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35. Jensen, E. V. et al. A two-step mechanism for the interaction of estradiol with rat uterus. Proc. Natl Acad. Sci. USA 59, 632638 (1968). 36. Jensen E. V. & De Sombre, E. R. Estrogenreceptor interaction: estrogenic hormones effect transformation of specific receptor proteins to a biochemically functional form. Science 182, 126134 (1973). 37. Baulieu, E.-E. et al. in Vitamins and Hormones 649736 (Academic Press, New York, 1975). 38. Pratt, W. B. & Toft, D. O. Steroid receptor interactions with heat shock proteins and immunophilin chaperones. Endocrine Rev. 18, 306360 (1997). 39. Parker, M. G. (ed.) Nuclear Hormone Receptors (Academic Press, London, 1991). 40. Kuiper, G. J. M. et al. Cloning of a novel estrogen receptor expressed in rat prostate. Proc. Natl Acad. Sci. USA 93, 59255930 (1996). 41. Laudet, V. & Gronemeyer, H. The Nuclear Receptor Facts Book (Academic Press, Orlando, 2002). 42. Chambon, P. The molecular and genetic dissection of the retinoid signaling pathway. Recent Prog. Horm. Res. 50, 317332 (1995). 43. Mansn, A., Yu, F., Forrest, D., Larsson, L. & Vennstrm, B. TRs have common and isoform-specific functions in regulation of the cardiac myosin heavy chain genes. Mol. Endocrinol. 15, 21062114 (2001). 44. Cheng, K. W., Cheng, C.-K. & Leung, P. C. K. Differential role of PR-A and -B isoforms in transcription regulation of human GnRH receptor gene. Mol. Endocrinol. 15, 20782092 (2001). 45. Gauthier, K. et al. Different functions of the thyroid hormone receptors Tr and Tr in the control of thyroid hormone production and post-natal development. EMBO J. 18, 623631 (1999). 46. Schwabe, J. W. R. et al. The crystal structure of the estrogen receptor DNA-binding domain bound to DNA: how receptors discriminate between their response elements. Cell 75, 567578 (1993). 47. Rastinejad, F. et al. Structural determinants of nuclear receptor assembly on DNA direct repeats. Nature 375, 203211 (1995). 48. Schule, R. et al. Many transcription factors interact synergistically with steroid receptors. Science 242, 14181420 (1988). 49. Papavassilou, A. Transcription Factors in Eukaryotes (Springer, Berlin, 1997). 50. Fondell, J. D., Roy, A. L. & Roeder, R. G. Unliganded thyroid hormone receptor inhibits formation of a functional preinitiation complex: implications for active repression. Genes Dev. 7, 14001410 (1993). 51. Chakravarti, D. et al. Role of CBP/P300 in nuclear receptor signalling. Nature 383, 99103 (1996). 52. Demarest, S. J. et al. Mutual synergistic folding in recruitment of CBP/p300 by p160 nuclear receptor cooactivators. Nature 415, 549553 (2002). 53. Halachmi, S. E. et al. Estrogen receptor-associated proteins: possible mediators of hormone-induced transcription. Science 264, 14551458 (1994). 54. Onate, S. A., Tsai, S. Y., Tsai, M. J. & OMalley, B. W. Sequence and characterization of a co-activator for the steroid hormone receptor superfamily. Science 270, 13541357 (1995). 55. Chambon, P. et al. Promoter elements of genes coding for proteins and modulation of transcription by estrogens and progesterone. Recent Prog. Horm. Res. 40, 139 (1984). 56. Beato, M. et al. Interaction of steroid hormone receptors with transcription factors involves chromatin remodelling. J. Ster. Biochem. Mol. Biol. 56, 4759 (1996). 57. Ashburner, M. et al. Temporal control of puffing activity in polytene chromosomes. Cold Spring Harbor Symp. Quant. Biol. 38, 655662 (1974). 58. Thummel, C. S. From embryogenesis to metamorphosis: the regulation and function of Drosophila nuclear receptor superfamily members. Cell 38, 871877 (1995). 59. Ito, M. & Roeder, R. G. The TRAP/SMCC/Mediator complex and thyroid hormone receptor function. Trends Endocrinol. Metab. 12, 127134 (2001). 60. Parker, P. (ed.) Cell Signalling (Cold Spring Harbor, New York, 1996). 61. Cohen, P. & Frame, S. The renaissance of GSK3. Nature Rev. Mol. Cell Biol. 2, 769776 (2001). 62. Sachs, L. M. & Shi, Y.-B. Targeted chromatin binding and histone acetylation in vivo by thyroid hormone receptor during amphibian development. Proc. Natl Acad. Sci. USA 97, 1313813143 (2000). 63. Elbashir, S. M. et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494498 (2001). 64. Brivanlou, A. H. & Darnell, J. E. Jr. Signal transduction and the control of gene expression. Science 295, 813818 (2002).

Online links
DATABASES The following terms in this article are linked online to: LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink/ androgen receptor | CBP | epidermal growth factor | growth hormone | insulin | Janus kinase | oestrogen receptor | PPAR | progesterone receptor | prolactin | RXR | thyroid hormone receptor | tyrosine aminotransferase | VDR OMIM: http://www.ncbi.nlm.nih.gov/Omim/ diabetes Access to this interactive links box is free online.

Acknowledgement
I wish to thank Debbie Duthie for expert help in preparing this review.

TIMELINE

Cyclic nucleotide research still expanding after half a century

Joseph A. Beavo and Laurence L. Brunton
Since the discovery in 1957 that cyclic AMP acts as a second messenger for the hormone adrenaline, interest in this molecule and its companion, cyclic GMP, has grown. Over a period of nearly 50 years, research into second messengers has provided a framework for understanding transmembrane signal transduction, receptoreffector coupling, protein-kinase cascades and downregulation of drug responsiveness. The breadth and impact of this work is reflected by five different Nobel prizes.

It is now dogma that many hormones and neurotransmitters (first messengers) bind to cell-surface receptors, and that their signals are transduced into an intracellular second messenger, such as cyclic AMP. However, this knowledge is a recent development. The lineage of cyclic-nucleotide research can be traced back to Carl and Gerty Cori, who received the Nobel Prize in 1947 for their work on glycogen metabolism. In their laboratory, a postdoctoral fellow, Earl Sutherland (BOX 1), absorbed what was known about the actions of glycogen phosphorylase, phosphoglucomutase and glucose-6-phosphatase, and set himself the goal of understanding how a hormone such as adrenaline caused glycogenolysis. He and a few colleagues at Washington University, and later at Western Reserve, made good progress in describing the hormonal activation of glycogen phosphorylase in slices of dog liver. However, in 1955 shortly before the discovery of cAMP Sutherland wrote, the precise mechanism of action of any hormone has not yet been clearly established (cited in REF. 1).

1956 and 1957 were red-letter years. Wosilait and Sutherland discovered that the release of inorganic phosphate accompanies the inactivation of liver phosphorylase2. Krebs and Fischer found that ATP is required for the conversion of phosphorylase B to phosphorylase A in soluble extracts of rabbit muscle3. Rall, Sutherland and Wosilait4 showed that the activation of liver phosphorylase was accompanied by the incorporation of phosphate into the enzyme. Rall, Sutherland and Berthet5 then showed this activation in a cellfree system. Three conclusions from that paper are noteworthy. First, the relative activities of sympathomimetic amines in homogenates were similar to those observed in liver slices and in intact animals (this care to be physiologically relevant was typical of Sutherland). Second, the response to the hormones in liver homogenates was separated into two phases: the formation of an active factor in particulate (membrane) fractions in the presence of hormones; and the stimulation by the factor of the formation of liver phosphorylase in the supernatant fractions of homogenates in which the hormones themselves had no effect. Third, the active factor was heat stable and could be dialysed, and was purified considerably by chromatography on anion- and cation-exchange resins. Shortly thereafter, the heat-stable factor was found to contain adenine, ribose and phosphate in a ratio of 1:1:1, and to be stable to acid and common phosphatases but to be enzymatically inactivated to 5-AMP6. So, by 1958, cAMP, the synthetic activity (adenylyl cyclase) and the degrading activity (phosphodiesterase (PDE)) had been described.

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Box 1 | Nobel Prizes for discoveries about cyclic nucleotides and signal transduction The importance of this regulatory system has been recognized by the award of no less than five different Nobel Prizes. These include the original prize to Earl W. Sutherland Jr for his pioneering initial discoveries, as well as more recent ones to Edwin G. Krebs and Edmond H. Fischer, largely for their discovery of the first molecular mechanism by which cyclic AMP works. The work of Martin Rodbell and Alfred G. Gilman on the G-protein-coupled receptors that regulate cyclic-nucleotide synthesis also formed the basis of an award. cGMP trailed behind a bit, but the pioneering work of Ferid Murad, Louis J. Ignarro and Robert F. Furchgott was also recognized by a Nobel Prize. Finally, although not given entirely for work on cyclic nucleotides, the recent award to Arvid Carlsson, Paul Greengard and Eric R. Kandel for their discoveries concerning signal transduction in the nervous system stems in large part from the work on functional effects of cAMP and cGMP in the nervous system. This is an amazing record for such simple second-messenger compounds, and emphasizes the widespread importance of the regulatory systems that underlie their functions. A list of the awards and a photograph and brief description of each of the awardees are shown. 1971 Earl W. Sutherland Jr, for his discoveries concerning the mechanisms of action of hormones through cAMP. 1992 Edmond H. Fischer and Edwin G. Krebs, for their discoveries concerning reversible protein phosphorylation as a biological regulatory mechanism. 1994 Alfred G. Gilman and Martin Rodbell, for their discovery of G proteins and the roles of these proteins in signal transduction in cells. 1998 Robert F. Furchgott, Louis J. Ignarro and Ferid Murad, for their discoveries concerning nitric oxide and cGMP as signalling molecules in the cardiovascular system. 2000 Arvid Carlsson, Paul Greengard and Eric R. Kandel, for their discoveries concerning signal transduction in the nervous system. Images The Nobel Foundation.
1971 1992 1994

Earl W. Sutherland 1998

Edmond Fisher

Edwin G. Krebs

Alfred G. Gilman 2000

Martin Rodbell

Robert Furchgott

Louis J. Ignarro

Ferid Murad

Arvid Carlsson

Paul Greengard

Eric R. Kandel

Moreover, the hormone receptors and adenylyl-cyclase activity were found to be confined to membranes. Rall and Sutherland were fortunate in their choice of preparations such results would not have been obtained easily in rat liver, in which the -adrenergic receptor pathway largely mediates the effect of adrenaline. Nonetheless, the validity of these early observations has not been challenged, and our ideas today follow the outline established by these first descriptions of the cAMP pathway (TIMELINE). Ted Rall and Henry Bourne

have offered interesting perspectives on this early work7. The discovery of cAMP and the formulation of the second-messenger concept were met with scepticism. In particular, the assertion that cAMP was a nucleotide that was stable to boiling in 0.1 N hydrochloric acid was considered heretical only acidlabile phosphates were known at the time. Not until the synthesis and determination of its structure by Lipkin and colleagues8,9 was the scepticism replaced by a measure of acceptance (FIG. 1).

Well into the 1960s, the second-messenger concept was not taught in most undergraduate biology courses. Indeed, in 1971, much of what was known about cyclic nucleotides could be summarized in two monographs1,10. Beginning around 1971, there was an explosion of activity and interest. We devote the remainder of this Perspective to the identification of some of the important advances that have stimulated progress in cyclic nucleotide research since the discoveries of cAMP and cGMP, and the initial descriptions of their basic regulatory

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Timeline | Major advances in cyclic nucleotide research

Discovery of cGMP as natural product in urine (Ashman and Price). 1963present. Expansion of cAMP effects beyond regulation of glycogenolysis (Butcher, Robison, Steinberg, Langan, Shultz, Fesenko, Bos, Montminy and others). Millipore filter assay for cAMP (Gilman). Immunoassay developed for cAMP (Steiner).

Somatic cell mutants for identification of cAMP pathways (Bourne, Coffino and Tompkins).

Role of cAMP in Aplesia memory (Kandel).

Description of EDRF (Furchgott).

Nucleotide-gated channels as receptors for cGMP and cAMP (Fesenko).

1958

1963

1968

1970

1971

1973

1975

1976

1978

1980

1983

1985

1986

Discovery of cAMP as factor that stimulates phosphorylase (Rall, Sutherland).

cAMP-dependent protein kinase identified (Krebs).

1971present. Multiple isozymes of G proteins, cyclase, kinases and PDEs allow cellular and subcellular specificity of cAMP and cGMP function (Bourne, Gilman, Storm, Garbers, Krebs, Greengard, Beavo, Houslay and others).

cGMP regulation of visual transduction by light activation of PDE (Bitensky; Yamazaki).

Reconstitution of hormonesensitive cyclase (Gilman).

Rapid turnover of cAMP and cGMP (Walseth and Goldberg).

cAMP regulation of transcription (Montminy; Hanson).

Only those discoveries of particular importance to the concepts being discussed in this Perspective are depicted. ANP, atrial natriuretic peptide; cAMP, cyclic AMP; cGMP, cyclic GMP; EDRF, endothelial-derived relaxant factor; NO, nitric oxide; NOS, nitric oxide synthase; PDE, phosphodiesterase; PKA, protein kinase A; PKG, protein kinase G.

systems. We emphasize data that, although often not considered particularly important when originally reported, in retrospect have greatly influenced our current understanding of cyclic nucleotide metabolism and function.
Importance of cyclic-nucleotide assays

The excitement surrounding the discovery of second messengers derived largely from the many physiological processes that these molecules regulate. However, some effects, such as regulation of cardiac rate and contractility by -adrenergic receptor agonists, were not so obvious initially and were, therefore, delayed in their acceptance. In retrospect, much of this delay revolved around the problems of measuring the rapid changes in cAMP that can occur in tissues. Researchers recognized early on that obtaining meaningful measurements of cAMP or cGMP required rapid tissue fixation. However, this fact did not translate into a realization that cyclic nucleotides could regulate physiological processes on a timescale of seconds to milliseconds. So, most early studies concentrated on how steady-state changes in cAMP and cGMP regulated slowly changing metabolic processes.
NH2 N N O 5 O 3 O N O N 5 O 3 O N N N O NH NH2

O P O

O P O

3, 5 -Cyclic AMP (cAMP)

3, 5 -Cyclic GMP (cGMP)

Figure 1 | Structures of cyclic AMP and cyclic GMP.

The problem of assaying cAMP became an issue almost immediately. Sutherlands original assay, which was the only one available for more than ten years, depended on the capacity of cAMP to activate glycogen breakdown in a test tube that contained inactive phosphorylase, liver homogenate and ATPMg2+. This assay was tedious at best, had a dynamic range of less than fourfold, depended on the purification of several enzymes and varied with the characteristics of the dog-liver supernatant fraction that contained the necessary protein kinases8. Subsequent assays relied on the direct transfer of 32P from [32P]ATP to muscle-phosphorylase kinase or casein11, or the dilution of 5AMP formation from radiolabelled cAMP in an assay of PDE activity12. In addition, there were enzyme-linked assays that used spectrophotometric or fluorescent output1,13, although these methods were also demanding. As a result, only a few laboratories were able to reproducibly measure cAMP, and these laboratories had a monopoly on studies of its metabolism. So, the development in 1970 of two competitive-binding assays a radioimmunoassay14 and a protein-binding assay15 made the assay of cAMP much more accessible. Gilmans procedure15 was the easier and cheaper of the two, and could detect 0.1 pmoles of cAMP, which is sensitive enough for most purposes, because basal levels of cAMP are ~5 pmoles mg1 of cell protein. The Gilman assay used a commercially available ligand: [3H]cAMP. Moreover, only one protein the cAMP-sensitive protein kinase that had been described by Krebs and colleagues11 needed to be prepared; a fresh beef steak from the butchers shop worked

well as starting material. This assay was a boon to many and an early triumph for Gilman, but he missed discovering how cAMP affects protein kinase A (PKA). That was left to others1618, who independently deduced that cAMP caused the dissociation of the holoenzyme on activation. A similar binding assay19 and radioimmunoassay20 for cGMP followed shortly. The radioimmunoassay developed by Harper and Brooker20 was noteworthy: acetylation of the cyclic nucleotide brought the sensitivity into the femtomole range. This is important for measuring cGMP, which often exists at onetenth of the tissue content of cAMP.
Cyclic nucleotide synthesis

Once the main enzymes of cyclic-nucleotide metabolism had been identified, there was still the question of how an agonist added to the outside of the cell membrane could raise cAMP levels within the cell (FIG. 2). In a series of studies between 1969 and 1971, Birnbaumer and Rodbell2125 used membranes purified from hepatocytes and adipocytes to address this issue. Their careful work showed that hormone-sensitive cAMP production required GTP, leading to the postulation of a transducer that somehow linked the receptor and the cyclase. This finding was not immediately accepted, in part because other investigators could measure hormonal responses in vitro without adding GTP. Rodbell and colleagues were right, however other investigators were using insufficiently washed membranes and ATP that was slightly, but sufficiently, contaminated with GTP, so that micromolar concentrations of GTP were actually present in the assays. Once

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we now recognize ~335 seven transmembrane (7TM) receptors in the G-protein-coupled receptor superfamily (non-sensory), 20 G-protein -subunits and 10 adenylyl cyclases35, not to mention the G subunits, which affect the activities of some isoforms of adenylyl cyclase and are involved in the localization of G-protein-receptor kinases (GRKs) that are important in inactivating G-proteinlinked signal transduction1 (see also the article on page 639 by K. L. Pierce, R. T. Premont and R. J. Lefkowitz).
Cyclic GMP
1987present. Knockouts of PKA, PKG and cyclases (McKnight, Hofmann, Storm and Garbers). Regulation of NOS by Ca2+/CaM (Bredt and Snyder). Crystal structure of adenylyl cyclase and Gs (Sprang and Gilman). Introduction of Viagra for treatment of erectile dysfunction (Pfizer). Crystal structure of PDE catalytic domain (Xu).

NO stimulates soluble guanylyl cyclase (Ignarro; Murad).

Receptor for ANP is particulate guanylyl cyclase (Chinkers and Garbers).

Crystal structure of PKA. (Taylor and Sowadski).

Cyclic nucleotides can also bind to and activate guanine-nucleotide exchange factors (GEFs) (Bos and Graybiel).

Crystal structure of GAF domain (Martinez).

1987

1989

1990

1991

1997

1998

2000

2002

these issues became known, the requirement for GTP was clear. What, exactly, GTP was doing was not clear, but several observations helped to form an understanding. Cassel and Selinger described a hormone-stimulated GTPase activity26. Rodbells lab found that non-hydrolysable analogues of GTP activated adenylyl-cyclase activity in the absence of hormone. Then, Maguire, van Arsdale and Gilman found that the addition of GTP selectively reduced the affinity of the 2receptor for agonists but did not alter the affinity for antagonists15. Adenylyl cyclase activity is exceedingly labile, defying efforts to solubilize and study it in a simple in vitro system. Progress was facilitated by the work of Gordon Tomkins and colleagues, who characterized a cytocidal effect of cAMP on clonal mouse S49 lymphoma cells27. Bourne et al.28 selected a variant of these cells that had 2-receptors but seemed to make no cAMP; it seemed to lack adenylyl cyclase and was named cyc. Analysis of cyc cells by Ross and Gilman provided biochemical evidence for the stimulatory G protein2931. They found a factor, sensitive to proteases and distinct from adenylyl cyclase, which could reconstitute hormone-sensitive adenylyl cyclase activity in cyc membranes. Clearly, cyc S49 cells were not missing adenylyl cyclase; rather, they were lacking this activating or coupling protein. Gilmans lab therefore proposed that hormone receptors regulated the interaction of the activating/coupling protein and adenylyl cyclase. At the same time, Thomas Pfeuffer partially resolved adenylyl-cyclase activity and the activating factor using a guanine-nucleotide affinity resin32.

The parallels of hormonal activation of cAMP production and phototransduction did not escape the attention of Mark Bitensky, who identified a light-activated cGMP PDE activity and a light-activated GTPase activity in retinal cells33. This work led to the identification of Gt or transducin, and to the understanding that a photon of light, interacting with the membrane receptor rhodopsin, activates transducin, which in turn activates PDE6 (REF. 34). With purification and the advent of molecular cloning, the family of G proteins began to expand, and Go, Gq, Gz, G12/13 and small G-proteins were discovered. In mammalian systems,
a
ANPs Guanylin, STa pGC cGMP PDEs 5-GMP

Although it was synthesized earlier, cGMP was not identified as a molecule of potential biological importance until 1963, when Ashman and Price identified the main organic-phosphate-containing molecules in urine as being cAMP and cGMP36; moreover, the amounts varied according to the hormonal state of the animal37. By 1969, the enzymes for the synthesis and breakdown of cGMP had also been identified38. Until the 1980s, however, it was unknown which hormone stimulated the synthesis of cGMP or which metabolic pathway it regulated. Two discoveries, both related to regulation of smooth-muscle contraction, were pivotal. The first was the discovery that endothelial-derived relaxant factor (EDRF) a small molecule made in endothelial cells that causes the adjacent smooth muscle to relax39 could stimulate the production of cGMP4042. The second was the discovery that a peptide made in the heart (atrial natriuretic peptide; ANP) could also increase cGMP in
b
7TM receptor Hormone, Neurotransmitter Adenylyl cyclase

GTP
NO sGC NO

Gs
5-AMP PDEs

G s AT P

cAMP

cAMP PDEs PKG PKA Protein phosphorylation Protein phosphorylation CNG channels GEFs CNG channels

cAMP

5-AMP

Na+, Ca2+

Na+, Ca2+

Figure 2 | Basic mechanisms of cyclic-nucleotide regulation and function. This cartoon shows the basic synthetic and regulatory pathways for a | cyclic GMP and b | cAMP metabolism. In both cases, the main mechanisms for regulation of synthesis by hormones and the main receptors for the nucleotide are depicted. Modified with permission from REF. 92 2000 The Company of Biologists Ltd. ANPs, atrial natriuretic peptides; CNG, cyclic-nucleotide gated; GEF, guanine-nucleotide exchange factor; NO, nitric oxide; pGC, particulate guanylyl cyclase; PKA, protein kinase A; PKG, protein kinase G; sGC, soluble guanylyl cyclase; STa, heat-stable enterotoxin.

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Cyclic-nucleotide-binding proteins. Every few years, another cyclic-nucleotide-binding protein family has been identified; we now recognize three other types of biologically important cyclic-nucleotide-binding protein. The first to be discovered were the high-affinity allosteric binding sites on cGMP-stimulated phosphodiesterase (PDE2)53, which now encompass a family of homologous cAMPbinding sites on several proteins54. The next receptors that were found were the cGMPand cAMP-binding sites on cyclicnucleotide-gated channels55, including the potassium channel on cardiac pacemaker cells. Originally thought to be present only in the retina and cardiac pacemaker cells, these channels have now been found in many other cell types56. Most recently, high-affinity cyclic-nucleotide-binding sites on guaninenucleotide exchange factors (GEFs) have been shown to have great biological importance57,58. It is probably worth emphasizing that we still do not know how many of the processes that are regulated by cyclic nucleotides are not driven by the PKA/PKG kinase system. GEFs and cyclic-nucleotidegated ion channels will probably assume increasing importance. Long-term effects: transcriptional regulation. In the late 1960s, Langan noted that raised cAMP levels enhanced the phosphorylation of nuclear histones50, giving rise to the speculation that histone phosphorylation could mediate hormonal regulation of messenger RNA levels. Shortly after, several laboratories developed systems in which elevated cAMP and the activation of PKA seemed to be coupled to the induction of specific proteins59. Delineating the molecular basis of this effect had to await a better understanding of the elements of transcription in mammalian cells. The effects of cAMP became clearer with the discovery of a cAMP-response element in the somatostatin gene60 and a cAMP regulatory domain in the phosphoenolpyruvate carboxykinase gene61. The first transcription factor found to be regulated by PKA was the cAMP-response-element-binding protein (CREB) and its family members activating transcription factor 1 (ATF1) and cAMPresponsive-element modulator (CREM) (BOX 3; see Mayr and Montminy62). These mechanisms of cyclic-nucleotide effects on transcription have been incorporated into our current ideas about everything from memory formation to immune-cell regulation63,64. PDEs: regulators of cyclic nucleotides. For the first few years after the discovery of cAMP and cGMP, many studies focused on PDEs.

Box 2 | Mechanism of action of the drug Viagra

Locally produced NO
Ca2+ export Ca2+ Ca2+ entry

sGC

Ca2+ mobilization

CaM MLCK MLCK (active)

5-GMP

PDEs

IP3R cGMP

Ca2+

(inactive) P MLCK (non-activatable)

Viagra

PKG

MLC phosphatase MLC

P MLC

Smooth muscle cell

Actin Contraction

Penile erection occurs when blood swells the corpus cavernosum, an effect facilitated by relaxation of regional smooth muscle. Smooth muscle tone is regulated by cellular Ca2+, which activates the Ca2+/calmodulin (CaM)-dependent enzyme myosin light chain kinase (MLCK), which leads to MLC phosphorylation and contraction. The nitric oxide (NO) pathway leads to relaxation of smooth muscle by stimulating the soluble guanylyl cyclase (sGC), which results in the production of cyclic GMP (cGMP) and the activation of cGMP-dependent protein kinase (PKG). PKG causes smooth-muscle relaxation by mechanisms that are still being defined and that might include a reduction in cytosolic Ca2+ (by enhanced Ca2+ export and/or by reduced inositol trisphosphate (InsP3) receptor-mediated Ca2+ mobilization) and dephosphorylation of myosin light chains (by activation of MLC phosphatase and/or by sequestration of MLCK in a phosphorylated form that is not readily activated by Ca2+/CaM). Viagra specifically inhibits the breakdown of cellular cGMP by PDE5 (an isoform of phosphodiesterase that is localized to erectile tissue), and thereby prolongs and enhances the effects of NO/cGMP.

smooth muscle, kidney and the adrenal gland4346. EDRF turned out to be nitric oxide (NO), a direct activator of the soluble form of guanylyl cyclase, and ANP to be a ligand for the transmembrane form of guanylyl cyclase. The idea that NO, a small reactive gas, functioned as a locally effective hormone was not immediately accepted. Even after it was shown that NO could stimulate the soluble guanylyl cyclase, the pathway remained in doubt until several hormones were shown to activate a calciumcalmodulin-dependent NO synthase and the resultant NO was shown to stimulate soluble guanylyl cyclase47. The studies leading to this concept formed the basis for the Nobel Prize that was awarded to Furchgott, Ignarro and Murad in 1998. The general public learned of NO and cGMP not from these advances in basic science but from the publicity that surrounded the drug Viagra. Viagra selectively increases cGMP in penile erectile tissue by inhibiting a specific form of phosphodiesterase, PDE5 (BOX 2). The resultant smooth-muscle relaxation allows blood to flow into the corpus cavernosum to cause an erection. This is a good example of the synergy between stimulators of cyclic-nucleotide synthesis (for example, NO)

and inhibitors of cyclic-nucleotide hydrolysis, an effect that was originally recognized by Sutherland and colleagues, but one that has only recently been exploited for drug action.
Effectors of cyclic-nucleotide action

Protein kinases A and G. One of the most important early discoveries about the mechanism of action of cyclic nucleotides was the identification of the cyclic nucleotide-dependent-protein kinases in virtually all phyla of the animal kingdom48,49. Krebs and colleagues originally found that adrenaline stimulated a cascade of kinases, the first of which was phosphorylase-kinase kinase. This kinase was directly stimulated by cAMP, thereby completing the pathway from hormone to glycogen breakdown. Because of this direct activation by cAMP and the findings by Langan, Greengard, Steinberg and others that many other substrates could be phosphorylated by it, the kinase kinase was soon renamed cAMP-dependent protein kinase or protein kinase A (PKA)5052. There followed a huge effort to identify the specific substrates of this enzyme both in vitro and in intact cells. This is a continuing process, and it is likely that new substrates will be identified.

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cGMP action is, likewise, compartmented within cells. Co-localization of substrates and specific isoforms of PKG contributes to this, as exemplified by the association of PKG1 with vimentin in smooth-muscle cells70 and the regulation of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel by membrane-targeted PKG2 (REF.71). The association of endothelial NO synthase with caveolin and the subcellular localization of other components of the cGMP system probably provide restricted signalling domains72,73. Olfactory cilia provide a good example of differential cellular and subcellular localization of both cAMP and cGMP regulatory enzymes (FIG. 3). Work such as this has given rise to four postulates about cAMP action in mammalian cells67,69: first, not all cAMP gains access to all PKA; second, not all PKA has access to all possible substrates; third, not all cAMP has access to all cellular PDEs; fourth, there is a fixed spatial relationship between a given receptorGsadenylyl-cyclase complex and a specific intracellular compartment. Only with the development of our knowledge of the structural basis of compartmentation have these postulates been accepted. Recent work shows that compartmentation occurs through specific binding or anchoring sites at crucial locations in the cell membrane and within the cell. Hormone receptors, G proteins and adenylyl cyclases target to, or might be excluded from, caveolae, in which signalling may be regulated by local differences in the stoichiometry of signalling components, by interactions with caveolin or by the proximity of regulators, such as the PKC isoforms that associate with caveolae. PKAs are anchored by A-kinase anchoring proteins (AKAPs) in specific locations74,75 that is, AKAPs are non-randomly distributed within cells. AKAPs are also organizing structures for PDEs, phosphatases, substrates of PKA and other proteins76. Different isoforms of PDE are tethered by several mechanisms at specific subcellular locations77. Such localized PDEs might limit the spatial domain of diffusible cAMP78. The result is that the activation of PKA, cyclic-nucleotide-gated channels or GEFs in response to a hormoneGs interaction, is effectively limited or channelled to a specific subcellular region. The spatial organization of the multiple components of cyclic-nucleotide pathways, coupled with the differential regulation of individual isoforms of adenylyl cyclase, guanylyl cyclase and PDEs, provides many permutations of this basic theme. Discerning the details of these signalling microdomains is a challenge for the future.

Box 3 | Mechanism of cAMP effects on CREB Ligand (L) binding to a receptor (R) coupling to C a G-protein (G) increases the activity of adenylyl C cyclase (AC), which results in increased cAMP production. Binding of cAMP to protein kinase C C C A (PKA) dissociates the catalytic subunits (C) of CBP Pol II R R PKA P PKA from the regulatory (R) subunits. When CREB phosphorylated by the catalytic subunit on PP-1 Ser133, nuclear cyclic-AMP-response-elementbinding protein (CREB) recruits a co-activator, AC R the CREB-binding protein (CBP). CBP has G intrinsic histone acetyltransferase activity and L interacts with RNA polymerase II (Pol II). The result is the enhanced transcription of about 105 genes that have the cAMP response element (CRE) motif in their promoter regions, including tyrosine hydroxylase, inducible nitric-oxide synthase, the aryl/aromatic hydrocarbon (Ah) receptor, angiotensinogen, insulin, the glucocorticoid receptor, BCL2 and the cystic fibrosis transmembrane conductance regulator (CFTR). Multiple kinases can phosphorylate Ser133, and this site therefore becomes a common link in many regulatory pathways. The result of the phosphorylation, however, depends on the context in which it occurs62. Target gene activation is terminated by the serine/threonine phosphatase (PP-1)-mediated dephosphorylation of CREB.

One of the early studies (on what we now know to be the PDE1 or calciumcalmodulin family of enzymes) resulted in the initial discovery and description of calmodulin as a calcium-binding modulator of enzyme function65. A few years later, the stimulatory effect of cGMP on another PDE (now known as PDE2) was described53. Even in early studies, it was appreciated that the maximal rates of degradation exceeded by more than an order of magnitude the maximal rates of synthesis. It was also known that the addition of PDE inhibitors to most cell or tissue preparations was necessary to see more than minor increases in levels of cAMP or cGMP in response to hormones. Similarly, rapid fixation of tissue was required to preserve elevations of cyclic-nucleotide content. Nevertheless, it was not broadly appreciated until recently that PDEs must be highly regulated in the cell. We now know that regulation of PDE activity occurs by many mechanisms and is often more important, quantitatively, than the regulation of synthesis.
Generation of specificity

response to signals. One manifestation of there being several isoforms of proteins in the synthesisresponsedegradation pathway is subcellular compartmentation of signalling. Compartmentation of cyclic nucleotides. We now know that both cAMP and cGMP function and are metabolized in discrete subcellular compartments. Commenting on the proteinkinase hypothesis of cAMP action, Ted Rall, the co-discoverer of cAMP, noted that this scheme presents the unsatisfying picture of the catalytic subunit of protein kinase swimming about, happily phosphorylating a variety of cellular constituents whether they need it or not66. PKA became an early focal point for studies on compartmentation when two groups reported that prostaglandin E1 (PGE1) and the -adrenergic receptor agonist isoprenaline caused an elevation of cardiac cAMP and PKA, but that only isoprenaline activated glycogen phosphorylase. This dichotomy correlated with the observation that much of the PKA was associated with membrane fractions of heart. PGE1 activated PKA only in a soluble fraction, whereas the expected effects of cAMP elevation occurred only when a particulate fraction of PKA was activated. On the basis of these data, it was postulated67, and later confirmed68, that differences in the responses of the heart to isoprenaline and PGE1 are the consequences of activation of cAMP signal-transduction pathways that are confined to distinct intracellular compartments. Many similar examples of distinct pools have recently been reviewed69.

Multiplicity of isoforms. An important concept in understanding signalling pathways has been the realization that regulation of the synthesis, action and degradation of intracellular messengers is usually catalysed by several isozymes. This diversity of enzymes carrying out the same reactions at different sites under distinct modes of regulation allows a cell to regulate selectively the amplitude, duration and localization of the

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channels, GEFs and PDEs could be as high as the local concentration of cyclic nucleotide, and concentrations of protein kinases and substrates could be comparable in subcellular regions. Perhaps these regulatory systems operate as rapid molecular switches that can respond at near-diffusionlimited rates rather than as slowly responding rheostats. Walseth and Goldberg79,80, using the novel method of 18O metabolic labelling, showed that, in many tissues, cellular levels of cAMP and cGMP turned over much more rapidly than previously supposed, owing largely to the robust phosphodiesterase activities. For example, the total pool of cGMP is turned over in 15 msecs in the retina81 and in 300 msecs in platelets82. These rapid rates have now been extended to other tissues83. The physiological consequences of this rapid turnover are only beginning to be understood, and will undoubtedly be the topic of future discoveries. Interestingly, each time a mole of cAMP or cGMP is hydrolyzed, more than 7 kcal of energy are released84. Resolution of rapid changes. To study these rapid processes in subcellular compartments of the cell in real time and with the necessary spatial resolution, new and better methods are needed. Unfortunately, most early methods that were used to study turnover did not give any information about spatial distribution of the cyclic nucleotides. However, a good start to tackling this problem has been made with the recent introduction of genetically encoded fluorescent probes that are sensitive to cyclic nucleotide concentrations in the cell (BOX 4). Others have also used various Ca2+-binding dyes as indirect measures of cAMP and cGMP levels, especially when coupled to cyclic nucleotide gated channels56,85.

Figure 3 | Immunocytochemical localization of three cyclic-nucleotide PDEs in rat olfactory epithelium. This photo shows how different cyclic-nucleotide regulatory enzymes and therefore different regulatory processes can be cellularly and subcellularly localized in a tissue. Blue staining shows phosphodiesterase 1C (PDE1C), a calciumcalmodulin-dependent phosphodiesterase that is found in the cilia of most olfactory neurons. In green is the distribution of PDE4A, a cAMP-specific isozyme. This isozyme is also present in most neurons, but is localized to a different region of the cell. In red is the expression of PDE2A, which is also called cGMP-stimulated PDE. This enzyme is expressed in yet another group of neurons. Clearly, different olfactory neurons express different subsets of PDEs. Even within the same cell, different regions control their cyclic-nucleotide levels in different ways.

Ramifications. The ramifications of cyclicnucleotide compartmentation are just beginning to be realized. For example, the concept that discrete pools of cyclic nucleotides and cyclic-nucleotide effector enzymes differentially modulate processes in subcellular regions greatly changes our ideas about what the effective concentrations of a cyclic nucleotide must be in the cell. Early measurements of cAMP made by many laboratories put the levels of cAMP at ~107 M in most tissues. Similar measurements for cGMP were in general one-tenth of that. It is now clear that a large proportion of cAMP or cGMP might reside within a limited number of cells in a tissue or within a limited volume of a cell. Therefore, we have to ask what the effective concentration is in this limited volume. Surely the old values must be revised upwards, perhaps as much as 100-fold in some cases. Two implications of this concept are striking. First, it solves the early paradox that some of the new effector mechanisms, such as cyclic-nucleotide-gated channels and GEFs, bind cyclic nucleotides with midmicromolar affinities. If we revise the level of cyclic nucleotide upwards then these affinities are clearly more appropriate for regulation by the cyclic nucleotides. On the

other hand, these data might indicate that the classical effector enzymes, such as PKA and PKG, which bind cyclic nucleotides in vitro with affinities in the 108107 range, would always be in a fully activated state. However, this is not necessarily true, as several factors might limit activation: levels of effector enzymes are probably also much higher than originally supposed, and inhibitory influences, such as the heat-stable inhibitor of PKA, are active. In fact, local concentrations of PKAs, PKGs, nucleotide-gated

Box 4 | Real-time measurements of cAMP and cGMP in cells Tsien and colleagues have constructed a series of genetically encoded probes that use the principles of fluorescence resonance energy transfer (FRET) to report on cyclic-nucleotide concentrations in regions within cells8690. They have bracketed protein kinase A (PKA) and protein kinase G (PKG) with the cyan (CFP) and yellow (YFP) mutants of green fluorescent protein (GFP). Activation produces a structural change (for PKG) or the dissociation of the regulatory and catalytic subunits (for PKA) such that, with irradiation (excitation) of CFP, energy transfer decreases, giving an increase in the signal from CFP at the expense of the emission from YFP. This strategy works well in various isolated cell systems and seems to distribute appropriately with respect to A-kinase-anchoring-protein interactions and nuclear translocation, and to reproduce cNMP signalling as assessed by other measures. In addition to its potential for reporting from microdomains, as shown recently91, the genetically encoded probe has advantages over chemically labelled PKA also developed by Tsiens lab, which needs to be microinjected into cells. The idea of CFP/YFP probes that use FRET is being usefully extended to other protein kinase systems79 and signalling cascades.

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Concluding remarks
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The past several years have brought a renaissance in interest in cyclic-nucleotide regulation. Much of this has sprung from the realization that many newly discovered pathways that regulate processes such as cellular growth, inflammation, sensory signalling, neuroplasticity and transcription are themselves regulated by cyclic-nucleotide-dependent processes. Even more startling is the fact that different species often use different molecular mechanisms to achieve the same end result. For researchers in the field who thought that all of the important reasons to study cyclic nucleotides had largely passed 10 years ago, it is exciting, but also sobering, to study these new systems. It is exciting because of the high interest in these areas of research. It is sobering because we now realize that we did not get all of it right the first time.
*Department of Pharmacology, University of Washington, Seattle, Washington 98195-7280, USA.Department of Pharmacology, University of California, San Diego, La Jolla, California 92093-0636, USA. e-mails: beavo@u.washington.edu; lbrunton@ucsd.edu
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Online links
DATABASES The following terms in this article are linked online to: LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink/ -adrenergic receptor | -adrenergic receptor | guanylyl cyclase | PDE1 | PDE2 | PDE5 | PDE6 | PKA | PKC | PKG Medscape DrugInfo: http://promini.medscape.com/drugdb/search.asp Viagra Swiss-Prot: http://www.expasy.ch/sprot/sprot-top.html adenylyl cyclase | ANF | angiotensinogen | ATF1 | BCL2 | casein | CFTR | CREB | CREM | GFP | glucocorticoid receptor | glucose-6-phosphatase | glycogen phosphorylase | insulin | MLCK | NO synthase | PDE4A | phosphoenolpyruvate carboxykinase | phosphoglucomutase | rhodopsin | transducin | tyrosine hydroxylase Access to this interactive links box is free online.

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| SEPTEMBER 2002 | VOLUME 3

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