Electronic Reviews

CYCLIC NUCLEOTIDE SIGNALING

An overview of signaling

The vast undertaking of integrating the body's many essential services required the evolution of specialized cells to synthesize and release hormones that encode changes in both our internal and external environments, and like a newspaper, hormones have a broad circulation. For hormones to finely control and tune the body's organs so that supply in matched with demand, they must be released and circulated in appropriate concentrations over carefully regulated periods of time. For example, a sudden exposure to an external threat or stress, such as a charging bull or an irate boss, and there is an immediate need for energy to accompany the newly found motivation. Adrenaline, the emergency 'fight or flight' hormone, is released from the chromaffin cells of the adrenal medulla after a delay of only a few thousandths of a second due to rapid stimulation from incoming sympathetic nerves. The adrenal medulla is an example of an endocrine gland, releasing its hormones into the bloodstream in response to a stimulus, in this case the perception of threat or danger. Within its sphere of influence adrenaline causes an increase in both the rate and output of the heart, changes that are accompanied by a reduction in the resistance of the blood vessels to ensure that the supply of oxygen and nutrients to the muscles, brain and periphery meets the sudden demand. Adrenaline is perhaps the body's simplest hormonal signaling system, where the levels of a single hormone increase and subside with the perception of threat, its sphere of cellular influence defined by the limits of the circulation and the number of cells that have an ear to listen.

However for efficient communication to take place an exchange of information must occur. The receptor must first pass its information onto the cell and the cell in turn must reply to the receptor (feedback) in order to confirm that the message has been received and understood. Just as the cell listens to the receptor, the receptor in turn listens to the cell, and the receptor's ears are to be found within its diverse intracellular regions which frequently contain stretches, or sequences that are rich in the amino acids threonine, serine or tyrosine. These three amino acids share in common side chains that have the chemical properties of water, allowing them to receive information from the cell in the form of dense negative charges carried by phosphate molecules. Such phosphate groups may be readily added onto or removed from these three amino acids by specialized enzymes known as 'kinases' and 'phosphatases' respectively, and the dense negative charge that they carry may switch on or off the signal carried into the cell by the receptor protein, or else modify it. Thus the phosphate modification, or phosphorylation of receptor proteins provides a feedback pathway to ensure that receptor sensitivity and information transfer is precisely tuned to mirror the activity of the cell and its capacity to respond to further stimulation. Such a persistent activation of receptors may cause them to desensitize, or become less effective at transferring signals across the membrane, rather as the ear becomes accustomed to background noise or the eye to background light. Alternatively the addition of phosphates may enhance signal transfer, for the down-regulation of one receptor by phosphorylation, such as the insulin receptor, may conveniently coincide with the up-regulation of the glucagon receptor to tip the balance of the seesaw with the general swing of events. There are in addition other mechanisms by which the receptor listens to the cell, but these are often secondary or additional to the key strategy of receptor phosphorylation and serve the same purpose:- to match the throughput of information to the cell's capacity to respond, and as importantly to ensure that a hormonal signal has an end as well as a beginning.

Receptors are classically imagined as a lock that can only be opened by a very specific key, namely a hormone or transmitter of a defined shape, or at the very least a very good skeleton key. However, to the discriminating mind this analogy does not survive scrutiny, as biological molecules such as hormones and transmitters differ from keys, and receptors from locks, in that the various regions of the receptor protein possess not only specific shape, but also a defining distribution of electrical charge, affinity for water and for one other. Thus as a biological lock the receptor responds not only to the shape of the hormone, but also to its distribution of charge and hydrophobicity, making biological receptors more akin to Cinderella's shoe than a multi-lever Union lock. Hormones don't just fit their receptors, they interact with them, just as the curvature of Cinderella's shoe was precisely molded to fit the delicate, unique and shapely contours of her foot thereby allowing her prince to find his one true partner.

Receptors serve as a molecular bridge by which water-borne signals cross the membrane, but before they can be understood by the cell, they must first be translated into a language that the cell understands: a change in the chemical balance of the cell. These signals are known as second messengers, and are generated following the activation of a receptor by the primary messenger, such as a hormone or transmitter, and may be generated by a startling variety of mechanisms. Some second messengers are metabolic derivatives of the cell's basic building blocks, synthesized by off-shoots of well-worn biochemical pathways, or they may take the form of specialized proteins that transfer phosphate groups to and from specific cell proteins and lipids, whilst some second messenger signals are provided by changes in the levels of charged ions or radicals in the cell, such as calcium, nitric oxide, or even in the form of the pH of the cell. Second messengers carry receptor signals into the cell's metabolic heartlands where they are integrated to direct changes in the activity of the cell's various engines that include its metabolic machinery, protein skeleton and its center of intelligence - its nucleus. Through carefully regulated changes in the levels of specific second messengers changes in the cell's environment can be transduced into signals that can be interpreted and acted upon by those cellular proteins that decipher and copy the genes. Genes are thus environmental sensors, responding to their external world by producing more or less of whatever protein is necessary to adapt and survive, allowing the cell to adapt to the pressures of change and contribute to the survival of the cellular nation.

For any given cell, as for any given animal, an inappropriate response to an event or stimulus within its immediate environment can spell disaster. An animal that fails to fight or take flight from a predator, to pay appropriate homage to a higher social animal or to take advantage of foraging opportunities will almost certainly fail within a competitive environment. In much the same way, a cell will fail and die if it does not respond quickly and appropriately to an infection, to positional cues within in a bustling and congested cellular society, or to fluctuations in the supply of nutrients and hormones that portend greater changes in the whole. Clearly receptor signals had to evolve to be sufficiently fast to enable us to respond rapidly to changes in our environment. For instance, those receptors which are present upon nerve cells and at the junction between nerve and skeletal muscle cells transfer the signal from an incoming nerve cell, or neuron within a few thousandths of a second. Such signals are, however, as short-lived as they rapid, requiring a highly specialized receptor design that is in sharp contrast to that of a second class of receptor which communicates trends rather than instantaneous decisions to the cell. This second class is typified by receptors that mediate the responses of cardiac muscle cells to noradrenaline released from the sympathetic nerves which supply the heart, and by those receptors which are found upon the surface of olfactory receptor neurons and confer our sense of smell. Noradrenaline augments the rate and output of the heart by enhancing the entry of calcium ions into the muscle cells that power the ventricles of the heart. In isolation cardiac muscle cells take many seconds to respond to noradrenaline, requiring more than ten minutes to reach the peak of their response to the hormone. Responses to this class of receptor are generally long lasting, and require many minutes to return to their original levels after the removal of this circulating catecholamine (adrenaline has much the same mode of action). There is however also a third class of receptor, employed by those hormones that we collectively refer to as growth factors. Receptor responses to growth factors are usually slow in onset and prolonged in action, mirroring those changes in cell function that they regulate. Growth factor receptors govern the survival, growth, division and specialization of cells, and part of their action involves activating programs that lie latent within the genes, thereby altering the release of information into the cell.
 
 

Adenylate and guanylate cyclases use the commonly available building blocks of DNA and units of energy currency ATP and GTP to create, within a single rate-limiting step, 3’-5’ cyclic forms of the monophosphate derivatives of these two purine nucleotides. Cyclic 3’,5 adenosine monophosphate and cyclic 3’,5 guanosine monophosphate are so unique in their structure that they are effective as highly specific second messengers, yet can be readily converted to and from freely available and energy containing cellular building blocks. This process is driven by the hydrolysis of a high-energy phosphate bond formation with the release of pyrophosphate (PPi). The pyrophosphate released is hydrolyzed by a pyrophosphatase, making the ‘forward’ reaction thermodynamically favorable, effectively consuming two high energy phosphate bonds and creating a new one in the process of cyclization. The catalytic domains of adenylate and guanylate cyclase are highly conserved, suggesting a common mechanism of catalysis.

Introducing the players:

(I) adenylate cyclase

Unlike guanylate cyclase, the primary regulators of adenylate cyclase (AC) activity are the free GTP-bound alpha subunits of G_s (and the related G_olf. All except AC9 are also activated by the plant diterpene forskolin, but not by its related analog dideoxyforskolin. However, there are at least nine different isoforms of AC that differ in their regulation by G-proteins, free Ca²⁺ ions and protein kinase phosphorylation. The cell and tissue-specific expression pattern of each of these isoforms allow AC to serve as a specialized integrator of relevant stimulatory and inhibitory signals in cells for different functions throughout the body.

AC is an integral membrane protein related to the P-glycoprotein (MDR1; an ATP-Binding Cassette (ABC)-transporter, an ATP-dependent pump, and the CFTR which is an ATP-dependent Cl^- channel, although so far in mammalian cells, no channel or pump function for AC has been detected). The structure of AC isoenzymes (isozymes) comprises five domains (each functional region or domain of a protein is usually encoded by one or more exons). These are in order (always presented by convention from N-terminus to C-terminus):

Adenylate cyclase structure and function

1. A cytoplasmic N-terminal domain

(II) A membrane-anchoring hydrophobic domain (M1) with 6 transmembrane helices

Anchors AC within membrane

(III) A large cytoplasmic domain (C1):

Required for catalysis.

(IV) A second 6 transmembrane-anchoring domain (M2):

Contains at least one-N-linked (asparagine-conjugated) glycosylation site (important in cell-cell recognition and signaling). AC that has been genetically engineered to be without M domains is soluble and regulated in a similar manner to the membrane-bound enzymes.

(V) A second catalytic cytoplasmic domain (C2) homologous to the first at the C-terminus

The two C2 domains pack head-to-tail into a dimer in a 'wreath-like' structure. On one side (ventral surface believed to face towards the cytoplasm for ease of substrate access) is a deep cleft lined with hydrophobic residues with forskolin-binding sites at either side. The likely ATP substrate-binding site is in a pocket on one side of the ventral cleft opposite the forskolin-binding site. The forskolin molecule serves as a stabilizing interdomain bridge in the G_sa complex with AC in the active 'crystallized' configuration of the enzyme.

Experiments with ADP-ribosylation with Pertussis toxin (PTX) suggest that it is the C-terminus of G_ia and G_oa (but PTX does not inhibit G_za) that interacts with and inhibits AC. There is some, but less apparent specificity for b and g subunits in the modulation of AC. Some bg subunits activate some isoforms of AC synergistically and inhibit others. The region N-terminal helix on the G_a subunit that mediates the stimulation of AC is also essential in assembly and interaction of co-assembled G_abg subunits in their GDP-bound form.

0 = not expressed, + expressed, + + differentially high expression, blank unknown
 
 

@ stimulates; i, inhibits; i/@ effect dependent upon conditions; x, no effect; blank ?

* The effect of bg subunits may be conditional upon their subunit composition and availability. The stimulatory effects of G_bg are dependent upon G_sa signaling properties of adenylate cyclase

(a) Synergistic regulation

AC shows synergistic (non-additive) regulation in combination with other modulators. For example forskolin and G_sa combine to activate AC2 in a non-linear fashion (the net output is NOT the direct sum of the inputs). AC2, AC4, AC5 and AC6, but not AC1 show synergistic activation between Ca²⁺-CaM and G_sa. This is also true of G_a and G_bg subunits, especially those originating from different classes of G-protein.

For example a Gs coupled receptor will activate AC2 present in lung tissue through G_sa, an action that will be enhanced by the availability of bg subunits from a nearby activated Go-coupled receptor. However, bg subunits from a Go or Gi-coupled receptor will antagonize stimulation by G_sa subunits from a Gs-coupled receptor if hippocampal AC1 is the target.

(b) Signal integration.

AC receives and integrates inputs from multiple receptors and also information in the form of calcium entry through channels and even changes in membrane potential. This allows AC to function as a net integrator and output signal after integrating the signaling inputs from a number of sources into a final effector message within a region of the cell.

For example, AC1 and AC8 are synthesized only in neurons, particularly those in regions involved in learning and memory, notably high levels being synthesized in the granule and pyramidal cells of the hippocampus. Both these isoforms are synergistically activated by G_sa and Ca²⁺-CaM. Signals that converge upon the receptive field of these neurons to increase Ca²⁺ when AC is stimulated through a Gs-coupled receptor will inevitably combine to enhance levels of cAMP and therefore PKA activity. These changes lead in alterations in gene transcription and hence long-term forms of plasticity associated with memory formation in these neurons.

(c) AC as a signal amplifier.

Each receptor that binds a hormone or ligand in turn stimulates hundreds of G-proteins for the duration that the receptor is in its active form. In turn each adenylate cyclase enzyme stimulated by an active G-protein alpha subunit generates hundreds of cyclic AMP molecules for each second of the G-protein's active lifetime. This staggering amplification causes an increase in cyclic AMP concentrations within the cell which are many thousands of times greater than the prevailing external concentration of the hormone or ligand that activate the receptor. Added to the phenomenon of receptor reserve, where only 10% or fewer of receptors on the surface of the cell need to bind ligand to achieve maximal effect and it is not difficult to appreciate the non-linearity and amplification achieved through hormone signaling systems.

In cardiac myocytes where AC5 and AC6 are most abundant isoforms, inhibition of AC activity results primarily from the activation of voltage-gated L-type Ca²⁺ channels and the resulting influx of this inhibitory ion. b-adrenergic agonists such as adrenaline (epinephrine) or noradrenaline (norepinephrine) act upon the myocytes of the heart to increase its inotropic effects (force of contraction). They do so by activating AC through Gs to increase cAMP levels and PKA activity. The resultant PKA activation increases L-type Ca²⁺ channel activity and directly inhibits AC5 and AC6 through phosphorylation. The consequent increase in Ca²⁺ concentration that results further inhibits AC5 and AC6 activity, resulting in a suitable negative feedback loop to limit the positive inotropic actions of these catecholamines upon the heart.
 
 

GUANYLATE CYCLASES ARE INTEGRAL MEMBRANE RECEPTORS, AND EACH IS SPECIFIC FOR A SINGLE HORMONE FAMILY

Receptor guanylate cyclases (at least 3 classes are known at present) are integral membrane-spanning receptors which contain an extracellular hormone binding domain and a cytoplasmic guanylate cyclase (GC) domain. As binding of the hormone to the receptor directly regulates GC activity, GC activation and consequent cGMP production is therefore BOTH hormone-specific and concentration-dependent.

All three membrane GC receptors are specific for hormones involved in cell body water and electrolyte homeostasis. This means that the cGMP signaling pathway has come to play a somewhat specialized role in the regulation of the excretion and uptake of water and salts through the kidneys and intestines. Additionally, in combination with another nitric oxide-regulated soluble guanylate cyclase, cGMP regulates vascular resistance, another essential component of maintaining appropriate blood supply and composition. In addition the soluble nitric oxide-regulated guanylate cyclases are important elsewhere in the body in the local (paracrine) regulation of processes as diverse as synaptic plasticity, smooth muscle contraction, immune cell function and sexual arousal. These four types of GC are discussed in detail below.

The atrial natriuretic peptide receptor family

(a) physiology

The atrial natriuretic peptide family consists of three related polypeptide hormones, Atrial Natriuretic Peptide (ANP, also called ANF); Brain (-derived) Natriuretic Peptide (BNP) and so-called C-type Natriuretic Peptide (CNP).

ANP is present at its highest concentrations within specific granules of atrial cardiac myocytes (heart muscle cells), and is released into the circulation within the heart in response to the stretching of the atrial wall due to an increased blood 'volume', which manifest itself as an increased filling pressure. (The water content, or 'dilution', of the blood is therefore increased). ANP released into the circulation causes diuresis (water excretion) and natriuresis (sodium excretion) by the kidneys, as well as causing a possible decrease in water absorption and increase in salt water secretion by the intestinal epithelium, which together combine to cause mild systemic hypotension (decreased blood volume and pressure).

BNP was first isolated from the brain, but is found at its highest concentrations within the myocardial tissue of the heart. BNP is secreted into the circulation not only from the atria of the heart like ANP, but predominantly from the ventricles. Like ANP, BNP is released in response to blood volume expansions detected by the stretch of the muscle layers that form the heart wall of both chambers. BNP's effects are like those of ANP, reducing vascular tone (resistance), inhibiting sodium and water reabsorption in the kidney tubules and inhibiting vascular cell growth. ANP and BNP are physiological antagonists of the renin-Angiotensin II signaling system, which has opposing systemic actions.

In contrast CNP is found at very low levels in the circulation and is predominantly found in the brain where it is synthesized by endothelial cells. CNP inhibits vascular cell growth and plays a paracrine (local intercellular) role in the regulation of vascular tone.

All three peptide hormones are cleared from the circulation by the actions of binding the non-cyclase receptor (NPR-C) and the cleavage of the peptide hormone by neutral endopeptidase.

(b) anp receptor family: relative affinities and structures

There are two subtypes of natriuretic peptide GC receptor. GC-A which binds to the peptides with rank affinity ANP>BNP>>CNP, and GC-B which binds to the three with an inverse affinity profile CNP>ANP>BNP.

The two receptors consist of a extracellular ligand binding domain, a single transmembrane spanning domain, a protein kinase-'like' domain (KLD) which has no actual kinase activity but does bind to ATP; a connecting region that causes adjacent receptors to dimerize upon ligand binding and activation of the guanylate cyclase domain.

Mutations in the capacity of the KLD to bind ATP block hormone-mediated GC activation. It is thought that binding of ANP to its receptor causes a conformational change which allows ATP to bind to the KLD which relieves the autoinhibition of the GC domain by the KLD. GC-A is normally believed to be present in dimeric (paired) form, but only the ANP binding-induced dimerization of the intracellular amphipathic helix region between the KLD and the GC domains causes full cyclase activation. Thus ANP binding is believed to stimulate intracellular dimerization that is dependent upon ATP-binding to the KLD. Thus both intracellular and extracellular dimerization appears necessary for a fully active GC-A ANP receptor.
 
 

(c) receptor desensitization

GC-A is not desensitized by the internalization of ligand-bound receptors as a means of turning off the signal. Rather GC-A is desensitized by a feed-back loop, wherein ANP stimulates an increase in cGMP levels which activates the serine-threonine protein kinase G (PKG), and this phosphorylates and activates a phosphatase PP5 which in turn dephosphorylates and desensitizes GC-A, leading to a decrease in cGMP production. Thus initial rates of cGMP formation upon ANP stimulation are higher than those observed upon sustained stimulation. Interestingly agents that activate PKC activity also stimulate the desensitization of the GC-A cyclase receptor.
 
 

ANP + GC-Aà ANP.R** (+Pi) à á cGMP à @PKGà @PP5à i ANP.GC-A*

The GC-C receptor was first identified to be the binding site for the heat-stable toxins (ST’s) released by the E.Coli bacterial strains that are responsible for traveler’s diarrhea. What was even more intriguing is that these toxins bind to the lumenal face of the enterocytes that make up the intestinal epithelium. These toxins achieve their effect by greatly elevating levels of cGMP in the enterocyte. This is in contrast to the mechanism of action of heat-labile enterotoxins such as Cholera Toxin (CTX) which act by increasing cAMP levels in the cell by irreversibly ADP-ribosylating Gs, and Pertussis toxin (PTX from Bordetella Pertussis) which inhibits Gi signaling through the same mechanism. The action of ST’s are reversible, unlike those of CTX and PTX, and less ubiquitous, the CTX Gs target G-protein being widely expressed.

Structure and function of the GC-C guanylate cyclase receptor

Molecular sequence and structure of GC-C receptor ligands

Guanylin was first isolated as a peptide 15 amino acids in length from rat mucosa in 1992 and the closely related uroguanylin (8 identical residues, with 4 cysteines and C-terminus conserved) from opossum urine. The four conserved cysteines form two intramolecular disulfide bridges which are important in determining the conformation and biological activity of these peptides.

Uroguanylin is more acidic than guanylin, and lacks the internal aromatic Phenylalanine (F) or Tyrosine (Y) of its guanylin relative. The differences in primary structure account for the pH-modulated affinities and potencies of these peptides in their action at GC-C, uroguanylin being more potent in the pH range 5.0-5.5, and guanylin more active in stimulating intestinal secretion and cGMP accumulation at pH 7.5. As the duodenum (the region of the small intestine closet to the stomach) is more acidic than the lower intestinal regions, uroguanylin is released and is effective at stimulating the secretion of fluid, electrolytes and bicarbonate from the duodenal mucosa to neutralize the acidic stomach chyme. This raises the pH of the intestinal lumen and increases the action of guanylin in causing secretion in lower regions of the intestine. In addition uroguanylin is likely more effective in modulating transport in the acidic regions of the intestinal villus epithelium in the jejunum (pH 6.4-6.8), whilst guanylin probably acts more potently in the crypt regions of the jejunum and ileum (pH7.6-8.6).

Uroguanylin is produced by some enterochromaffin cells of the crypt and its expression is higher in the duodenum than for guanylin. Guanylin is found predominantly in the mucus-secreting goblet cells of the rat ileum and jejunum. It may be that uroguanylin (and perhaps guanylin) are secreted into the circulation by enterochromaffin cells and enteroendocrine cells, whilst goblet cells and enterocytes release them into the intestinal lumen. There is evidence to suggest that guanylin is released into the intestinal lumen following cholinergic stimulation of the enteric (gut) nervous system.

Circulating uroguanylin may have a direct diuretic, natriuretic and kaliuretic action on the kidney, increasing water, sodium and potassium excretion, thus providing a feedback hormonal signal to regulate salt and water homeostasis.

The signaling molecule that was released from endothelial cells of blood vessels that caused relaxation of the surrounding vascular smooth muscle (endothelium-derived relaxing factor, EDRF) was ultimately identified as the free radical nitric oxide (NO^.).

NO causes vasorelaxation by increasing cGMP levels in smooth muscles by activating a soluble guanylate cyclase (sGC). To date no soluble mammalian adenylate cyclase has been identified, and no membrane bound G-protein regulated guanylate cyclases have been identified either. Thus AC and GC differ not only in their product, but also in the nature of the signaling molecules that directly activate them.

Domain structure and function of guanylate cyclase

(I) Haem-binding domain

The N-terminal portion within each subunit contains a haem-binding domain and is the least conserved region of the protein. Haem is an iron-containing metalloporphyrin. Haem-deficient sGC cannot be activated by NO. Haem-reconstituted sGC can be activated nearly 100-fold by NO. As with haemoglobin (Hb) the Fe moiety must be in its ferrous (Fe²⁺) state, not only for enzyme activity but also for the haem group to bind. Oxidation to the ferric (Fe³⁺) state results in the loss of both. Reducing agents such as thiols and ascorbate maintain the iron in a reduced state and maintain sGC activity. In fact S-nitrosothiols, which are formed when cytoplasmic thiols react with free NO radicals, may be the physiological donor form of NO to the sGC haem domain.

The ‘axial’ imidazole side chain of His105 in the b1 subunit binds the haem moiety in conjunction with other cysteine residues (especially Cys78 and Cys214) within the b1 subunit. It is thought that it is the b1 subunit, rather than the a1 subunit, that co-ordinates binding to the haem group, although both subunits are required for haem co-ordination and NO sensitivity.

(II) Catalytic domain

The catalytic domain is present at the C-terminus of each subunit, and are highly homologous between subunits and the C-terminus regions of both particulate GC and AC. Even though both subunits contains catalytic domains, the expression of both subunits is necessary for cyclase activity.

Between the N-terminal Haem-binding domains and the C-terminal Cyclase domains is a dimerization domain which mediates the association between b and a subunits that appears necessary for activity.

Mechanism of sGC activation

The haem of sGC binds to N-O in a fashion similar to the way in which the haem groups of Hb and myoglobin (Mb) bind to O-O (O₂), except that, unlike Hb and Mb which obviously require an aerobic environment, sGC has a very low affinity for oxygen due to the existence of a negative polarity in the distal pocket of the haem, which is thought to be due to the presence of an oscillating ligand at the sixth co-ordinate position that confers specificity to NO. The ferrous iron of haem is a ‘high-spin’ atom, which in the inactivated state oscillates between a five and six co-ordinate form.

The first step in activation is the formation of an intermediate hexa-co-ordinate complex between Fe³⁺, His105 and NO. This hexa-co-ordinate complex then converts either spontaneously or via an interaction of NO with an as yet unidentified non-haem binding site to a penta-co-ordinate complex. This induces the conformational activation of the sGC catalytic domain. It is this disruption of the histidine- Fe³⁺ linkage which results in the conformational change and the consequent exposure of the catalytic site to its GTP substrate.

Catalytic conversion of GTP to cGMP by the cyclase is dependent upon the presence of divalent cations (Mg²⁺ or Mn²⁺ are the preferred species). Conformational stimulation of the enzyme by NO binding results in a decrease in km for the GTP substrate from 100 to 50 mM with a concomitant 200-fold increase in V_max.

Cu (I) is also believed to be an essential cofactor for catalytic activity of sGC, and Cu (I) may be necessary to catalyze the release of NO bound to S-nitroso-thiols, by catalyzing the decomposition of the S-nitrosothiols. In fact, animals fed a copper-deficient diet exhibit a loss of endothelial-dependent relaxation. Carbon monoxide may also activate sGC by binding to haem, but is far less potent (4-5-fold stimulation).

Pharmacology of the NO-sGC-cGMP pathway

NO donors activate sGC

Inhibition by superoxide free-radical donors

Other inhibitors: N-methylhydroxylamine, ODQ (specific to haem groups)

Selective sGC activators

Pathophysiology of the NO-sGC-cGMP pathway

Specific activators and inhibitors yield clues to the physiology and pathology of a given pathway:

Insufficient activity causes impotence (hence viagra), hypotension and asthma.

ODQ blocks NO-dependent smooth muscle relaxation in the respiratory and urinary tracts and vasculature.

The inhibitor ODQ blocks the NO-mediated reduction in platelet reactivity (enhances platelet aggregation, also known as thrombosis or clot formation).

The NO-sGC-cGMP activators YC-1 and isoliquiritigenin inhibit platelet aggregation and enhance vascular smooth muscle relaxation in response to vasodilators.

NO (EDRF) release is important in memory formation across synapses in the nervous system and in mediating light adaptation in the retina
 
 

Phosphodiesterases terminate cyclic nucleotide signals

Some important examples of cyclic nucleotide-regulated signaling pathways

(1) The olfactory receptor uses adenylate cyclase to turn a smell into an electrical signal.

Inhaling through the nostrils causes air to rush in and over the tufted patches of olfactory epithelium that resemble a pile carpet under the electron microscope. The odorant receptors that lie upon the tufts comb the air for odors, and when a population of olfactory receptors capture their favored odorant, the receptor triggers a chain of enzymatic events that ultimately causes the opening of another type of sodium channel which is gated by cyclic nucleotides. Thus the olfactory receptor neuron first detects odors through the binding of odorant to its battery of receptors, and then converts this chemical signal into an electrical signal that can eventually be received and interpreted by the olfactory cortex within the brain.

When odorant binds to a receptor there is a change in the binding energies within the olfactory receptor that leads to the movement of the receptor's internal regions. Through these movements odorant signals cross the plasma membrane where they subsequently reappear as changes in the conformation of the internal regions of the receptor. Here the relative positions of amino acid residues change in the presence of odorant, becoming either freshly exposed or hidden from the view of other cellular proteins. As far as the other blind, but feeling proteins of the cell are concerned, the receptor protein has changed its face and correspondingly the news that it bears, and this change now dictates a fresh set of instructions to the internal messenger proteins in the olfactory cell. Consequently a chain reaction of altered enzyme activities occurs within a key second messenger pathway of the olfactory receptor neuron that eventually results in the opening of a population of sodium channels within the membrane of olfactory receptor neuron. Therefore, within a single sensory neuron a chemical signal is detected and turned, or transduced, into an electrical one. The evolution of the olfactory receptor has enabled chemical information to be discriminated, sampled and converted into the universal language of the nervous system - changes in the cell’s electrical potential. The odorant receptor functions as both a sensor and a transducer, and there can be little doubt that the odorant receptor is one of the finest examples of nanotechnology in action.

When bound to a chemical, the activated odorant receptor triggers nearby G-proteins to exchange their bound cargo of guanosine diphosphate (GDP) for guanosine triphosphate (GTP) upon their 'alpha' subunits. The nucleotide GTP differs from GDP in that it contains an extra phosphate molecule and therefore an additional high energy bond between phosphates and serves as an essential building block used in the synthesis of DNA. The bulky presence of this additional negatively charged phosphate group, bound deep within the heart of the alpha subunit, commands a change in the conformation of the protein subunit, causing a molecular separation of the alpha subunit from its partner beta and gamma subunits. Once separated the alpha and beta-gamma components go their separate ways to form a forked signaling pathway, each molecule attending to its own duties until another built-in time switch, this time within the alpha subunit causes the third phosphate of GTP to be cleaved, returning the alpha subunit to its inactive GDP-bound state. The inactivated alpha subunit then reassociates with any stray beta-gamma subunits that it can find, and once their union is rejoined the G-protein has returned to its resting conformation in readiness for another cycle.

What then is, or are, the molecular targets for the G-protein's migrant and 'active' alpha subunit? The answer to this question in the olfactory receptor neuron is adenylate cyclase, an enzyme that mass produces the second messenger molecule cyclic adenosine monophosphate (cyclic AMP) by catalyzing the formation of a ring-like structure from the cell's reservoir of ATP. Adenylate cyclase reflects the essence of many second messenger pathways in that it converts the common metabolic building block ATP into a short-lived messenger molecule that is itself a highly specific and carefully regulated signal, the whole transformation occurring through a single metabolic step. This is nowhere more elegantly demonstrated than in the conversion of the linear structure of ATP into the ring-like structure of cyclic AMP, the entire process being driven by the liberation of the energy stored between phosphate molecules in ATP. In response to the binding of an odorant the concentration of cyclic AMP increases within the olfactory receptor neuron and binds to the intracellular face of an ion channel which serves as the molecular sensor of the ambient concentration of cyclic AMP, and therefore odorant. The ion channel is thus the end point of the signaling pathway, and represents the final step in the process of converting changes in odorant concentration into changes in membrane potential that the nervous system can utilize.

So why doesn't the odorant receptor simply activate the ion channel directly and cut out the energy consuming middle men from the business of detecting odors, along with all the delay and expense that goes with them? The answer to this piece of evolutionary wisdom lies in the remarkable amplification of the receptor signal that takes place through the various elements of the G-protein cascade. Each receptor that binds an odorant molecule in turn stimulates hundreds of G-proteins for the duration that odorant is bound to it, and each adenylate cyclase enzyme that is stimulated by a G-protein generates hundreds of cyclic AMP molecules for each second of its active lifetime. This staggering amplification causes an increase in cyclic AMP levels that is thousands times greater than the prevailing change in the concentration of odorant, a signal which is sufficient to open many hundreds of ion channels rather than the one integral channel that is opened by a molecule of acetylcholine binding to an electroplax acetylcholine receptor. The resulting depolarization is enough to cause an increase in neurotransmitter release from the terminal of the olfactory receptor neuron which we perceive as a smell. If each odorant molecule only activated one ion channel, then the tiny traces of odorant within our environment that signal food or danger would be invisible to our impoverished sense of smell.

So why doesn't cyclic AMP simply continue to accumulate inside the olfactory receptor neuron resulting in a lingering sense of smell? Molecules talk to one another, not just in rigid linear pathways like troops lined up along a World War I trench. They talk with considered pauses, and feedback messenger molecules leave the trench to inform the originator of the message that it has been clearly received and understood. Indeed without such feedback, no complex system for the integration of information could function efficiently. In the olfactory receptor the role of feedback messenger is undertaken by the beta-gamma subunits, the migrant partners of the alpha subunits. This lone pairing of proteins activates another feedback messenger, a G-protein receptor kinase, whose duty it is to inform the olfactory receptor to stop stimulating further G-proteins, a task it achieves by the transfer of phosphate molecules onto the odorant receptor itself, a process known as phosphorylation. The addition of this bolus of negative charge directs the receptor towards its inactive, or 'red light' conformation, but just to tidy up affairs, it would seem only sensible after the departure of the odorant molecule to terminate the cyclic AMP signal as well. Just as cyclic AMP was created in a single step by cleaving a bond between the first and second phosphate molecules of ATP, so in a single step it is then converted from a ring structure back into a more linear one in the form of its cousin adenosine monophosphate (AMP), from which the ATP supply can subsequently be regenerated. The enzyme responsible for cleaving the phosphate bridge that maintains the ring structure is known as a phosphodiesterase, an enzyme that mediates the reverse step in this 'dead-end' pathway. In terms of their atomic make-up, AMP and cyclic AMP are almost identical, but only the latter has substantial second messenger activity in opening the receptor's sodium channels, illustrating the great importance of structure, or stereochemistry in determining the biological activity of molecules.

Odorant detection by the olfactory receptor neuron is just one of many cellular processes that are regulated by cyclic AMP, and cyclic AMP is only one of many second messengers whose levels are in some way governed by G-proteins. The fasting hormone glucagon, adrenaline, the luteinising and follicle-stimulating hormones that control mammalian reproductive cycles and the calcitonin and parathyroid hormones which regulate bone metabolism are but some of the many hormone-receptor systems that employ G-proteins and cyclic AMP to amplify receptor signals. However, in contrast to the olfactory receptor neuron, in many of these different types of cell cyclic AMP binds to an enzyme rather than an ion channel, and this enzyme goes by the name of protein kinase A. Two molecules of cyclic AMP bind to protein kinase A, causing the liberation of active kinase subunits which then transfer phosphate molecules from ATP onto a number of target enzymes and proteins, further amplifying and spreading the receptor's signal. Yet a significant part of the puzzle is still missing. Hormones such as glucagon and insulin compete with one another to establish the balance of cellular activity within many signaling systems, but so far G-proteins have been mentioned only in the context of increasing second messenger levels, where one species of G-protein, known as Gs, acts to increase adenylate cyclase activity. However, without ying there cannot be yang, as all life is a balance of opposing forces. In order to mediate the balance between antagonistic hormones another class of G-proteins evolved to inhibit adenylate cyclase, and this contrary species of G-protein is known as Gi. Thus each class of G-protein is in turn regulated by a distinct triad of hormone, receptor and G-protein. In consequence the cell's response to hormones is determined by what amounts to a see-saw mechanism, where the prevailing concentrations of hormone and the relative numbers of receptors collaborate to determine the balance of cellular activity.

(2) The visual system uses a cGMP-phosphodiesterase to detect single photons of light.

We can perhaps now begin to appreciate how the process of phototransduction serves to encode changes in the intensity of light into electrical signals that represent the language of the nervous system. The outer segments of rod photoreceptors contain the light-capturing pigment rhodopsin, which is composed of the chromophore 11-cis-retinal chemically bound to the protein opsin. Rhodopsin is present in the membrane discs of the outer segment, spanning the membrane seven times in all. Selig Hecht showed as long ago as 1938 that the absorption of single photon of light by a human rod cell is sufficient to cause a perceptible one milliVolt change in the potential across the photoreceptor membrane. This was later shown through the classical experiments of George Wald and co-workers to result from a photoisomerization, or geometrical change in the structure of 11-cis-retinal to trans-retinal, converting the energy of light into atomic motion. This atomic motion is in turn transmitted through the chemical bond linking retinal to opsin, causing a structural change in its protein partner within milliseconds. The activated form of the protein, metarhodopsin is then in the right 'shape' or conformation to activate a universal cellular signaling mechanism which translates the atomic motion of receptors into changes in the energy stored in chemical bonds. This mechanism is provided by 'G'-proteins, which are in essence a type of 'chemical switch' that couples signals received by receptors in the cell membrane into biochemical processes that take place both inside the cell and elsewhere in the cell membrane. G-proteins are found anchored to the membrane when unstimulated and are formed from two components; an alpha subunit and a seemingly inseparable combination of beta and gamma subunits. Metarhodopsin causes the alpha subunit to exchange the nucleotide guanosine diphosphate (GDP) for guanosine triphosphate (GTP) which contains an extra high energy bond between its second and third phosphate molecules. The presence of this additional negatively charged phosphate bound to the alpha subunit commands a change in the conformation of the alpha subunit which causes it to lose its affinity for the beta-gamma subunit. The two components then separate to form a bifurcating, or forked signaling pathway. The alpha subunit, now in its stimulated GTP-bound state is then able to activate the enzyme phosphodiesterase by pulling away its inhibitory subunit.
 
 

The phosphodiesterase of the photoreceptor cleaves the high energy phosphate bond that is formed within the second messenger molecule cyclic guanosine monophosphate, or cyclic GMP which is produced by the action of the enzyme guanylate cyclase upon GTP. A second messenger is any ion, molecule or protein that mediates the transfer of information within a cell following the arrival of a signal carried by a primary messenger to the cell surface, which may be a hormone, transmitter or in the case of the photoreceptor, light. The regulation of cGMP levels in the photoreceptor reflects the essence of many second messenger pathways where a metabolic building block such as the nucleotide component of GTP used in the assembly of DNA, is tranformed through a one-step dead-end biochemical pathway into a short-lived messenger molecule that is itself a highly specific and carefully regulated signal. This is elegantly demonstrated by the process of converting GTP into cyclic GMP through the action of a guanylyl cyclase and then back into the 'linear' non-messenger form guanosine monophosphate (GMP) by cleaving the phosphate bridge formed by the phosphate molecule by the action of a phosphodiesterase enzyme, the entire process being driven by the release of free energy stored in the bonds formed between phosphate molecules. The concentration of cGMP becomes the critical signal informing the visual system as to relative changes in light intensity in the photoreceptor at a point in time, and is itself precisely regulated by the balance of phosphodiesterase and guanylate cyclase enzyme activities in the photoreceptor. However, if changes in the cGMP concentration provide an almost instantaneous measure of changes in relative light intensity, other signaling pathways must have evolved to ensure that the sensitivity of elements that detect and control the concentration of cGMP are continuously adapted, or modulated to the absolute background level of light.

But what role do the beta-gamma subunits play in the light response? It was once thought that they served only as anchors for alpha subunits in the membrane, but they have now been implicated as major players in a number of signaling pathways. In the photoreceptor dissociated beta-gamma subunits migrate to activate rhodopsin kinase which is instrumental in the termination of the light response. Rhodopsin kinase belongs to another class of enymes involved in cell signaling called protein kinases which participate in another ubiquitous 'dead end' signaling pathway of cell metabolism. Protein kinases facilitate the transfer of the terminal phosphate molecule from adenosine triphosphate (ATP), the universal energy currency of the cell, onto specific amino acids of the target protein. These are amino acids which possess 'water-like' hydroxyl (-OH) groups and are surrounded by a specific signal sequence of amino acids that are recognised by the kinase before the transfer of a phosphate molecule onto the amino acid can occur, a process known as phosphorylation. Protein kinases are classified into two broad families according to whether they phosphorylate the hydroxyl groups of the amino acids serine or threonine, like rhodopsin kinase, or the larger amino acid tyrosine, an event typical of many pathways involved in the regulation of cell growth and division. The transfer of a highly negatively charged phosphate molecule onto a target protein has several effects. Firstly it dramatically alters interactions between charged residues within the target protein resulting in a change in its conformation and consequently its activity, and secondly it alters its interactions with other proteins either as a result of these conformational changes or from direct interactions with the added phosphate groups. In some proteins phosphorylation alters the affinity of a protein for the cell membrane, which may sometimes lead to a change in its location within the cell.

This does not fully explain how rhodopsin kinase inactivates metarhodopsin. Once rhodopsin kinase has performed multiple phosphorylations at the terminal portion of metarhodopsin, another protein aptly named arrestin binds to metarhodopsin to prevent it from further stimulating G-protein activity. This in cell talk is known as a feedback pathway, where the duration of a signal is regulated by downstream elements in the pathway; in this case the receptor that stimulates the G-protein is itself switched off by an element downstream of G-protein activation. The light response finally ends for metarhodopsin when it is regenerated by the replacement of the spent trans-retinal chromophore with fresh 11-cis-retinal, whereupon arrestin can no longer bind to phosphorylated rhodopsin and leaves. A protein phosphatase, the reverse component of the 'dead-end' kinase signaling pathway, is then able to strip the phosphates off the terminal portion of rhodopsin in preparation for another cycle. As for the alpha subunit, it possesses an intrinsic time switch which is triggered by its interaction with the cGMP phosphodiesterase, resulting in the cleavage of the terminal phosphate of its bound GTP molecule in a second example of a feedback pathway. This returns the alpha subunit to its inactive GDP-bound conformation and allows it to reassociate with beta-gamma subunits, and the G-protein cycle is ready to start again.

But how is the concentration of cyclic GMP finally translated into an electrical signal ? It seemed reasonable to predict that the photoreceptor possesses a sensor that couples changes in the cyclic GMP concentration to the observed changes in membrane potential. Fesenko and colleagues were the first to be able to show that cGMP binds directly to a population of ion channels causing them to open. These cGMP-regulated channels were shown to be selectively permeable to calcium and sodium ions, which in the absence of light-stimulated phosphodiesterase activity flood into the cell and depolarize the photoreceptor membrane. This depolarization is in turn is propagated and translated into an increase in the rate of glutamate release from the photoreceptor terminal. Conversely, in response to light phosphodiesterase reduces the cyclic GMP concentration, cyclic GMP-regulated channels close, the intracellular face of the membrane becomes more negatively charged, or hyperpolarized and there is a subsequent decrease in glutamate release from the photoreceptor terminal. So how does a single photon generate as much as a milliVolt change in membrane potential ? The answer lies in the remarkable amplification of the light signal by the elements of the phototransduction cascade. A metarhodopsin receptor activated by a single photon has been estimated to activate about five hundred G-proteins during its lifetime. In turn, each phosphodiesterase enzyme activated by a G-protein can cleave more than four thousand molecules of cGMP for each second of its active lifetime. The resulting fall in the cGMP concentration due to the activation of this biochemical cascade is sufficient to close hundreds of cation channels and evoke a membrane hyperpolarization as large as a milliVolt, which we sense as a reduction in the rate of release of glutamate from the photoreceptor terminal.

But what happens to the calcium ions that flood into the photoreceptor outer segment through the cyclic GMP-regulated channel ? Calcium in its free ionic form is normally kept at very low concentrations inside cells, in fact as much as one hundred thousand times lower than the external concentration. In the photoreceptor this gradient is maintained by a membrane transport protein that continuously exports calcium and potassium ions from the cell interior in exchange for entry sodium ions from outside the cell. Following the influx of calcium ions through the cGMP-regulated channels specialized intracellular proteins bind to free calcium ions with high affinity to prevent the internal calcium concentration from reaching levels that will destroy the cell. King-Wai Yau and Nakatani were the first to show that this membrane transport protein continues to export calcium ions after the cGMP-regulated channels that mediate calcium entry have closed in response to light, which Peter McNaughton and colleagues subsequently showed causes a fall in the intracellular calcium concentration which is a critical signal in adaptation.

But at what points in the phototransduction cascade is the degree of amplification, or sensitivity to light controled to provide a mechanism for adaptation to lower background levels of light ? The first clue was provided by the observation that after a prolonged period in the dark calcium levels in the unstimulated rod photoreceptor become elevated due to the continuous influx of calcium through GMP-regulated channels. The team of Mark Gray-Keller, Arthur Polans, Kris Palczewski and Peter Detwiler demonstrated that the calcium-binding protein recoverin prolongs the duration of the light response by preventing rhodopsin kinase from phosphorylating metarhodopsin. Denis Baylor and Leon Lagnado showed that in addition to recoverin, a number of other calcium-regulated factors combine to amplify light-stimulated increases in phosphodiesterase activity as much as thirty-fold. However Koch and Stryer, and later Yiannis Koutalos and colleagues provided evidence that increases in free calcium that occur in dark-adapted rod photoreceptors also inhibit the production of cyclic GMP by guanylate cyclase through another unidentified calcium-binding protein, decreasing the size of the current carried through cyclic GMP-regulated channels. Lastly, but by no means finally, Yi-Te Hsu and Robert Molday showed that calcium binds to a ubiquitous calcium-regulated protein known as calmodulin, causing a reduction in the affinity of the channel for cyclic GMP. This in turn results in a decrease in the sensitivity of the channel to changes in cyclic GMP concentration, and may serve to extend the range of cGMP concentrations over which the channel sensor operates. Combining the actions of these various calcium-binding proteins of the rod photoreceptor into the larger picture of adaptation, Yiannis Koutalos and King-Wai Yau were able to make a number of predictions about the consequences that a fall in intracellular free calcium levels will have upon the degree of amplification of the light signal in broad daylight. The decrease in free calcium levels will increase the cGMP concentration in the rod photoreceptor by both inhibiting phosphodiesterase activity and stimulating guanylate cyclase activity. More cation channels will open due to the elevated cGMP concentrations and their increased affinity for cGMP, because calmodulin is no longer activated by calcium. The combined effect of these decreases in calcium during light adaptation is to reduce the sensitivity of the rod photoreceptor to a given increase in light intensity, because each excited metarhodopsin protein hydrolyses a smaller fraction of the available pool of cyclic GMP molecules due to the reduced level of gain in the phototransduction cascade and the increased cGMP concentration. The net advantage that all of these biochemical adjustments confer is to trade a reduction in the absolute sensitivity of the rod photoreceptor to light for an increase the range of background light levels over which the photoreceptor is able to detect a change in light intensity.

Is calcium the only second messenger that plays a role in light adaptation in the photoreceptor? It would appear not. The guanylate cyclase present in the photoreceptor has a built-in sensor that detect increased nitric oxide levels, and when this sensor binds to nitric oxide it stimulates an increase in cGMP production. A team led by Steve Barnes and Dmitry Kurrenny provided evidence that nitric oxide opens both cGMP-regulated cation and excitable calcium channels in rod photoreceptors. Their findings strongly suggest that nitric oxide production in response to increased levels of background light serves to amplify changes in the rate of glutamate release from the rod photoreceptor by enhancing the rate of calcium entry into the terminal. Further, increases in cyclic GMP concentration stimulated by nitric oxide will help to extend the range of light intensities over which the photoreceptor is responsive to changes in light intensity.

(3) An example of interplay (cross-talk) between cyclic nucleotide-signaling pathways : The regulation of small intestinal secretion.

Nowhere is this fine regulatory balance more dramatically illustrated than in the intestine. The single layer of epithelial cells that lines the small intestine is capable of both the net secretion and absorption of salt and water, a balance that is kept in check by the graded release of various hormones and transmitters from the terminals of the intestine's enteric nervous system. Hormones that increase cyclic AMP levels within these intestinal epithelial cells favour a balance of salt and water secretion over absorption, whilst hormones that act by decreasing cyclic AMP levels favour absorption. A delicate balance of cyclic AMP elevating and depressing hormones compete to regulate the ebb and flow of the intestinal tide, a balance which is lost with fatal consequences in the inherited disease Cystic Fibrosis, where there is insufficient secretion to support the proper absorption of fluid and nutrients, and also in infectious diarrhoea where the body becomes dehydrated through the uncontrolled loss of salt and water from the intestines. Thus the net transfer of salt and water across the intestine is the outcome of a fine interplay between the contradictory processes of absorption and secretion, a balance of hormonal signaling that is effectively integrated at the molecular level by adenylate cyclase.

Watery diarrhoea may be induced by bacterial or viral infection and is due to the 'hypersecretion' of salt and water across the surface of the intestine, a condition that accounts for the deaths of some five million children a year through dehydration. Two of the principle bacterial culprits responsible are cholera and Bordetella pertussis, both of which produce toxins that effectively hijack the cyclic AMP second messenger system of the intestinal epithelial cells. Their toxins sabotage the balance by persistently activating adenylate cyclase through the chemical modification of the G-protein alpha subunits that govern cyclic AMP production. Cholera toxin irreversibly modifies the alpha subunit of Gs by adding the cellular metabolite ADP-ribose, preventing it from returning to its quiescent state, resulting in a 'runaway' production of cyclic AMP and the uncontrolled loss of salt and water from the intestine. In a perhaps not so subtle contrast, pertussis toxin modifies and permanently inactivates adenylate cyclase's suppressor Gi through much the same mechanism, thereby preventing it from down-regulating cyclic AMP production with the same devastating consequences. Concentrations of intracellular second messengers are thus a delicate balance set by competing hormonal influences, and as is illustrated by the intestine, a perturbation in the pathways that govern their production may have dramatic (and fatal) consequences.

Receptors that talk to the cell through G-proteins are termed metabotropic receptors because they directly influence the metabolic status of the cell. G-proteins have been shown to govern a great number of cellular processes including the contraction of smooth muscle cells, the control of glycogen reserves in liver cells, platelet aggregation in blood vessels and the secretion of inflammatory mediators by mast cells. The control of such a diverse number of cellular processes is achieved not only through the modulation of ion channels and adenylate cyclase, but also through other second messenger generating enzymes. In his book the Extended Phenotype, Professor Richard Dawkins refers to the meme, which he defines as a cultural or technological theme that becomes self-perpetuating in a society simply because it is a good idea. Biological systems of interacting genes, such as those that form G-protein signaling systems have similarly been preserved throughout evolution because they provide a simple and effective biochemical strategy for the amplification of signals. Consequently G-protein systems are ubiquitous, regulating all manner of systems from intracellular cellular calcium levels to the direct regulation of ion channels, and the list of processes known to be controlled by G-proteins grows ever larger with investigation. The advent of the G-protein switch represented a quantum leap in evolution, an advance that allowed small signals to become greatly amplified, endowing us with otherwise unimaginable levels of acuity in our senses of vision, smell and taste.

So we have now identified two receptor mechanisms, one for the fast, localized transmission of information in nerves and muscle, and another for the graded amplification of signals that are carried by otherwise undetectable levels of hormone. Two quite different problems of information transfer that were solved by two intriguingly different strategies, demonstrating once again that time and Nature are the greatest engineers. Yet one further problem of information transfer remains unelucidated. Many cellular processes such as growth and division, regulated by the family of hormones known as growth factors, take place over many hours or even days, demanding co-ordinated changes within the cell's many diverse metabolic and signaling pathways. Whereas a receptor that governs the opening of an ion channel or the activity of a G-protein may elicit a brief and highly localized change in the activity of the cell, the nature of a receptor that governs long-lasting changes in both the expression of the cell's genetic program and its metabolic and structural machinery is likely to be very different. Clearly such a receptor signal must be not only be amplified, but also spread widely through the cell to co-ordinate such complex changes in activity. In fact this class of receptor communicates through not just one, but indeed several distinct second messenger pathways that serve to disperse the receptor signal.