0031-6997/00/5203-0375$03.00/0
`PHARMACOLOGICAL REVIEWS
`Copyright © 2000 by The American Society for Pharmacology and Experimental Therapeutics
`Pharmacol Rev 52:375–413, 2000
`
`Vol. 52, No. 3
`33/845272
`Printed in U.S.A
`
`Guanylyl Cyclases and Signaling by Cyclic GMP
`
`KIMBERLY A. LUCAS, GIOVANNI M. PITARI, SHIVA KAZEROUNIAN, INEZ RUIZ-STEWART, JASON PARK,
`STEPHANIE SCHULZ, KENNETH P. CHEPENIK, AND SCOTT A. WALDMAN1
`Division of Clinical Pharmacology, Departments of Medicine and Biochemistry and Molecular Pharmacology, Thomas Jefferson
`University, Philadelphia, Pennsylvania (K.A.L., G.M.P., S.K., I.R.-S., J.P., S.S., K.P.C., S.A.W.); and Institute of Pharmacology,
`University of Catania Medical School, Catania, Italy (G.M.P.)
`This paper is available online at http://www.pharmrev.org
`
`Downloaded from
`
`at Thomas Jefferson University on December 2, 2021
`
`Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 376
`I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 376
`II. Guanylyl cyclases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 377
`A. Molecular biology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 377
`1. Identification of the members of the guanylyl cyclase family . . . . . . . . . . . . . . . . . . . . . . . . . . . 377
`2. Structure and location of guanylyl cyclase genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 378
`3. Genetic disorders associated with guanylyl cyclases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 379
`B. Membrane-bound guanylyl cyclases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 380
`1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 380
`2. Isotypes of particulate guanylyl cyclases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 380
`a. Natriuretic peptide receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 380
`b. Intestinal peptide receptor guanylyl cyclase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 382
`c. Orphan receptor guanylyl cyclases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383
`3. Structure of particulate guanylyl cyclases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383
`a. Extracellular domain. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383
`i. Glycosylation of receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383
`ii. Cysteines and oligomerization of receptors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 384
`b. Transmembrane domain. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 384
`c. Juxtamembrane domain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 385
`d. Kinase homology domain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 385
`i. Structure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 385
`ii. Kinase activity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 385
`iii. Phosphorylation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 386
`e. Hinge region . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 386
`f. Catalytic domain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 386
`i. Dimerization of catalytic domains is required for enzymatic activity. . . . . . . . . . . . . . 386
`ii. Determinants of purine specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 386
`iii. Configuration of the catalytic site. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 387
`g. Carboxyl terminal tail. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 388
`4. Receptor-effector coupling and particulate guanylyl cyclase function . . . . . . . . . . . . . . . . . . . . 388
`a. Interaction of ligand and receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 388
`b. Oligomerization of receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389
`c. Regulation by adenine nucleotides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389
`i. Allosteric activation of guanylyl cyclases by nucleotides . . . . . . . . . . . . . . . . . . . . . . . . . 389
`ii. Allosteric inhibition of guanylyl cyclases by nucleotides . . . . . . . . . . . . . . . . . . . . . . . . . 390
`d. Kinase homology domain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 391
`e. Phosphorylation and homologous and heterologous desensitization . . . . . . . . . . . . . . . . . . 391
`f. Accessory protein regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 392
`g. Model for coupling of particulate guanylyl cyclase receptor and effector . . . . . . . . . . . . . . 392
`C. Soluble guanylyl cyclase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393
`1. Subunit structure and isotypes of soluble guanylyl cyclase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 394
`2. Domain structure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 395
`
`1 Address for correspondence: Scott A. Waldman, MD, PhD, FCP, Division of Clinical Pharmacology, Thomas Jefferson University, 132
`South 10th St., 1170 Main, Philadelphia, PA 19107. E-mail: scott.waldman@mail.tju.edu
`375
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`3. Regulation of soluble guanylyl cyclase by ligands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 395
`a. Nitric oxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 395
`b. Protoporphyrin IX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 396
`c. Catalytic mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 396
`d. Divalent cations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 396
`III. Cyclic GMP and cell signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 397
`A. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 397
`B. Protein kinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 398
`1. Cyclic GMP-dependent protein kinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 398
`2. Cyclic AMP-dependent protein kinases and cyclic GMP signaling. . . . . . . . . . . . . . . . . . . . . . . 399
`C. Cyclic nucleotide-gated channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 400
`D. Cyclic GMP-regulated phosphodiesterases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 401
`E. Cyclic GMP and cell physiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 401
`1. Motility of vascular smooth muscle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 401
`2. Intestinal fluid and electrolyte homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 403
`3. Phototransduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 405
`IV. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 406
`Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 407
`References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 407
`
`Abstract——Guanylyl cyclases are a family of en-
`zymes that catalyze the conversion of GTP to cGMP.
`The family comprises both membrane-bound and sol-
`uble isoforms that are expressed in nearly all cell
`types. They are regulated by diverse extracellular ago-
`nists that include peptide hormones, bacterial toxins,
`and free radicals, as well as intracellular molecules,
`such as calcium and adenine nucleotides. Stimulation
`of guanylyl cyclases and the resultant accumulation of
`cGMP regulates complex signaling cascades through
`immediate downstream effectors, including cGMP-de-
`pendent protein kinases, cGMP-regulated phosphodi-
`esterases, and cyclic nucleotide-gated ion channels.
`Guanylyl cyclases and cGMP-mediated signaling cas-
`
`cades play a central role in the regulation of diverse
`(patho)physiological processes,
`including vascular
`smooth muscle motility, intestinal fluid and electro-
`lyte homeostasis, and retinal phototransduction. Top-
`ics addressed in this review include the structure and
`chromosomal localization of the genes for guanylyl
`cyclases, structure and function of the members of the
`guanylyl cyclase family, molecular mechanisms regu-
`lating enzymatic activity, and molecular sequences
`coupling ligand binding to catalytic activity. A brief
`overview is presented of the downstream events con-
`trolled by guanylyl cyclases, including the effectors
`that are regulated by cGMP and the role that guanylyl
`cyclases play in cell physiology and pathophysiology.
`
`I. Introduction
`Guanylyl cyclases have evolved to synthesize cGMP in
`response to diverse signals, such as nitric oxide (NO),2
`2 Abbreviations: NO, nitric oxide; [Ca21]i, intracellular calcium;
`AMPS, adenosine-59-O-(3-thiomonophosphate); ANP, atrial natri-
`uretic peptide; ANPCR, ANP clearance receptor; ATPgS, adenosine-
`59-O-(3-thiotriphosphate); BNP, brain natriuretic peptide; CFTR,
`cystic fibrosis transmembrane conductance regulator; [cGMP]i, in-
`tracellular cGMP; CNG channel, cyclic nucleotide-gated channel;
`CNP, C-type natriuretic peptide; CO, carbon monoxide; EGFR, epi-
`dermal growth factor receptor; EC50, concentration of ligand yielding
`a half-maximum response; eNOS, endothelial nitric oxide synthase;
`GC, guanylyl cyclase; GCAP, guanylyl cyclase activating protein;
`PKA, cAMP-dependent protein kinase; PKG, cGMP-dependent pro-
`tein kinase; G protein, heterotrimeric G protein; GST, glutathione
`S-transferase; Kd, concentration of ligand yielding half-maximum
`binding; KHD, kinase homology domain; Ki, concentration of ligand
`yielding half-maximum inhibition; Km, concentration of substrate
`yielding half-maximum velocity; PCR, polymerase chain reaction;
`PDE, phosphodiesterase; pGC, particulate guanylyl cyclase; PKC,
`protein kinase C; PMA, phorbol 12-myristate 13-acetate; PPIX, pro-
`toporphyrin IX; PAGE, polyacrylamide gel electrophoresis; sGC, sol-
`uble guanylyl cyclase; SMC, smooth muscle cell; SNP, sodium nitro-
`
`peptide ligands, and fluxes in intracellular Ca21
`([Ca21]i). These signals use specific guanylyl cyclase-
`coupled receptors and cofactors to initiate the conversion
`of the cytosolic purine nucleotide GTP to cGMP. Intra-
`cellular cGMP ([cGMP]i) regulates cellular physiology
`by activating protein kinases, directly gating specific ion
`channels, or altering intracellular cyclic nucleotide con-
`centrations through regulation of phosphodiesterases
`(PDEs). The structure and function of the family of gua-
`nylyl cyclases, the molecular mechanisms regulating
`their activities, and the downstream effectors that un-
`derlie the physiology of cGMP-dependent processes are
`summarized in this review.
`
`prusside; ST, heat-stable enterotoxin; Vmax, maximum enzyme
`velocity; NOS, NO synthase; HCN, hyperpolarization-activated cy-
`clic nucleotide-gated.
`
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`GUANYLYL CYCLASES AND SIGNALING BY CYCLIC GMP
`
`377
`
`II. Guanylyl Cyclases
`A. Molecular Biology
`1. Identification of the Members of the Guanylyl Cyclase
`It was established by the mid-1970s that guany-
`Family.
`lyl cyclase activity was found in both the soluble and par-
`ticulate fractions of most cells (Hardman and Sutherland,
`1969; Ishikawa et al., 1969; Schultz et al., 1969; White and
`Aurbach, 1969), and that these activities were due to dif-
`ferent proteins (Garbers and Gray, 1974; Kimura and Mu-
`rad, 1974; Chrisman et al., 1975). However, only with the
`development of molecular cloning techniques more than a
`decade later could the breadth of this enzyme family be
`fully explored (Tables 1 and 2). Purification of guanylyl
`cyclase from the cytosolic compartment revealed the solu-
`ble isoform was a heterodimer composed of a- and b-sub-
`units. The b-subunit had a molecular mass of ;70 kDa,
`whereas the a-subunit was reported to be 73 to 82 kDa
`(Gerzer et al., 1981c; Kamisaki et al., 1986). Soluble gua-
`nylyl cyclase (sGC) was purified to apparent homogeneity
`from bovine or rat lungs (Koesling et al., 1988, 1990; Na-
`kane et al., 1988, 1990). Degenerate oligonucleotide probes
`based on the structure of purified subunits were used to
`screen cDNA libraries and thereby clone a1- and b1-sub-
`units. The C-terminal region of both subunits had a high
`degree of sequence identity with cloned adenylyl and par-
`ticulate guanylyl cyclases (pGCs), suggesting this was the
`catalytic domain. Sodium nitroprusside (SNP)-sensitive
`guanylyl cyclase activity was expressed when the cloned
`cDNAs for a1 and b1 were cotransfected into a heterolo-
`gous cell system, but not when transfected individually
`(Harteneck et al., 1990; Nakane et al., 1990). These data
`demonstrated both subunits of sGC are required for basal
`and nitrovasodilator-stimulated catalytic activity.
`Studies of pGCs suggested a new paradigm for signal
`transduction. Sea urchin sperm is one of the richest
`sources of pGC. In echinoderms, peptides secreted by eggs
`activate pGC of sperm in a species-specific manner (Suzuki
`et al., 1984; Ramarao and Garbers, 1985). Moreover, ra-
`diolabeled egg peptides could be chemically cross-linked to
`a sperm cell surface protein of the same size as that rec-
`ognized by antiserum against guanylyl cyclase (Shimo-
`mura et al., 1986). These observations suggested that pGC
`might also serve as a receptor for peptide ligands. While
`these studies were being conducted in the sea urchin,
`atrial natriuretic peptide (ANP) was demonstrated to ac-
`tivate guanylyl cyclase and to increase [cGMP]i in mam-
`
`malian tissues (Hamet et al., 1984; Waldman et al., 1984;
`Winquist et al., 1984). Subsequently, ANP binding and
`guanylyl cyclase activity were copurified, strongly suggest-
`ing the two activities reside in a single molecule (Kuno et
`al., 1986; Paul et al., 1987; Shimonaka et al., 1987; Me-
`loche et al., 1988). In 1988, pGC was first cloned from a sea
`urchin testis cDNA library using probes based on tryptic
`peptides obtained from the purified protein (Singh et al.,
`1988). This clone provided the necessary probe for isolating
`mammalian cDNAs encoding pGCs. The natriuretic pep-
`tide receptors, guanylyl cyclase A (GC-A) and B (GC-B),
`were the first pGCs cloned from mammalian tissues
`(Chang et al., 1989; Chinkers et al., 1989; Lowe et al., 1989;
`Schulz et al., 1989). The deduced primary sequences of the
`natriuretic peptide receptors predicted a protein with a
`single transmembrane domain that divides an extracellu-
`lar ligand-binding domain from an intracellular domain.
`Deletion mutagenesis studies have demonstrated that the
`intracellular domain serves regulatory, dimerization, and
`catalytic functions (Chinkers and Garbers, 1989). This reg-
`ulatory domain has sequence similarity with protein ki-
`nases, particularly the protein tyrosine kinases, which are
`also single transmembrane domain receptors (Singh et al.,
`1988). The sequences of the C-terminal catalytic domains
`are highly homologous to those of the a- and b-subunits of
`sGC and have limited identity with the two catalytic do-
`mains of adenylyl cyclases (Krupinski et al., 1989; Thorpe
`and Garbers, 1989).
`Development of the polymerase chain reaction (PCR)
`facilitated the search for new members of the guanylyl
`cyclase family. Degenerate PCR primers based on con-
`served amino acid sequences in the catalytic domains of
`both sGCs and pGCs were used to preferentially amplify
`guanylyl, as opposed to adenylyl, cyclases and yielded
`sequences of a second a- and a second b-subunit of sGC
`and five unique pGC sequences (GC-C to GC-G) (Yuen et
`al., 1990; Harteneck et al., 1991). A third pair of sGC
`subunits, cloned by screening a human cDNA library
`with rat cDNA clones, is most likely the human ortholog
`of a1/b1 (Giuili et al., 1992). Guanylyl cyclase C (GC-C)
`is the receptor for the bacterial heat-stable enterotoxins
`(STs) (Schulz et al., 1990; de Sauvage et al., 1991), and
`for the endogenous peptides guanylin and uroguanylin
`(Currie et al., 1992; Hamra et al., 1993). The remaining
`cloned mammalian pGCs are orphan receptors without
`known extracellular ligands. Guanylyl cyclase D (GC-D)
`
`TABLE 1
`Soluble guanylyl cyclase isoform chromosomal localization and tissue distribution
`
`Subunit
`
`Chromosome Locationa
`
`a1 (a3)
`
`4q31.3–q33 (Giuili et al., 1993)b
`
`a2
`b1 (b3)
`
`11q21–q22 (Yu et al., 1996)
`4q31.3–q33 (Giuili et al., 1993)
`
`13q14.3 (Behrends et al., 1999)
`b2
`a Human chromosome location.
`b References.
`
`Tissue Distribution
`
`Lung (Koesling et al., 1990); cerebellum, cerebrum, heart, kidney, liver, lung, skeletal muscle
`(Nakane et al., 1990); kidney (Ujiie et al., 1993)
`Brain, retina (Harteneck et al., 1991); kidney (Ujiie et al., 1993); placenta (Russwurm et al., 1998)
`Lung (Koesling et al., 1988); cerebellum, cerebrum, heart, kidney, liver, lung, skeletal muscle
`(Nakane et al., 1990); kidney (Ujiie et al., 1993); placenta (Russwurm et al., 1998)
`Kidney, liver (Yuen et al., 1990); kidney (Ujiie et al., 1993)
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`TABLE 2
`Particulate guanylyl cyclase isoforms, ligand and cofactor specificities, chromosomal localization, and tissue distribution
`
`Receptor
`
`Ligand(s)
`
`Cofactors
`
`Chromosome Locationa
`
`Tissue Distribution
`
`GC-A (NPR-A)
`
`ANP, BNP
`
`ATP
`
`1q21–q22 (Lowe et al., 1990b)b
`
`GC-B (NPR-B)
`
`CNP
`
`ATP
`
`9p12–p21 (Lowe et al., 1990b)
`
`GC-C
`
`GC-D
`
`ST, Guanylin,
`Uroguanylin
`?
`
`GC-E (Ret GC-1)
`GC-F (Ret GC-2)
`GC-G
`
`?
`?
`?
`
`GCAP 1,2,3
`GCAP 2,3
`
`a Human chromosome location.
`b References.
`c Inferred from mouse chromosomal assignment.
`
`12p12 (Mann et al., 1996b)
`
`11p15.4 or 11q13–q14.1c (Yang
`et al., 1996)
`17p13.1 (Oliveira et al., 1994)
`Xq22 (Yang et al., 1996)
`10q24–q26c (Schulz et al.,
`1998b)
`
`Adipose tissue, adrenal gland, ileum, kidney,
`placenta (Lowe et al., 1989); adrenal gland,
`cerebellum, heart, kidney, pituitary
`(Wilcox et al., 1991); lamina propria (Li
`and Goy, 1993); cochlea (Furuta et al.,
`1995); thymus (Vollmar et al., 1996); ovary
`(Jankowski et al., 1997)
`Placenta (Chang et al., 1989); adrenal
`medulla, cerebellum, pituitary (Wilcox et
`al., 1991); adrenal gland, aorta, atrium,
`cerebellum, lung, intestine, pituitary,
`testis, ventricle (Ohyama et al., 1992);
`uterus/oviduct (Chrisman et al., 1993);
`thymus (Vollmar et al., 1996); ovary
`(Jankowski et al., 1997)
`Intestinal mucosa (Li and Goy, 1993);
`regenerating liver (Laney et al., 1994)
`Olfactory epithelium (Fu¨ lle et al., 1995)
`
`Retina, pineal gland (Yang et al., 1995)
`Retina (Yang et al., 1995)
`Intestine, kidney, lung, skeletal muscle
`(Schulz et al., 1998b)
`
`is expressed in the olfactory neuroepithelium in a zonal
`pattern resembling that of the seven-transmembrane
`domain odorant receptors (Fu¨ lle et al., 1995). Two other
`members of the sensory tissue subfamily of guanylyl
`cyclases, guanylyl cyclase E (GC-E, retGC-1) and gua-
`nylyl cyclase F (GC-F, retGC-2), are expressed in retina
`(Shyjan et al., 1992; Lowe et al., 1995; Yang et al., 1995).
`GC-E also is expressed in the pineal gland (Yang et al.,
`1995). Although these enzymes are orphan receptors,
`their extracellular domains are homologous to that of
`GC-D and share a similar arrangement of cysteine res-
`idues in the extracellular domain with the other pGCs.
`This suggests they may have an extracellular ligand,
`although the catalytic activity of the retinal cyclases is
`regulated by [Ca21]i through guanylyl cyclase-activating
`proteins (GCAPs). The recently cloned GC-G most
`closely resembles the natriuretic peptide receptors, al-
`though it is not activated by natriuretic peptides (Schulz
`et al., 1998b). Apparently, the family of mammalian
`guanylyl cyclases is relatively small because low strin-
`gency library screening and degenerate PCR have not
`yielded an abundance of unique cDNAs. In contrast,
`Caenorhabditis elegans has approximately 30 genes en-
`coding guanylyl cyclase-like sequences and is seemingly
`rich in cGMP-coupled pathways (Yu et al., 1997).
`2. Structure and Location of Guanylyl Cyclase Genes.
`The chromosomal loci of the genes encoding isoforms
`of guanylyl cyclase and their ligands have been mapped
`in the human and/or the mouse (Tables 1, 2) and are
`unlinked and scattered throughout the genome, with
`notable exceptions. Thus, the genes encoding the natri-
`uretic peptide ligands for GC-A, ANP and brain natri-
`uretic peptide (BNP) are organized in tandem in both
`the human and the mouse (Huang et al., 1996; Tamura
`
`et al., 1996b). Similarly, guanylin and uroguanylin, the
`endogenous activators of GC-C, are encoded by closely
`linked genes (Whitaker et al., 1997). Retinal guanylyl
`cyclase activity is regulated by GCAPs, which are calci-
`um-binding proteins. To date, three members of the
`GCAP family have been identified. GCAP1 and GCAP2
`are found in a tail-to-tail arrangement on human chro-
`mosome 6, whereas GCAP3 is located on chromosome 3
`(Subbaraya et al., 1994; Haeseleer et al., 1999).
`The genes encoding human sGC subunits a3 (equiva-
`lent to a1) and b3 (equivalent to b1) have been mapped
`to chromosome 4q32 (Giuili et al., 1993). Because both
`subunits are required in a 1:1 stoichiometry for activity,
`their common chromosomal locus may imply a coordi-
`nated regulation of gene expression. The genes encoding
`a1 and b1 sGC subunits in the medaka fish are orga-
`nized in tandem within a 34-kb span (Mikami et al.,
`1999). The activity of the 59-upstream region of each of
`the medaka fish genes was analyzed using green fluo-
`rescent protein reporter constructs expressed in medaka
`embryos (Mikami et al., 1999). Although the a1 up-
`stream region promoted expression of green fluorescent
`protein, the b1 59 region was insufficient, suggesting
`expression of the a1- and b1-genes is coordinated. How-
`ever, the a2-subunit, which also can form an active
`dimer in vitro with b1, is encoded by a gene on chromo-
`some 11 (Yu et al., 1996). That a2- and b1-subunits
`dimerize under physiological conditions argues against
`the requirement for coordinated regulation of expression
`of a- and b-subunits (Russwurm et al., 1998).
`The structure of several genes for pGC has been de-
`termined, and the organization of their domains is re-
`flected in the conservation of the intron/exon arrange-
`ment. This arrangement is most highly conserved in the
`
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`
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`GUANYLYL CYCLASES AND SIGNALING BY CYCLIC GMP
`
`379
`
`portion of the gene encoding the catalytic and kinase
`homology domains. The extracellular domains of the
`guanylyl cyclases are conserved among, but not be-
`tween, subfamilies and the structure varies most in
`those parts of the genes. Genes for GC-A and -B are
`similar in size (16.5–17.5 kb) and structure, with 22
`exons and virtually identical intron/exon boundaries
`(Yamaguchi et al., 1990; Rehemudula et al., 1999). How-
`ever, the size of introns is not conserved between these
`genes. Similarly, the guanylyl cyclases in sensory tissue
`share a conserved gene structure and have only 20 exons
`(Yang et al., 1996). The gene for GC-C is much larger
`(.50 kb) than genes encoding the other guanylyl cycla-
`ses and has a unique intron/exon arrangement (S.
`Schulz, J. Park, and S. A. Waldman, unpublished data).
`The structures of the genes for sGC subunits have not
`yet been reported.
`Little is known regarding the regulation of expression
`of the genes for guanylyl cyclase. The 59 regulatory re-
`gions of genes that have been sequenced (GC-A, -C, -E)
`have no typical TATA box and an absent or inverted
`CAAT box. While consensus binding sites for many gen-
`eral transcription factors are present, the elements con-
`trolling tissue-specific expression are only now begin-
`ning to be explored. The GC-A gene promoter has at
`least three consensus binding sites for Sp1, a transcrip-
`tion factor that is implicated in the expression of a
`number of genes in the vasculature (Liang et al., 1999).
`Assays using electromobility shift and reporter gene
`techniques have demonstrated all three sites bind Sp1
`and are essential for basal transcription of the GC-A
`gene (Liang et al., 1999). Expression of the gene for
`GC-A also is regulated by its ligand, ANP. Levels of
`GC-A mRNA were suppressed by ANP in a time- and
`concentration-dependent manner in cultured aortic
`smooth muscle cells (SMCs) and primary cultures of
`inner medullary collecting duct cells (Cao et al., 1995,
`1998). A cell-permeable analog of cGMP also inhibited
`transcription of GC-A, suggesting the second messenger,
`rather than the natriuretic peptide, is responsible for
`modulating gene activity (Cao et al., 1995, 1998). The
`ANP/cGMP-responsive element in the promoter for
`GC-A has not been identified.
`Whereas GC-A is expressed in a variety of cell types
`and in many tissues, expression of GC-C in the adult
`human appears to be confined to the intestinal epithe-
`lium and primary and metastatic colorectal cancers
`(Carrithers et al., 1996). In the marsupial North Amer-
`ican opossum, a guanylyl cyclase-coupled ST receptor,
`possibly the opossum ortholog of GC-C, is expressed in
`epithelial cells of the kidney, liver, testis, trachea, and
`intestine (Forte et al., 1989; London et al., 1999). The
`mRNA for GC-C and binding of radiolabeled ST are
`detectable in neonatal and weanling mouse liver, and in
`fetal, neonatal, and regenerating rat liver (Laney et al.,
`1992, 1994; Scheving and Russell, 1996; Swenson et al.,
`1996). Although the sensitive reverse transcription-PCR
`
`technique has been used to amplify the mRNA for GC-C
`in a number of tissues, production of cGMP in response
`to ST has only been observed outside the intestine in
`rodent stomach and inner ear (Krause et al., 1997; Lon-
`don et al., 1997).
`An initial characterization of the 59 flanking region of
`the gene for GC-C, using reporter gene constructs, sug-
`gested intestine-specific transcriptional activity lies
`within the proximal 128 bp (Mann et al., 1996a). An
`analysis of this region, which is conserved between the
`human and the mouse, revealed potential binding sites
`for several transcription factors. Hepatocyte nuclear fac-
`tor-4 (HNF-4) binds to a specific element in the proximal
`promoter for GC-C and stimulates expression of GC-C
`when transfected into a cell line that normally expresses
`neither GC-C nor HNF-4 (Swenson et al., 1999). Muta-
`tion of the HNF-4 binding site abolished activity of the
`promoter for GC-C in intestinal cells, demonstrating
`that HNF-4 is necessary for basal gene expression
`(Swenson et al., 1999).
`Recent observations suggest the transcription factor
`Cdx2 mediates the intestine-specific expression of GC-C.
`Cdx2 is a member of the homeodomain family of tran-
`scription factors related to caudal, a Drosophila protein,
`and is required for the selective expression of several
`other genes in intestinal tissues (Traber and Silberg,
`1996). Deletion, or mutation, of a Cdx2 consensus bind-
`ing site in the proximal GC-C gene promoter reduced the
`activity of a reporter gene construct expressed in intes-
`tinal cells to the level observed in extraintestinal cells
`(Park et al., 2000).
`3. Genetic Disorders Associated with Guanylyl Cycla-
`ses. The only human diseases mapped to a gene for
`guanylyl cyclase involve retinal dystrophies. Leber’s
`congenital amaurosis (LCA1), dominant cone-rod dys-
`trophy (CORD6), cone dystrophy (CORD5), and central
`areolar choroidal dystrophy have been mapped to chro-
`mosome 17p12-p13, the interval containing the gene for
`GC-E (Balciuniene et al., 1995; Perrault et al., 1996;
`Hughes et al., 1998; Kelsell et al., 1998). In LCA1, the
`gene for GC-E contains mutations, including frame-
`shifts, which result in truncated proteins that lack the
`kinase-like and catalytic domains due to premature ter-
`mination of translation or a missense mutation in the
`kinase-like domain (Perrault et al., 1996). Expression of
`GC-E with this missense mutation in a heterologous cell
`line demonstrated that the mutant protein is stable but
`not activated by GCAP1 (Duda et al., 1999). In CORD6,
`GC-E contains mutations in the intracellular dimeriza-
`tion domain (Kelsell et al., 1998). It was postulated these
`mutations might cause a steric change in the protein
`that affects both mutant/mutant and mutant/wild-type
`dimers and thereby results in the dominant phenotype
`of CORD6. Indeed, one of the mutants has an increased
`affinity for GCAP-1, producing an enzyme that is stim-
`ulated at higher [Ca21]i than wild-type GC-E (Tucker et
`al., 1999). Thus, an abnormal increase in [cGMP]i in
`
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`
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`

`380
`
`LUCAS ET AL.
`
`dark-adapted photoreceptor cells may be the cause of
`their degeneration. When the gene encoding GC-E was
`eliminated in mice by targeted disruption, cones disap-
`peared by 5 weeks of age (Yang et al., 1999). Although
`the numbers and morphology of rods from GC-E null
`mice were similar to those from wild-type mice and the
`dark current was normal, retinas from null mice had a
`decreased response to light. The reason for the paradox-
`ical rod behavior is not known.
`Genetic alterations in other members of the guanylyl
`cyclase family are not associated with any described
`disease phenotype in humans. Several of the genes en-
`coding guanylyl cyclases have been functionally elimi-
`nated in mice by targeted disruption. This approach can
`provide insight into the normal physiological role of a
`gene product. Targeted disruption of the GC-A gene
`resulted in mice with salt-resistant hypertension (Lopez
`et al., 1995; Oliver et al., 1997). GC-A null mice were
`unable to respond either to an infusion of ANP or to
`acute volume expansion, stimuli that induced diuresis
`and natriuresis in their wild-type littermates (Kishi-
`moto et al., 1996). Null mice developed cardiac hyper-
`trophy. In one study, all GC-A null male mice died of
`congestive heart failure or aortic dissection by 6 months
`of age (Oliver et al., 1997; Franco et al., 1998). Thus, the
`GC-A nul