 26-12), and lipid-soluble DAG activates PKC. In contrast to Ca²⁺, which diffuses slowly and acts locally, IP₃ diffuses rapidly through the cytoplasm, triggering Ca²⁺ release. DAG is confined to membranes but diffuses laterally to bind and activate PKC. Enzymes inactivate both IP₃ and DAG. DAG is inactivated by phosphorylation to make phosphatidic acid, a second messenger in its own right but also an intermediate in the resynthesis of phosphoinositides. IP₃ is dephosphorylated to inositol, which is inactive as a second messenger. LiCl inhibits the final step: dephosphorylation of inositol-1-phosphate. Remarkably, Li⁺ is clinically useful as a treatment for bipolar disorder, presumably by interfering with phosphoinositide signaling in the brain. In some tissues, IP₃ is inactivated by phosphorylation to inositol 1,3,4,5-tetrakisphosphate (IP₄).