Drawing of a human fetus in utero, by Leonardo da
Vinci. Human sexuality and embryonic development
represent two hormonally regulated processes of
universal interest. (Florentine Royal Collection.
Windsor, England A.K.G., Berlin/Superstock, International)

SPECIAL TOPIC

Chapter 34

The Reception and Transmission of Extracellular Information

Higher life forms must have molecular mechanisms for detecting environmental information as well as mechanisms that allow for communication at the cell and tissue levels. Sensory systems detect and integrate physical and chemical information from the environment and pass this information along by the process of neurotransmission. Control and coordination of processes at the cell and tissue levels are achieved not only by neurotransmission but also by chemical signals in the form of hormones that are secreted by one set of cells to direct the activity of other cells. In this chapter, we will address these mechanisms of information transfer, beginning with the molecular basis of hormone action and then moving to a special focus on excitable membranes, neurotransmission, and sensory systems.
            Hormones are secreted by certain cells, usually located in glands, and travel, either by simple diffusion or circulation in the bloodstream, to specific target cells. As we shall see, some hormones bind to specialized receptors on the plasma membrane and induce responses within the cell without themselves entering the target cell. Other hormones actually enter the target cell and interact with specific receptors there. By these mechanisms, hormones regulate the metabolic processes of various organs and tissues; facilitate and control growth, differentiation, reproductive activities, and learning and memory; and help the organism cope with changing conditions and stresses in its environment.

Many different chemical species act as hormones. Steroid hormones, all derived from cholesterol, regulate metabolism, salt and water balances, inflammatory processes, and sexual function. Several hormones are amino acid derivatives. Among these are epinephrine and norepinephrine, which regulate smooth muscle contraction and relaxation, blood pressure, cardiac rate, and the processes of lipolysis and glycogenolysis, and the thyroid hormones, which stimulate metabolism. Peptide hormones are a large and still expanding group of hormones that appear to regulate processes in all body tissues, including the release of yet other hormones.
            Hormones and other signal molecules in biological systems bind with very high affinities to their receptors, displaying K_D values in the range of 10^-12 to 10^-6 M. The concentrations of hormones are maintained at levels equivalent to or slightly above these K_D values. Once hormonal effects have been induced, the hormone is usually rapidly metabolized.
            Hormonal regulation depends upon the transduction of the hormonal signal across the plasma membrane to specific intracellular sites, particularly the nucleus. Many steps in these signaling pathways involve phosphorylation of serine, threonine, and tyrosine residues on target proteins. The complexity of signal transduction is thus manifested in the estimates that the human genome may contain between 1000 and 3000 protein kinases. Although only a few of these kinases—and other signaling proteins and molecules—have been identified, the principles that govern these pathways are now becoming apparent (Chapter 15).
            Signaling pathways must operate with speed and precision, facilitating the accurate relay of intracellular signals to specific targets. But how does this happen? Many protein kinases and phosphatases have relatively broad specificities, but they depend upon interactions between specialized recognition domains on the involved proteins and also upon cellular strategies for localization of the signaling molecules. As we shall see later, modular proteins having one or more protein-protein or protein-lipid recognition domains mediate these events.

34.2 · Signal-Transducing Receptors Transmit the Hormonal Message

Very often in life, the message is more important than the messenger, and this is certainly true for hormones. The structure and chemical properties of a hormone are only important for specific binding of the hormone to its appropriate receptor. Of much greater interest and importance, however, is the metabolic information carried by the hormonal signal. The information implicit in the hormonal signal is interpreted by the cell, and an intricate pattern of cellular responses ensues.
   Figure 34.1 · Nonsteroid hormones bind exclusively to plasma membrane receptors, which mediate the cellular responses to the hormone. Steroid hormones exert their effects either by binding to plasma membrane receptors or by diffusing to the nucleus, where they modulate transcriptional events.

         Steroid hormones may either bind to plasma membrane receptors or exert their effects within target cells, entering the cell and migrating to their sites of action via specific cytoplasmic receptor proteins (Figure 34.1). The nonsteroid hormones, which act by binding to outward-facing plasma membrane receptors, activate various signal transduction pathways that mobilize various second messengers—cyclic nucleotides, Ca²⁺ ions, and other substances that activate or inhibit enzymes or cascades of enzymes in very specific ways. These hormonally activated processes are the focus of this chapter.

            All receptors that mediate transmembrane signaling processes fit into one of three receptor superfamilies:

1.  The 7-transmembrane segment (7-TMS) receptors are integral membrane proteins with seven transmembrane (helical) segments, an extracellular recognition site for ligands, and an intracellular recognition site for a GTP-binding protein (see following discussion).

2.  The single-transmembrane segment (1-TMS) catalytic receptors are proteins with only a single transmembrane segment and substantial globular domains on both the extracellular and intracellular faces of the membrane. The extracellular domain in the ligand recognition site and the intracellular catalytic domain is either a tyrosine kinase or a guanylyl cyclase.

3.  Oligomeric ion channels consist of associations of protein subunits, each of which contains several transmembrane segments. These oligomeric structures are ligand-gated ion channels. Binding of the specific ligand typically opens the ion channel. The ligands for these ion channels are neurotransmitters.

34.3 · Intracellular Second Messengers

Epinephrine and glucagon activate glycogen breakdown in the liver (Chapters 15 and 23), but the mechanism of this activation was a mystery until Earl Sutherland and his colleagues showed that the glycogen phosphorylase reaction (Figure 23.16), the initial step in glycogen breakdown, was stimulated by epinephrine and glucagon. Activation of phosphorylase was eventually shown to be due to an ATP-dependent phosphorylation of the enzyme. Sutherland also showed that a phosphatase from the liver cells inactivated phosphorylase, eliminating the activation due to phosphorylation. A significant breakthrough was achieved with Sutherland’s demonstration that hormones activated phosphorylase only in the presence of plasma membrane fragments. He hypothesized that binding of epinephrine or glucagon to a receptor in the membrane activated synthesis or release of a substance that activated phosphorylation of phosphorylase. This crucial substance was eventually shown (in 1956) to be adenosine 3',5'-cyclic monophosphate, now known as cyclic AMP, denoted cAMP (Figure 34.2).

Figure 34.2 · Cyclic AMP is synthesized by membrane-bound adenylyl cyclase and degraded by soluble phosphodiesterase.

Cyclic AMP is produced by an integral membrane enzyme, adenylyl cyclase (Figure 34.2). The strain and bond distortion inherent in the bicyclic ring structure of cAMP make its formation an endergonic process, but spontaneous hydrolysis of pyrophosphate formed in this reaction drives the synthesis of cAMP forward. The phosphodiesterase reaction hydrolyzes cAMP to AMP as shown. Because this reaction relieves the strain in the cAMP structure, it is highly exergonic (DG°' = 250.4 kJ/mol; see Table 3.1). Figure 34.3 · Earl Sutherland’s simple model of hormone action, circa 1967. Phosphorylase a was eventually shown to be a phosphorylated form of glycogen phosphorylase. Although Sutherland didn’t realize it, the interaction between the hormone receptor and adenylyl cyclase is not direct, but instead involves GTP-binding proteins.

Sutherland thought of the hormone as the first messenger signaling a need for glycogen breakdown, and he called cAMP a second messenger. The basic process, as Sutherland understood it in the 1960s, is shown in Figure 34.3. In this second messenger model, the hormone causes a variety of intracellular effects without itself entering the cell. It was eventually shown that many hormonally activated processes use cAMP as a second messenger. For his remarkable achievements in the elucidation of this model, Earl Sutherland received the Nobel Prize in physiology or medicine in 1971.
            Since Sutherland’s discovery of cAMP, many other second messengers have been identified (Table 34.1). The mediators of second messenger formation for all 7-TMS receptors are GTP-binding proteins.

34.4 · GTP-Binding Proteins: The Hormonal Missing Link

Figure 34.4 · The structure of guanylylimidodiphosphate.

Two observations in the early 1970s implicated another protein in the hormonal activation of adenylyl cyclase. First, purification of adenylyl cyclase and the hormone receptor resulted in a loss of hormone stimulation of cyclase activity. Second, Martin Rodbell and his colleagues showed that GTP was necessary for hormone activation of adenylyl cyclase. Interestingly, 5'-guanylylimidodiphosphate (GMP-PNP), a nonhydrolyzable analog of GTP (Figure 34.4), was a “superactivator” of adenylyl cyclase, giving higher cyclase activities than GTP itself. This prompted Rodbell to suggest that the GTP-binding site was the active site of a GTPase. In 1977, Elliott Ross and Alfred Gilman at the University of Virginia reported the partial purification of a GTP-binding protein, which, when reconstituted with the cyclase and hormone receptor, restored hormone stimulation to the adenylyl cyclase reaction. Thus, adenylyl cyclase is not directly activated by the hormone-receptor complex. Instead, binding of hormone to the receptor stimulates a GTP-binding protein (abbreviated now to G protein), which in turn activates adenylyl cyclase.

Figure 34.5 · Activation of adenylyl cyclase by heterotrimeric G proteins. Binding of hormone to its receptor causes a conformational change that induces the receptor to catalyze a replacement of GDP by GTP on G_a. The G_a(GTP) complex dissociates from G_bg and binds to adenylyl cyclase, stimulating synthesis of cAMP. Bound GTP is slowly hydrolyzed to GDP by the intrinsic GTPase activity of G_a. G_a(GDP) dissociates from adenylyl cyclase and reassociates with G_bg. G_a and G_g are lipid-anchored proteins. Adenylyl cyclase is an integral membrane protein consisting of 12 transmembrane a-helical segments.

Typically, G proteins are heterotrimers consisting of a- (45 to 47 kD), b- (35 kD), and g- (7 to 9 kD) subunits. The a-subunit binds GDP or GTP and has an intrinsic, slow GTPase activity. The Gabg complex in the unactivated state has GDP at the nucleotide site (Figure 34.5). Binding of hormone to receptor stimulates a rapid exchange of GTP for GDP on Ga. The binding of GTP causes Ga to dissociate from Gbg and to associate with an effector protein such as adenylyl cyclase. Binding of Ga (GTP) activates adenylyl cyclase. The adenylyl cyclase actively synthesizes cAMP as long as Ga(GTP) remains bound to it. However, the intrinsic GTPase activity of Ga eventually hydrolyzes GTP to GDP, leading to dissociation of Ga(GDP) from adenylyl cyclase and reassociation with the Gbg dimer, regenerating the inactive heterotrimeric Gabg complex.
Figure 34.6 · (a) Adenylyl cyclase activity is modulated by the interplay of stimulatory (G_s) and inhibitory (G_i) G proteins. Binding of hormones to b₁- and b₂-adrenergic receptors activates adenylyl cyclase via G_s, whereas hormone binding to a₂ receptors leads to the inhibition of adenylyl cyclase. Inhibition may occur by direct inhibition of cyclase activity by G_ia or by binding of G_ibg to G_sa(GTP). (b) Two views of the complex of the VC₁-IIC₂ catalytic domain of adenylyl cyclase and G_sa (c) Details of the G_sa complex in the same orientation as the structures above. (Courtesy of Alfred Gilman, University of Texas Southwestern Medical Center.)

            Two stages of amplification occur in the G-protein-mediated hormone response. First, a single hormone-receptor complex can activate many G proteins before the hormone dissociates from the receptor. Second, and more obvious, the Ga-activated adenylyl cyclase synthesizes many cAMP molecules. Thus, binding of hormone to a very small number of membrane receptors stimulates a large increase in concentration of cAMP within the cell. The hormone receptor, G protein, and cyclase constitute a complete hormone signal transduction unit (Figure 34.6a).
            A given G protein can be activated by several different hormone-receptor complexes. For example, either glucagon or epinephrine, binding to their distinctive receptor proteins, can activate the same species of G protein in liver cells. The effects are additive, and stimulation by glucagon and epinephrine together leads to higher cytoplasmic concentrations of cAMP than activation by either hormone alone.
            G proteins are a universal means of signal transduction in higher organisms, activating many hormone-receptor-initiated cellular processes in addition to adenylyl cyclase. Such processes include, but are not limited to, activation of phospholipases C and A₂, and the opening or closing of transmembrane channels for K⁺, Na⁺, and Ca²⁺ in brain, muscle, heart, and other organs (Table 34.2). G proteins are integral components of sensory pathways such as vision and olfaction. More than 100 different G protein-coupled receptors and at least 21 distinct G proteins are known. At least a dozen different G protein effectors have been identified, including a variety of enzymes and ion channels.

Hormone-receptor-mediated processes regulated by G proteins may be stimulatory (as we have seen) or inhibitory. Each hormone receptor protein interacts specifically with either a stimulatory G protein, denoted G_s, or with an inhibitory G protein, denoted G_i. For example, the catecholamine hormones (such as epinephrine) bind to four different types of adrenergic receptor proteins, designated a₁, a₂, b₁, or b₂. Binding to a₁ receptors has no effect on adenylyl cyclase, whereas binding to b₁ or b₂ receptors is stimulatory and binding to a₂ receptors is inhibitory. The b₁ and b₂ receptors interact specifically with a G_s complex, and a₂ receptors interact only with G_i proteins. The “G protein” described in Figure 34.5 is in fact a G_s-type G protein. Two possibilities exist (Figure 34.6a). Binding of a hormone to its receptor triggers GDP^zyGTP exchange and dissociation of G_{ia(GTP) from G}ibg. Inhibition may then occur either by binding of G_ia_{(GTP) to adenylyl cyclase to directly inhibit the cyclase, or by action of G}ibg, which can compete with the cyclase for G_sa_{(GTP) complexes. The presence in liver cell membranes of much more G}i than G_s favors the competitive role for G_ibg.
            Alfred Gilman, Stephen Sprang, and coworkers have determined the structure of a complex of G_sa _{(with bound GTP) with the cytoplasmic domains (VC}1 and IIC₂) of adenylyl cyclase (Figure 34.6b). The G_sa _{complex binds to a cleft at one corner of the C}2 domain, and the surface of G_sa_{-GTP that contacts adenylyl cyclase is the same surface that binds the G}bg dimer. The catalytic site, where ATP is converted to cyclic AMP, is far removed from the bound G protein.

Figure 34.7 · ADP-ribosylation of G_sa by cholera toxin.

Many of the details of G protein mediation of hormonal effects have been elucidated through studies of the potent effects of bacterial toxins, including cholera toxin and pertussis toxin. Vibrio cholerae, the Gram-negative bacterium that causes cholera, induces severe diarrhea in its victims, leading to death if the fluids are not replenished. Cholera toxin is an 87-kD protein consisting of an A subunit and five B subunits. The B subunits act in host-cell recognition. The A subunit consists of A₁ and A₂ peptides linked by a disulfide bond. The 22-kD A₁ peptide catalyzes the ADP-ribosylation of Arg²⁰¹ in the a-subunit of G_s (Figure 34.7), using NAD⁺ as a substrate. ADP-ribosylation strongly inhibits the GTPase activity of G_sa_{, effectively trapping G}sa in the activated state and causing prolonged activation of adenylyl cyclase. Elevated levels of cyclic AMP in turn cause intestinal epithelial cells to secrete high volumes of fluid. Both the bacteria and the cholera toxin remain localized in the intestines through the course of the disease, but if fluids are actively replaced, the body’s immune system eventually gains control and destroys the bacteria.

ADP-ribosylation of a Cys residue on G_ia _{is catalyzed by pertussis toxin, a product of Bordetella pertussis, the bacterium that causes whooping cough. Pertussis toxin is a 110-kD hexameric protein (a 28-kD A subunit and five B-like subunits.) In this case, the ADP-ribosylation inhibits exchange of GDP for GTP, thereby preventing G}ia from inhibiting adenylyl cyclase. Pertussis, in contrast to cholera, is a systemic infection, and the breakdown of adenylyl cyclase regulation is felt by tissues throughout the body.

Figure 34.8 · The structure of ras p21-GTP complexed with (a) GDP and (b) GMP-PNP. The ras p21-GMP-PNP complex is the active conformation of this protein.

GTP-binding proteins are implicated in growth control mechanisms in higher organisms. Certain tumor virus genomes contain genes encoding 21-kD proteins that bind GTP and show regions of homology with other G proteins. The first of these genes to be identified was found in rat sarcoma virus, and was dubbed the ras gene. Genes implicated in tumor formation are known as oncogenes; often they are mutated versions of normal, noncancerous genes involved in growth regulation, so-called proto-oncogenes. The normal, cellular ras protein is a GTP-binding protein that functions similarly to other G proteins described above, activating metabolic processes when GTP is bound and becoming inactive when GTP is hydrolyzed to GDP. The GTPase activity of the normal ras p21 is very low, as appropriate for a G protein that regulates long-term effects like growth and differentiation. A specific GTPase-activating protein (GAP) increases the GTPase activity of the ras protein. Mutant (oncogenic) ras proteins have severely impaired GTPase activity, which apparently causes serious alterations of cellular growth and metabolism in tumor cells. Several crystallographic studies have determined the structures of ras proteins (Figure 34.8) in complexes with GDP, GMP-PNP, and GMP-PCP (nonhydrolyzable analogs of GTP in which the b-P and g-P are linked by N or C rather than by O). ras consists of a six-stranded b-sheet surrounded by a-helices. The five peptide loops (G-1 through G-5) that comprise the guanine nucleotide binding site are the most highly conserved elements in this structure. Two regions of the ras structure change conformation upon GTP hydrolysis. One of these, designated switch I, corresponds to the G-2 loop that forms part of the Mg²⁺-binding site. This segment plays a role in binding of GAP proteins (see A Deeper Look) and ras target proteins and is therefore called the effector loop. The second flexible element, switch II, includes G-3 (which comprises the GTP g-P binding site) and the a2 helix that follows.
Figure 34.9 · A complex between raps (a ras analog, green) and the ras-binding domain of c-raf-1, purple.

            A glimpse of the molecular interactions between a ras protein and an effector (target) protein is revealed in the structure (Figure 34.9) of a complex between a ras analog called raps and the ras-binding domain from a ras effector known as c-raf-1. The principal interactions in this complex involve an interprotein b-sheet formed by two antiparallel strands from the two proteins. GTP hydrolysis induces a coil-to-helix transition at the N-terminus of the switch II domain and a subsequent reorientation of the entire a2 helix, which destroys the effector-binding site.

A Deeper Look
RGSs and GAPs—Switches That Turn Off G Proteins
Nature has made ras p21 and Gsa very poor enzymes by design. For example, ras p21 hydrolyzes GTP with a rate constant of only 0.02 min21. These G proteins are active only in the GTP-bound state, and downstream targets will dissociate upon GTP hydrolysis. If ras p21 and Gsa were efficient enzymes, the GTP-bound state would be short-lived, and G protein-mediated signaling would be ineffective.             But how can G proteins be switched off if they are inherently poor GTPases? The answer is provided by GTPase-activating proteins (GAPs) and regulators of G protein signaling (RGS), which cause dramatic increases in GTPase activity when bound to G proteins.	The left figure shows ras p21 (white) with a fragment of a GAP (blue) bound to it. GAPs increase the GTPase activity of ras p21 by a factor of 105. The right figure shows RGS (blue) bound to Gai (yellow). RGS proteins accelerate Gsa-catalyzed GTP hydrolysis by nearly 100-fold. In both ras p21 and Gsa, GTPase activity and the conversion from GTP-bound to GDP-bound forms of the protein involve conformation changes in the switch regions, portions of the G protein structure that surround the GTP/GDP binding site. GAPs and RGS increase GTPase activities of their respective G proteins by binding near the active site and stabilizing the transition state of the GTP hydrolysis reaction.

34.5 · The 7-TMS Receptors

Figure 34.10 · The arrangements of the b₂- and a₂-adrenergic receptors in the membrane. Substitution of Asp¹¹³ in the third hydrophobic domain of the b-adrenergic receptor with an Asn or Gln by site-directed mutagenesis results in a dramatic decrease in affinity of the receptor for both agonists and antagonists. Significantly, this Asp residue is conserved in all other G protein-coupled receptors that bind biogenic amines, but is absent in receptors whose ligands are not amines. Asp¹¹³ appears to be the counter-ion for the amine moiety of adrenergic ligands.

The primary and secondary structures of the 7-transmembrane segment (7-TMS) receptors are similar to those of bacteriorhodopsin (see Chapter 9) and rhodopsin. The a- and b-adrenergic receptors, for which epinephrine is a ligand, are good examples (Figure 34.10). Hydropathy analysis of these receptors is consistent with seven transmembrane a-helical segments. The extracellular N-terminal segment has two glycosylation sites. The hydrophilic loops connecting the seven hydrophobic domains are not required for ligand binding. Instead, the ligand-binding site (for the cationic catecholamines) is located within the hydrophobic core of the receptor.
            The binding of epinephrine to a b-adrenergic receptor initiates the above-described G protein activation of adenylyl cyclase. a₁-Adrenergic receptors stimulate inositol phospholipid metabolism when activated (see following section). Stimulation of a₂-adrenergic receptors appears to counteract hormone-stimulated increases in [cAMP]. The b-adrenergic receptors act through G proteins. These G proteins are coupled to several pathways, including adenylyl and guanylyl cyclases, phospholipases A and C, Ca²⁺ and K⁺ channels, and phosphodiesterases.
            The b-adrenergic receptor is desensitized by phosphorylation either by a specific b-adrenergic receptor kinase (bARK) or by protein kinase A (PKA), the cAMP-dependent protein kinase. Phosphorylation sites for both kinases are located in the receptors’ C-terminal domain. The sites phosphorylated by protein kinase A are adjacent to segments of the receptor that mediate coupling to G proteins, implying that phosphorylation at these sites interrupts this coupling. The desensitization due to bARK occurs by a different mechanism; it may involve another as yet unidentified protein.

34.6 · Specific Phospholipases Release Second Messengers

Figure 34.11 · (a) The general action of phospholipase A₂ (PLA₂), phospholipase C (PLC), and phospholipase D (PLD). (b) The synthesis of second messengers from phospholipids by the action of phospholipases and sphingomyelinase.

A diverse array of second messengers are generated by breakdown of membrane phospholipids. Binding of certain hormones and growth factors to their respective receptors triggers a sequence of events that can lead to the activation of specific phospholipases. Action of these phospholipases on membrane lipids produces the second messengers shown in Figure 34.11.

Figure 34.12 · The family of second messengers produced by phosphorylation and breakdown of phosphatidylinositol. PLC action instigates a bifurcating pathway culminating in two distinct and independent second messengers: DAG and IP₃.

Breakdown of phosphatidylinositol (PI) and its derivatives by phospholipase C produces a family of second messengers. In the best-understood pathway, successive phosphorylations of PI produce phosphatidylinositol-4-P (PIP) and phosphatidylinositol-4,5-bisphosphate (PIP₂) (Figure 34.12). Four isozymes of phospholipase C (denoted a, b, g, and d) hydrolyze PI, PIP, and PIP₂. Hydrolysis of PIP₂ by phospholipase C yields the second messenger inositol-1,4,5-trisphosphate (IP₃), as well as another second messenger, diacylglycerol (DAG). IP₃ is water-soluble and diffuses to intracellular organelles where release of Ca²⁺ is activated. DAG, on the other hand, is lipophilic and remains in the plasma membrane where it activates a Ca²⁺-dependent protein kinase known as protein kinase C (see following discussion).

Activation of Phospholipase C Is Mediated by G Proteins or by Tyrosine Kinases

Figure 34.13 · Phospholipase C-b is activated specifically by G_q, a GTP-binding protein, and also by Ca²⁺.

The different phospholipase C isozymes are activated by different intracellular events (Figure 34.13). Phospholipase C-b, -g, and -d are all Ca²⁺-dependent. In addition, phospholipase C-b is stimulated by a class of G proteins known as G_q. Binding of polypeptide hormones such as vasopressin or bradykinin to 7-TMS receptors releases G_{qa(GTP) from a G}qabg trimer. In turn, G_qa_{(GTP) activates phospholipase C-b (Figure 34.13). On the other hand, phospholipase C-g is activated by receptor tyrosine kinases (Figure}

Figure 34.14 · Phospholipase C-g is activated by receptor tyrosine kinases and by Ca²⁺.

_{34.14). The primary structures of phospholipase C-b and -g are shown in Figure 34.15. The X and Y domains of phospholipase C-b and -g are highly homologous, and both of these domains are required for phospholipase C activation. The other}

Figure 34.15 · The amino acid sequences of phospholipase C isozymes b, g, and d share two homologous domains, denoted X and Y. The sequence of g isozyme contains src homology domains, denoted SH2 and SH3. SH2 domains (approximately 100 residues in length) interact with phosphotyrosine-containing proteins (such as RTKs), whereas SH3 domains mediate interactions with cytoskeletal proteins. (Adapted from Dennis, E., Rhee, S., Gillah, M., and Hannun, E., 1991. Role of phospholipases in generating lipid second messengers in signal transduction. The FASEB Journal 5:2068-2077.)

_{domains of these isozymes confer specificity for G protein activation or tyrosine kinase activation.}

The Metabolism of Inositol-Derived Second Messengers

Figure 34.16 · The pathways of phosphoinositide biosynthesis and metabolism. The Li¹-sensitive steps are shown as red lines.

A summary of PIP₂-related signaling processes is shown in Figure 34.16. IP₃ has a cellular half-life of only a few seconds. It is rapidly processed along two principal paths: (a) it can be catabolized by a series of phosphatases to yield inositol-1,4-bisphosphate, inositol-4-phosphate, and free myo-inositol, which can be subsequently reincorporated into new inositol phospholipids; or (b) it can be phosphorylated to yield inositol-1,3,4,5-tetraphosphate, which then undergoes a complex series of phosphorylations and dephosphorylations to at least six other inositol phosphate compounds. Interestingly, many of these inositol phosphates have also been shown to behave as second messengers in various cellular processes. Some of these compounds may serve as extracellular signals in the regulation of specific neural mechanisms.

Phosphatidylcholine, Sphingomyelin, and Glycosphingolipids Also Generate Second Messengers

In addition to PI, other phospholipids serve as sources of second messengers. Breakdown of phosphatidylcholine by phospholipases yields a variety of second messengers, including diacylglycerol, phosphatidic acid, and prostaglan-dins. The action of sphingomyelinase on sphingomyelin produces ceramide, which stimulates ceramide-activated protein kinase. Similarly, gangliosides (such as ganglioside G_M3 (Chapter 8)) and their breakdown products modulate the activity of protein kinases and G protein-coupled receptors.

34.7 · Calcium as a Second Messenger

Figure 34.17 · Cytoplasmic [Ca²⁺] increases occur via the opening of Ca²⁺ channels in the membranes of calciosomes, the endoplasmic reticulum, and the plasma membrane.

Calcium ion is an important intracellular signal. Binding of certain hormones and signal molecules to plasma membrane receptors can cause transient increases in cytoplasmic Ca²⁺ levels, which in turn can activate a wide variety of enzymatic processes, including smooth muscle contraction, exocytosis, and glycogen metabolism. (Most of these activation processes depend on special Ca²⁺-binding proteins discussed in the following section.) Cytoplasmic [Ca²⁺] can be increased in two ways (Figure 34.17). As mentioned briefly earlier, cAMP can activate the opening of plasma membrane Ca²⁺ channels, allowing extracellular Ca²⁺ to stream in. On the other hand, cells also contain intracellular reservoirs of Ca²⁺, within the endoplasmic reticulum and calciosomes, small membrane vesicles that are similar in some ways to muscle sarcoplasmic reticulum. These special intracellular Ca²⁺ stores are not released by cAMP. They respond to IP₃, a second messenger derived from phosphatidylinositol (PI).

Calcium-Induced Calcium Release

Figure 34.18 · IP₃ mediates Ca²⁺-induced Ca²⁺ release. Binding of IP₃ to the ER IP₃ receptor opens ER Ca²⁺ channels. Flow of Ca²⁺ through these channels induces a conformational change that opens plasma membrane Ca²⁺ channels. (Adapted from Berridge, M., 1990. Calcium oscillations. Journal of Biological Chemistry 265:9583-9586.)

Much of the Ca²⁺ entering the cytoplasm by action of IP₃ appears to come from two sources: (a) parts of the endoplasmic reticulum that are closely associated with the plasma membrane, and (b) the extracellular environment. As shown in Figure 34.18, Ca²⁺ release appears to be a two-step process. IP₃ binding to receptors on the ER membrane opens Ca²⁺ channels releasing Ca²⁺ from the ER. Released Ca²⁺ binds to the ER IP₃ receptor, causing a conformational change that induces the opening of adjacent plasma membrane Ca²⁺ channels. This conformational coupling between a receptor on one membrane and a Ca²⁺ channel on an adjacent membrane is remarkably similar to the operation of the ryanodine receptor in sarcoplasmic reticulum (Chapter 17), and the IP₃ receptor shares remarkable structural similarities with the ryanodine receptor.
Figure 34.19 · IP₃-mediated signal transduction pathways. Increased [Ca²⁺] activates protein kinases, which phosphorylate target proteins. Ca²⁺/CaM represents calci-calmodulin (Ca²⁺ complexed with the regulatory protein calmodulin).

        The actions of the two second messengers IP₃ and DAG are complementary (Figure 34.19). IP₃ elevates cytoplasmic Ca²⁺ levels, and DAG activates protein kinase C in a Ca²⁺- and phosphatidylserine-dependent manner.

Calcium Oscillations

Figure 34.20 · Induced oscillations of cytoplasmic [Ca²⁺]. (a) Carbachol-dependent oscillations in the parotid gland. (b) Response of hepatocytes to norepinephrine. (c) Endothelial cell response to histamine. (Adapted from Berridge, M., 1990. Calcium oscillations.Journal of Biological Chemistry 265:9583-9586.)

One of the most intriguing and exciting developments in the field of Ca²⁺ regulation has been the discovery by Michael Berridge and others that the increases in intracellular [Ca²⁺] induced by IP₃ are oscillatory in nature! Several examples of Ca²⁺ oscillatory behavior are shown in Figure 34.20, in-cluding patterns of near-sinusoidal oscillations and baseline spikes. Ca²⁺ oscillations are induced most often by activation of receptors that act through the phosphoinositide pathway. Examples include a₁-adrenergic receptors, vasopressin and angiotensin receptors in liver, histamine receptors in endothelial cells, cholecystokinin receptors in pancreatic cells, and B₂ bradykinin receptors in chromaffin cells.
        Several models have been proposed to account for oscillatory behavior, including models involving oscillations in IP₃ and models in which a constant IP₃ level induces fluctuations in the uptake and release of Ca²⁺. The purpose of Ca²⁺ oscillations in cells is not understood, but two possibilities include (a) the need to protect sensitive intracellular processes from prolonged high levels of Ca²⁺, and (b) the need to create spatial “waves” of Ca²⁺ in the cell. Many Ca²⁺ oscillatory systems display a spatial organization, so that Ca²⁺ transients such as those of Figure 34.20 actually spread through a cell in the form of a wave of Ca²⁺ that propagates at a rate of 10 to 100 mm/sec. There is also evidence that Ca²⁺ waves may spread from one cell to the next. For example, activated cells in the ciliated epithelium of the lung excite neighboring cells through a signal, thought to be Ca²⁺, that radiates outward in a wavelike manner at approximately 10 mm/sec.

Intracellular Calcium-Binding Proteins

Given the central importance of Ca²⁺ as an intracellular messenger, it should not be surprising that complex mechanisms exist in cells to manage and control Ca²⁺. When Ca²⁺ signals are generated by cAMP, IP₃, and other agents, these signals are translated into the desired intracellular responses by calcium-binding proteins, which in turn regulate many cellular processes. One of these, protein kinase C, is described in Section 34.8. The other important Ca²⁺-binding proteins can, for the most part, be divided into two groups on the basis of structure and function: (a) the calcium-modulated proteins, including calmodulin, parvalbumin, troponin C, and many others, all of which have in common a structural feature called the EF hand (Figure 6.26 and Figure 17.30), and (b) the annexin proteins, a family of homologous proteins that interact with membranes and phospholipids in a Ca²⁺-dependent manner.
    

Figure 34.21 · (a) Structure of uncomplexed calmodulin (gold). Calmodulin, with four Ca²⁺-binding domains, forms a dumbbell-shaped structure with two globular domains joined by an extended, central helix. Each globular domain juxtaposes two Ca²⁺-binding EF hand domains. An intriguing feature of these EF hand domains is their nearly identical three-dimensional structure, despite a relatively low degree of sequence homology (only 25% in some cases). (b, c) Complex of calmodulin (gold) with a peptide from myosin light chain kinase (white); (b) side view; (c) top view.

    More than 170 calcium-modulated proteins are known (Table 34.3). All possess a characteristic peptide domain consisting of a short a-helix, a loop of 12 amino acids, and a second a-helix. Robert Kretsinger at the University of Virginia initially discovered this pattern in parvalbumin, a protein first identified in the carp fish and later in neurons possessing a high firing rate and a high oxidative metabolism. Kretsinger lettered the six helices of parvalbumin A through F. He noticed that the E and F helices, joined by a loop, resembled the thumb and forefinger of a right hand (Figure 6.26), and named this structure the EF hand, a name in common use today to identify the helix-loop-helix motif in calcium-binding proteins. In the EF hand, Ca²⁺ is coordinated by six carboxyl oxygens contributed by a glutamate and three aspartates, by a carbonyl oxygen from a peptide bond, and by the oxygen of a coordinated water molecule. The EF hand was subsequently identified in calmodulin, troponin C, and calbindin-9K (Figure 34.21). Most of the known EF-hand proteins possess two or more (as many as eight) EF-hand domains, usually arranged so that two EF-hand domains may directly contact each other.

Calmodulin Target Proteins Possess a Basic Amphiphilic Helix

Figure 34.22 · Helical wheel representations of (a) a model peptide, Ac-WKKLLKLLKKLLKL-CONH₂, and (b) the calmodulin-binding domain of spectrin. Positively charged and polar residues are indicated in green and hydrophobic residues are orange. (Adapted from O’Neil, K., and DeGrado, W., 1990. How calmodulin binds its targets: Sequence independent recognition of amphiphilic a-helices. Trends in Biochemical Sciences 15:59-64.)

Circular dichroism measurements show that the conformations of EF-hand proteins change dramatically upon binding of Ca²⁺ ions. This change promotes binding of the EF-hand protein with its target protein(s). For example, calmodulin (CaM), a 148-residue protein found in many cell types, modulates the activities of a large number of target proteins, including Ca²⁺-ATPases, protein kinases, phosphodiesterases, and NAD⁺ kinase, as well as several proteins involved in intracellular motility. CaM binds to these and to many other proteins with extremely high affinities (K_D values typically in the high picomolar to low nanomolar range). All CaM target proteins possess a basic amphiphilic alpha helix (a Baa helix), to which CaM binds specifically and with high affinity. Viewed end-on, in the so-called helical wheel representation (Figure 34.22), a Baa helix has mostly hydrophobic residues on one face; basic residues are collected on the opposite face. However, the Baa helices of CaM target proteins, although conforming to the model, show extreme variability in sequence. How does CaM, itself a highly conserved protein, accommodate such variety of sequence and structure? Each globular domain consists of a large hydrophobic surface flanked by regions of highly negative electrostatic potential—a surface suitable for interacting with a Baa helix. The long central helix joining the two globular regions behaves as a long, flexible tether. When the target protein is bound, the two globular domains fold together, forming a single binding site for target peptides (Figure 34.21b). The flexible nature of the tethering helix allows the two globular domains to adjust their orientation synergistically for maximal binding of the target protein or peptide.

34.8 · Protein Kinase C Transduces the Signals of Two Second Messengers

Protein kinase C (PKC) elicits a variety of cellular responses by phosphorylation of various target proteins at Ser and Thr residues. PKC is specifically activated by two intracellular second messengers: diacylglycerol and Ca²⁺ (the “C” in PKC stands for Ca²⁺). Because Ca²⁺ levels increase in the cell in response to IP₃, the activation of PKC depends upon both of the second messengers released by hydrolysis of PIP₂. PKC is a cellular transducer, translating the hormonal message and the signals of second messengers into the protein phosphorylation events that control growth and development.
Figure 34.23 · The primary structure of the a-isozyme of protein kinase C. V indicates variable regions; C indicates conserved domains. The phorbol-binding domain is identical with the DAG-binding domain.

        Protein kinase C (PKC) is an 80-kD polypeptide with four conserved domains and five variable regions. The conserved regions include an ATP-binding domain, a substrate-binding domain, a calcium-binding domain, and a DAG-binding domain (Figure 34.23). The DAG-binding domain is often referred to as the “pseudosubstrate domain” because it has an amino acid sequence that closely resembles protein substrates for the enzyme. With somewhat variable sequence, there are at least eleven different members of the protein kinase C family, which probably have separate and distinguishable functions. At low levels of Ca²⁺ and in the absence of DAG, protein kinase C is inactive and is a soluble protein in the cytoplasm. In this state, the pseudosubstrate domain occupies the substrate-binding site, keeping the enzyme inactive (Figure 34.24). DAG binding causes conformation changes that dissociate the pseudosubstrate domain from the substrate-binding site and increase the affinity of the enzyme for Ca²⁺ and lipid, causing protein kinase C domain C₁ to bind to the cytoplasmic surface of the plasma membrane, whereupon the kinase becomes active. Control of enzymatic activity by the insertion of a pseudosubstrate domain into the active site has been referred to as intrasteric control, in contrast to allosteric control, in which an enzyme regulator with a structure unrelated to the substrate binds at a site separate from the active site. Many protein kinases and protein phosphatases are regulated by intrasteric control (Chapter 15).

Figure 34.24 · A model for Ca²⁺- and DAG-dependent activation of protein kinase C. C₁ through C₄ refer to domains indicated in Figure 34.23. (Adapted from Sando, J. J., Maurer, M. C., ^{Bolen, E. J., and Grisham, C. M., 1992. Role of cofactors in protein kinase C activation. Cellular Signalling 4:595-609.)}

Figure 34.25 · The structure of a phorbol ester. Long-chain fatty acids predominate at the 12-position, whereas acetate is usually found at the 13-position.

        PKC phosphorylates serine and threonine residues on a wide range of protein substrates. A role for protein kinase C in cellular growth and division is demonstrated by its strong activation by phorbol esters (Figure 34.25). These compounds, from the seeds of Croton tiglium, are tumor promoters, agents that do not themselves cause tumorigenesis but that potentiate the effects of carcinogens. The phorbol esters mimic DAG, bind to the regulatory pseudosubstrate domain of the enzyme, and activate protein kinase C.

Cellular Target Proteins Are Dephosphorylated by Phosphoprotein Phosphatases

Along with the growing appreciation of the importance of protein phosphor-ylation in the response of cells to hormones, growth factors, and other cellular control signals has come an equal appreciation for the roles of phosphoprotein phosphatases. Many phosphoprotein phosphatases, specific either for serine/threonine phosphates or for tyrosine phosphates, have now been characterized. Four different classes of serine/threonine phosphatases are known. Type 1 phosphoprotein phosphatases (PP1) dephosphorylate the b-subunit of phosphorylase kinase. Phosphoprotein phosphatase 1 is inhibited by nanomolar concentrations of two heat-stable proteins, inhibitor-1 and inhibitor-2. The other three classes of serine/threonine phosphatases are the type 2 phosphatases, and they are designated PP2A, PP2B, and PP2C. These enzymes are distinguished in part by their sensitivities to divalent cations. PP1 and PP2A do not require divalent cations, whereas PP2B is dependent on Ca²⁺ and calmodulin, and PP2C requires Mg²⁺.
        The phosphatases PP2A and PP2C are predominantly cytosolic, but PP1 binds to membranes within the cell. Evidence is accumulating that PP1 (and possibly other serine/threonine phosphatases) interacts with specialized regulatory subunits that target the enzyme to particular locations in the cell and enhance its activity toward selected substrates.

A Deeper Look
Okadaic Acid: A Marine Toxin and Tumor Promoter
A novel agent for evaluating the serine/threonine phosphatases is okadaic acid (see figure), a complex fatty acid polyketal produced by marine dinoflagellates (plankton) that accumulates in the digestive glands of shellfish and marine sponges such as Halichondria okadaii, from which it gets its name. Okadaic acid is the major cause of diarrhetic shellfish poisoning, and it is a potent tumor promoter. It is a specific inhibitor of PP1 and PP2A, but has no effect on other phosphatases or on protein kinases, including protein kinase C. Because PP1 and PP2A are the enzymes that reverse the actions of protein kinase C, it is no surprise that okadaic acid is as potent a tumor promoter as the phorbol esters. Okadaic acid is a hydrophobic molecule and easily enters cells, where it causes prolonged phosphorylation of	many cellular proteins. In so doing, it causes long-lasting contraction of vascular smooth muscle and also mimics the stimulatory behavior of insulin on glucose metabolism. The role of okadaic acid in diarrhetic shellfish poisoning appears to be related to the diarrhetic action of cholera toxin, which activates adenylyl cyclase, and the cAMP-dependent protein kinase, which phosphorylates proteins that control sodium secretion by intestinal cells. Okadaic acid probably causes diarrhea by stimulating the phosphorylation of the same sodium channel-regulating proteins. Another toxin with similar properties is calyculin A, isolated from the sponge  Discodermia calyx. It is also a strong inhibitor of PP1 and PP2 and a potent tumor promoter, although its structure differs significantly from that of okadaic acid.
Structures of okadaic acid, calyculin A, and related toxins.

34.9 · The Single-TMS Receptors

Two principal classes of hormone receptors display intrinsic enzymatic activity: receptor tyrosine kinases and receptor guanylyl cyclases. Interestingly, each of these enzyme activities is manifested in two different cellular forms. Thus, guanylyl cyclase activity is found both in membrane-bound receptors and in soluble, cytoplasmic proteins. Tyrosine kinase activity, on the other hand, is exhibited by two different types of membrane proteins. The receptor tyrosine kinases are integral transmembrane proteins, whereas the nonreceptor tyrosine kinases, which are related to a family of retroviral transforming proteins, are peripheral, lipid-anchored proteins.

Nonreceptor Tyrosine Kinases

Figure 34.26 · The soluble tyrosine kinase pp60^v-src is anchored to the plasma membrane via an N-terminal myristyl group.

The first tyrosine kinases to be discovered were associated with viral transforming proteins. These proteins, produced by oncogenic viruses, enable the virus to transform animal cells, that is, to convert them to the cancerous state. A prime example is the tyrosine kinase expressed by the src gene of Rous or avian sarcoma virus. The protein product of this gene is pp60^v-src(the abbreviation refers to phospho-protein, 60 kD, viral origin, sarcoma-causing). The v-src gene was derived from the avian proto-oncogenic gene c-src during the original formation of the virus. The cellular proto-oncogene homolog of pp60^v-src is referred to as pp60^c-src. pp60^v-src is a 526-residue peripheral membrane protein. It undergoes two post-translational modifications: (a) the amino group of the NH₂-terminal glycine is modified by the covalent attachment of a myristyl group (this modification is required for membrane association of the kinase; see Figure 34.26), and (b) Ser¹⁷ and Tyr⁴¹⁶ are phosphorylated. The phosphorylation at Tyr⁴¹⁶, which increases kinase activity two- to threefold, appears to be an autophosphorylation. The significance of nonreceptor tyrosine kinase activity to cell growth and transformation is only partially understood, but many, many cellular proteins (of mostly undetermined function) are phosphorylated by such kinases.

Receptor Tyrosine Kinases

Figure 34.27 · The three classes of receptor tyrosine kinases. Class I receptors are monomeric and contain a pair of Cys-rich repeat sequences. The insulin receptor, a typical Class II receptor, is a glycoprotein composed of two kinds of subunits in an a₂b₂ tetramer. The a- and b-subunits are synthesized as a single peptide chain, together with an N-terminal signal sequence. Subsequent proteolytic processing yields the separate a- and b-subunits. The b-subunits of 620 residues each are integral transmembrane proteins, with only a single transmembrane a-helix, and with the amino terminus outside the cell and the carboxyl terminus inside. The a-subunits of 735 residues each are extracellular proteins that are linked to the b-subunits and to each other by disulfide bonds. The insulin-binding domain is located in a cysteine-rich region on the a-subunits. Class III receptors contain multiple immunoglobulin-like domains. Shown here is fibroblast growth factor (FGF) receptor, which has three immunoglobulin-like domains. (Adapted from Ullrich A., and Schlessinger, J., 1990. Cell 61:203-212.)

The binding of polypeptide hormones and growth factors to receptor tyrosine kinases activates the tyrosine kinase activity of these proteins. These catalytic receptors are composed of three domains (Figure 34.27): a glycosylated extracellular receptor-binding domain, a transmembrane domain consisting of a single transmembrane a-helix, and an intracellular domain that includes a tyrosine kinase domain that mediates the biological response to the hormone or growth factor via its catalytic activity and a regulatory domain that contains multiple autophosphorylation sites.
        There are three classes of receptor tyrosine kinases (Figure 34.27). Class I, exemplified by the epidermal growth factor (EGF) receptor, has an extracellular domain containing two Cys-rich repeat sequences. Class II, typified by the insulin receptor, has an a₂b₂ tetrameric structure with transmembrane b-subunits and a Cys-rich domain in the extracellular a-subunit. Class III receptors, such as the platelet-derived growth factor (PDGF) receptor, have five (or sometimes three) immunoglobulin-like extracellular domains.

Receptor Tyrosine Kinases Are Membrane-Associated Allosteric Enzymes

Figure 34.28 · Ligand (hormone)-stimulated oligomeric association of receptor tyrosine kinases.

Given that the extracellular and intracellular domains of receptor tyrosine kinases are joined only by a single transmembrane helical segment, how does extracellular hormone binding activate intracellular tyrosine kinase activity? How is the signal transduced? As shown in Figure 34.28, signal tranduction occurs by hormone-induced oligomeric association of receptors. Binding of hormone triggers a conformational change in the extracellular domain, which induces oligomeric association. Oligomeric association allows adjacent cytoplasmic domains to interact, leading to phosphorylation of the cytoplasmic domains, and stimulation of cytoplasmic tyrosine kinase activity. In the case of the class II receptors (for example, the insulin receptor), hormone binding induces interactions between the two ab halves within the disulfide-linked receptor complex. By virtue of these ligand-induced conformation changes and oligomeric interactions, receptor tyrosine kinases are membrane-associated allosteric enzymes.
        Autophosphorylation at Tyr residues allows the receptor tyrosine kinase to remain active even after the activating hormone has dissociated from the receptor. On the other hand, the tyrosine kinase can be inactivated by phosphorylation of intracellular Ser/Thr residues. These inactivating phosphorylations are catalyzed by protein kinase C and by cAMP-dependent protein kinases, providing a direct linkage between receptor tyrosine kinases and several key second messengers, including cAMP, IP₃, DAG, and Ca²⁺.

Receptor Tyrosine Kinases Phosphorylate a Variety of Cellular Target Proteins

Receptor tyrosine kinases catalyze the phosphorylation of numerous cellular target proteins, producing coordinated changes in cell behavior, including alterations in membrane transport of ions and amino acids, the transcription of genes, and the synthesis of proteins. Many individual phosphorylation targets have been characterized, including the g-isozymes of phospholipase C and phosphatidylinositol-3-kinase. The latter enzyme phosphorylates PI at the 3-position, producing several phosphoinositide metabolites that are not hydrolyzed by known phospholipase C enzymes. These PI-3-P species may either act as membrane-associated second messengers or be hydrolyzed by other phospholipases.

The Polypeptide Hormones

The largest class of hormones in vertebrate organisms is that of the polypeptide hormones (Table 34.4). One of the first polypeptide hormones to be discovered, insulin, was described by Banting and Best in 1921. Insulin, a secretion of the pancreas, controls glucose utilization and promotes the synthesis of proteins, fatty acids, and glycogen. Insulin, which is typical of the secreted polypeptide hormones, is discussed in detail in Chapters 5, 15, and 23.
        Many other polypeptide hormones are produced and processed in a manner similar to that of insulin. Three unifying features of their synthesis and cellular processing should be noted. First, all secreted polypeptide hormones are originally synthesized with a signal sequence, which facilitates their eventual direction to secretory granules, and thence to the extracellular milieu. Second, peptide hormones are usually synthesized from mRNA as inactive precursors, termed preprohormones, which become activated by proteolysis. Third, a single polypeptide precursor or preprohormone may produce several different peptide hormones by suitable proteolytic processing. The following processing events are common to all preprohormones:

1.  Proteolytic cleavage of a hydrophobic N-terminal signal peptide sequence
2.  Proteolytic cleavage at a site defined by pairs of basic amino acid residues
3.  Proteolytic cleavage at the site of single Arg residues
4.  Post-translational modification of individual amino acids, including a-amidation of the C-terminal residue, phosphorylation, glycosylation, or acetylation of the N-terminal residue

Figure 34.29 · The major pathway of gastrin biosynthesis in human gastric mucosal cells. The asterisk (*) indicates a sulfate group, and P indicates a phosphorylation site. (Adapted from Dockray, G, et al., 1989. Gastrin and CCK-related peptides. In Martinez, J., ed. Peptide Hormones as Prohormones. New York, Halstead Press.)

A good example of these processing events is the biosynthesis of gastrin, a heptadecapeptide (17 residues) secreted by the antral mucosa in the stomach. Gastrin activates acid secretion in the stomach, and also stimulates growth of the acid-secreting mucosa. It is a product of preprogastrin, a peptide of 101 (human) or 104 (pig) residues (Figure 34.29). As expected for a secretory hormone, it has a signal peptide (21 residues), cleavage of which leaves progastrin. Cleavage at pairs of basic residues (noted in the figure) followed by C-terminal amidation produces gastrin (G17) and three other peptides. Several post-translational modifications occur in progastrin, including incorporation of a sulfate at Tyr¹², phosphorylation of Ser²¹, and protective modifications of both the C- and N-termini of gastrin itself. The N-terminal residue of gastrin is a pyroglutamate (a pyrrolidone carboxylic acid), formed by cyclization of the N-terminal glutamine after proteolytic cleavage of the flanking peptide. An identical N-terminal modification is found in bacteriorhodopsin (Chapter 9). At the C-terminal end, three enzymatic steps result in amidation, which protects the peptide hormone from action of carboxypeptidases. The steps involve a trypsin-like cleavage on the C-terminal side of the second arginine, a carboxypeptidase-like action to cleave the two arginines, and degradation of the glycine residue to leave a C-terminal phenylalanine amide (Figure 34.30).

Figure 34.30 · The enzyme-catalyzed proteolysis and amidation reactions at the C-terminus of progastrin that lead to gastrin.

Figure 34.31 · The conversion of prepro-opiomelanocortin to a family of peptide hormones, including corticotropin, b- and g-lipotropin, a- and b-MSH, and endorphin.

            An impressive example of the production of many hormone products from a single precursor is the case of prepro-opiomelanocortin, a 250-residue precursor peptide synthesized in the pituitary gland. A cascade of proteolytic steps produces, as the name implies, a natural opiate substance (endorphin), melanocyte-stimulating hormones (a- and b-MSH), and corticotropin (also known as adrenocorticotrophic hormone, ACTH) as shown in Figure 34.31. As noted, these proteolytic cleavages actually take place in different tissues. Cleavage of pro-opiomelanocortin in the anterior pituitary yields corticotropin and b-lipotropin, which proceed to cells of the central nervous system for the final proteolytic steps.

Figure 34.32 · (a) The primary structures of cytoplasmic and membrane-bound protein-tyrosine phosphatases (PTPases) and cell adhesion molecules (CAM). Key: S-S loops are immunoglobulin domains; blue squares are fibronectin domains; gold rectangles are PTPase catalytic domains; green and red rectangles are membrane-associated domains; blue squares on SH-PTPase are SH2 domains; olive rectangle on PTPase H1 is a cytoskeletal domain. (b) Molecular graphic of PTPase 1B. (Adapted from Streuli, M., et al., 1989. A family of receptor-linked protein tyrosine phosphatases in humans and Drosophila. Proceedings of the National Academy of Sciences, USA 80:8698-8702; and Mauro, I. J., and Dixon, J. E., 1994. Zip codes direct intracellular protein tyrosine phosphatases to the correct cellular “address.” Trends in Biochemical Sciences 19:151-155. Molecular graphic courtesy of David Barford, Oxford University.)

Phosphatases specific for phosphorylated tyrosines on proteins are different from their serine/threonine-specific cousins, and display a wide variety of structures. Some of these protein-tyrosine phosphatases (PTPases) are integral membrane proteins, whereas others are cytoplasmic in nature (Figure 34.32 and Table 34.5). The cytoplasmic PTPases consist of N-terminal catalytic domains with C-terminal regulatory regions that may guide the enzyme to specific intracellular locations and substrates and/or specifically activate the PTPase toward those substrates. The membrane-bound PTPases all consist of a cytoplasmic catalytic domain, a transmembrane domain, and various extracellular domains that are designed to convey signals across the membrane. The extracellular domains bear considerable similarity to cell adhesion molecules (CAM), with repeated immunoglobulin domains and fibronectin type II