I. Introduction

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Guanylyl cyclases have evolved to synthesize cGMP in response to diverse signals, such as nitric oxide (NO),² peptide ligands, and fluxes in intracellular Ca²⁺ ([Ca²⁺]_i). These signals use specific guanylyl cyclase-coupled receptors and cofactors to initiate the conversion of the cytosolic purine nucleotide GTP to cGMP. Intracellular cGMP ([cGMP]_i) regulates cellular physiology by activating protein kinases, directly gating specific ion channels, or altering intracellular cyclic nucleotide concentrations through regulation of phosphodiesterases (PDEs). The structure and function of the family of guanylyl cyclases, the molecular mechanisms regulating their activities, and the downstream effectors that underlie the physiology of cGMP-dependent processes are summarized in this review.

	II. Guanylyl Cyclases

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A. Molecular Biology

1. Identification of the Members of the Guanylyl Cyclase Family. It was established by the mid-1970s that guanylyl cyclase activity was found in both the soluble and particulate 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 different proteins (Garbers and Gray, 1974; Kimura and Murad, 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 soluble isoform was a heterodimer composed of - and -subunits. The -subunit had a molecular mass of ~70 kDa, whereas the -subunit was reported to be 73 to 82 kDa (Gerzer et al., 1981c; Kamisaki et al., 1986). Soluble guanylyl cyclase (sGC) was purified to apparent homogeneity from bovine or rat lungs (Koesling et al., 1988, 1990; Nakane et al., 1988, 1990). Degenerate oligonucleotide probes based on the structure of purified subunits were used to screen cDNA libraries and thereby clone 1- and 1-subunits. The C-terminal region of both subunits had a high degree of sequence identity with cloned adenylyl and particulate guanylyl cyclases (pGCs), suggesting this was the catalytic domain. Sodium nitroprusside (SNP)-sensitive guanylyl cyclase activity was expressed when the cloned cDNAs for 1 and 1 were cotransfected into a heterologous 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.

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 natriuretic peptide ligands for GC-A, ANP and brain natriuretic 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 calcium-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 chromosome 6, whereas GCAP3 is located on chromosome 3 (Subbaraya et al., 1994; Haeseleer et al., 1999).

3. Genetic Disorders Associated with Guanylyl Cyclases. The only human diseases mapped to a gene for guanylyl cyclase involve retinal dystrophies. Leber's congenital amaurosis (LCA1), dominant cone-rod dystrophy (CORD6), cone dystrophy (CORD5), and central areolar choroidal dystrophy have been mapped to chromosome 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 frameshifts, which result in truncated proteins that lack the kinase-like and catalytic domains due to premature termination 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 dimerization 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 stimulated at higher [Ca²⁺]_i than wild-type GC-E (Tucker et al., 1999). Thus, an abnormal increase in [cGMP]_i in 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 disappeared 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 paradoxical rod behavior is not known.

B. Membrane-Bound Guanylyl Cyclases

1. Introduction. Seven eutherian mammalian pGC isoforms (GC-A to GC-G) have been identified (Table 2). They exhibit highly conserved domain structures, including (1) an extracellular binding domain at the N terminus that in some cases binds defined ligands (GC-A, -B, -C), (2) a single transmembrane domain, (3) a cytoplasmic juxtamembrane domain, (4) a regulatory domain that shares significant homology with protein kinases, (5) a hinge region, and (6) a C-terminal catalytic domain (Fig. 1). Isoforms expressed in intestinal mucosal cells (GC-C) and in sensory organs (GC-D, -E, -F) also possess a C-terminal tail.

View larger version (36K): [in this window] [in a new window]   Fig. 1.   Domain structure of guanylyl cyclases. The cognate domains of ANPCRs, pGCs, and sGCs are compared. ANPCRs are homodimeric truncated guanylyl cyclases that possess extracellular ligand binding, transmembrane, and juxtamembrane domains but lack kinase homology, hinge, and catalytic domains. The pGC illustrated is a homodimer modeled after GC-A and -B and possesses a single ligand-binding site formed by two extracellular amino terminal domains. In addition to the domains present in ANPCRs, pGCs also possess kinase homology domains, hinge regions, and catalytic domains that form two functional catalytic sites. GC-C, -D, -E, and -F possess a carboxyl terminal tail that is not depicted here. sGCs are heterodimers possessing amino terminal regulatory domains containing a heme prosthetic group with a ferrous (Fe2+) core that forms an imidazole axial bond with His105 of the -subunit. In addition, sGCs possess dimerization domains and carboxyl terminal catalytic domains that form one active and one inactive (flat blue) catalytic site. Colors identify domains with significant homology.

2. Isotypes of Particulate Guanylyl Cyclases. a. Natriuretic Peptide Receptors. Mammalian atrial cardiomyocytes produce ANP, which mediates a pleiotropic response designed to maintain cardiovascular homeostasis in the face of a pressure or volume challenge (de Bold, 1985). Thus, ANP induces natriuresis, diuresis, and hypotension and inhibits secretion of renin and aldosterone (de Bold et al., 1981; Atarashi et al., 1984a,b). Indeed, ANP appears to mediate short- and long-term control of blood pressure and fluid and electrolyte balance (de Bold, 1985; John et al., 1995, 1996; Lopez et al., 1995; Kishimoto et al., 1996; Oliver et al., 1997; Franco et al., 1998). In addition to ANP, other natriuretic peptides identified to date include BNP and CNP. ANP is synthesized as a prepro-polypeptide of 151 residues in which the C-terminal portion contains the biologically active sequences (de Bold, 1985). The circulating form of this hormone is the mature 28-amino acid peptide consisting of a 17-amino acid loop stabilized by a single intrachain disulfide bridge and N- and C-terminal extensions, all of which are required for biological activity. BNP is a 26-amino acid peptide that was first isolated from acid extracts of porcine brain (Sudoh et al., 1988). Subsequently, this peptide also was identified in heart and blood (Aburaya et al., 1989). The disulfide-stabilized 17-amino acid ring and C-terminal sequence, essential for ANP bioactivity, are conserved in BNP. The genes encoding ANP and BNP are organized in tandem, and in humans, the BNP gene is located upstream of ANP (Huang et al., 1996; Tamura et al., 1996b). CNP is a 22-amino acid peptide that was first identified in acid extracts of porcine brain. CNP contains the disulfide-stabilized 17-residue ring structure found in ANP and BNP. In contrast, CNP lacks a C-terminal extension and the N-terminal region is not homologous with that of ANP and BNP. Although CNP induces natriuresis, diuresis, and vascular smooth muscle relaxation, it is significantly less potent than ANP or BNP (Sudoh et al., 1990).

3. Structure of Particulate Guanylyl Cyclases. a. Extracellular Domain. Extracellular domains of pGCs exhibit the least homology among the members of the family. This diversity in structure presumably reflects the functional specificity of binding and induction of transmembrane signaling by different ligands. The precise molecular mechanisms mediating interaction of ligand with the extracellular domain and coupling of ligand binding with activation of the catalytic domain remain to be defined. In GC-B, Glu₃₃₂ appears to be required for CNP binding and signaling because its removal or substitution with His or Lys results in complete loss of cyclase activity (Duda et al., 1994). However, it is not known whether Glu₃₂₂ is essential for proper folding of receptor protein required for ligand binding, or if it is located in the binding site for the ligand (Duda et al., 1994). Similarly, amino acids 387 to 393 in the extracellular domain of GC-C interact directly with and are required for binding to ST (Hasegawa et al., 1999).

i. Glycosylation of Receptors. All mammalian pGCs, except GC-F, contain at least one N-linked glycosylation site in the extracellular domain, although the extent of glycosylation varies among receptors (Chinkers et al., 1989). Glycosylation results in heterogeneity in the size of guanylyl cyclase receptors. Thus, pulse-chase studies revealed two species of GC-A that are synthesized during the first 7.5 h of incubation, but only one species in incubations longer than 7.5 h. The two species identified in shorter incubations were heterogeneously glycosylated GC-A, and the less glycosylated receptor served as a precursor for the fully glycosylated protein (Lowe and Fendly, 1992). Similarly, affinity labeling of GC-C with radiolabeled ST revealed multiple, specifically labeled proteins that could be resolved by SDS-polyacrylamide gel electrophoresis (PAGE) (Thompson and Giannella, 1990; Hugues et al., 1992). Heterologously expressed GC-C also yielded multiple receptor proteins specifically labeled with ST, demonstrating they were derived from a single transcript by post-translational processing (de Sauvage et al., 1992; Vaandrager et al., 1993a,b). Receptors of different sizes were converted to a single molecular weight by treating membranes with endoglycosidase, which removes N-linked carbohydrates (Vaandrager et al., 1993a). Similarly, cells grown in tunicamycin, an inhibitor of glycosylation, generated a single receptor species (Vaandrager et al., 1993a).

ii. Cysteines and Oligomerization of Receptors. All mammalian pGCs have two conserved cysteine residues at the N terminus and one midway in the extracellular domain (Chinkers et al., 1989; Schulz et al., 1989, 1990, 1998b; Fülle et al., 1995; Yang et al., 1995; Foster et al., 1999). All pGCs, except GC-C, also contain two conserved cysteine residues at the C-terminus of the extracellular domain proximal to the membrane-spanning domain (Chinkers et al., 1989; Schulz et al., 1989, 1990, 1998b; Fülle et al., 1995; Yang et al., 1995). GC-C shares two cysteine residues that are located midway in the extracellular domain with GC-A, but shares only one cysteine residue with other pGCs (Chinkers et al., 1989; Schulz et al., 1989, 1990, 1998b; Fülle et al., 1995; Yang et al., 1995). Historically, it has been presumed that the cysteines in the extracellular domain form intrachain disulfide bonds important for stabilizing the tertiary structure of the receptor, similar to their function in other members of the superfamily of growth factor receptors (Itakura et al., 1994; Stults et al., 1994). The ANP clearance receptor (ANPCR), a truncated isoform of GC-A that lacks the cytoplasmic domain beyond the juxtamembrane domain, possesses five cysteines in the extracellular domain (Lowe et al., 1990a) (Fig. 1). Site-specific mutagenesis demonstrated the first four cysteines are joined sequentially, forming Cys₁₀₄-Cys₁₃₂ and Cys₂₀₉-Cys₂₅₇ intrachain disulfide bridges (Itakura et al., 1994; Iwashina et al., 1994). The precise role of these intrachain disulfide bridges in the function of the extracellular domain and in transmembrane signaling remains to be defined.

b. Transmembrane Domain. All pGCs have a single transmembrane domain similar to that in other members of the superfamily of growth factor receptors. The -helix found in the transmembrane domain creates a hydrophobic region that permits insertion into the hydrophobic membrane lipid bilayer. Deletion of hydrophobic amino acids in the transmembrane domain of the epidermal growth factor receptor (EGFR) did not alter ligand binding or dimerization, suggesting this domain is required for localization to the membrane, but not for signal transduction (Kashles et al., 1988

i. Structure. All pGCs possess between the juxtamembrane and catalytic domains a ~250-residue kinase homology domain (KHD) that is absent in sGCs. The KHDs of pGCs are ~30% homologous with a wide range of protein kinases (Koller et al., 1992). Generally, protein kinases contain 11 conserved subdomains and 33 invariant amino acids that are critical for kinase activity (Hanks et al., 1988). Within the natriuretic peptide receptor-like cyclases, GC-A, GC-B, and GC-G, the KHD possesses 9, 9, or 8 of the conserved subdomains and 28, 27, or 22 of the invariant residues, respectively. The intestinal receptor cyclase GC-C contains 8 of the conserved subdomains and 25 of the 33 invariant residues and is 30% conserved relative to GC-A and GC-B (Koller et al., 1992). The sensory organ cyclases, GC-D, -E, and -F contain 9, 7, or 8 of the conserved subdomains and 22, 21, or 22 of the invariant residues, respectively. An invariant aspartate residue found in subdomain VI of protein kinases, which functions as the catalytic base, is substituted in all pGCs (Knighton et al., 1991; Taylor et al., 1992). Similarly, the glycine-rich region of subdomain I (GXGXXG) that mediates nucleotide binding to protein kinases is present in GC-A and -B, but absent in GC-C. This structural difference may underlie some of the functional differences in regulation of these receptors by adenine nucleotides (Koller et al., 1992).

ii. Kinase Activity. All protein kinases contain an HRD consensus sequence in subdomain VI in which the acidic Asp mediates the transfer of a phosphate group from ATP to the appropriate substrate (Hanks et al., 1988). The HRD consensus sequence is absent from the JH2 domain of the JAK protein kinase family, which does not exhibit kinase activity (Foster et al., 1999). Similarly, mutation of the invariant Asp to Asn in c-Kit and v-Fps results in loss of kinase activity (Moran et al., 1988; Tan et al., 1990). This catalytic Asp residue is replaced in all pGCs, commonly by Ser, Arg, or Asn. Thus, the prevailing hypothesis is that pGCs do not possess kinase activity, reflecting the absence of the catalytic Asp residue in subdomain VI (Potter and Hunter, 1998b). Although no other guanylyl cyclase possesses protein kinase activity, retinal pGC exhibits intrinsic kinase activity (Aparicio and Applebury, 1996). Thus, ATP binds to purified retinal guanylyl cyclase at a site distinct from the catalytic GTP substrate-binding site. The protein kinase activity is magnesium-dependent, autophosphorylates serine residues, and transfers phosphate groups to myelin basic protein. The K_m for ATP is 81 µM, and the kinase activity is unaffected by cyclic nucleotides, phorbol 12-myristate 13-acetate (PMA), L--phosphatidylserine, or Ca²⁺, but is inhibited by staurosporine. These properties are distinct from other Ser/Thr kinases identified in rod outer segments including protein kinase A, protein kinase C (PKC), and rhodopsin kinase. This observation of intrinsic kinase and autophosphorylation activity in a pGC remains an isolated result, and its functional significance continues to be unclear.

iii. Phosphorylation. Receptor guanylyl cyclases exist as phosphoproteins in the basal state. Upon binding of ligand, dephosphorylation of the receptor occurs, resulting in desensitization and a reduction in ligand-induced guanylyl cyclase activity. GC-A possesses six phosphorylation sites within the KHD, including Ser₄₉₇, Thr₅₀₀, Ser₅₀₂, Ser₅₀₆, Ser₅₁₀, and Thr₅₁₃, that are necessary for ligand-dependent activation of GC-A (Potter and Garbers, 1992; Koller et al., 1993; Potter and Hunter, 1998b). Mutation of these sites individually to Ala reduced GC-A activation by ANP, and replacement of five of the sites simultaneously yielded a receptor that was unresponsive to ligand. Similarly, GC-B exists as a phosphoprotein in the basal state when no ligand is bound to the receptor. GC-B possesses five residues within the KHD, including Ser₅₁₃, Thr₅₁₆, Ser₅₁₈, Ser₅₂₃, and Ser₅₂₆, that are phosphorylated (Potter and Hunter, 1998a). As in GC-A, the phosphorylation state of GC-B at these residues correlates with catalytic responsiveness, and dephosphorylation results in desensitization. Mutation of these five residues to Ala results in a 90% decrease in CNP-dependent cyclase activity, demonstrating that phosphorylation of these sites is necessary for ligand-induced activation of GC-B.

i. Dimerization of Catalytic Domains Is Required for Enzymatic Activity. Guanylyl cyclases must undergo dimerization to express catalytic activity. Heterodimeric sGCs require coexpression of both - and -subunits for catalytic activity, and expression of either subunit individually yields catalytically inactive protein (Harteneck et al., 1990; Buechler et al., 1991). Expression of truncated  and  catalytic domains in Sf9 cells yielded catalytically active heterodimers (Wedel et al., 1995). Mammalian adenylyl cyclases contain two catalytic domains in a single polypeptide chain (Krupinski et al., 1989). The hinge region in GC-A, discussed above, is absolutely required for GC-A catalytic subunits to dimerize and express catalytic activity (Thompson and Garbers, 1995; Wilson and Chinkers, 1995). Similarly, GC-C forms oligomers in a ligand-independent fashion that are important for producing catalytically active protein (Hasegawa et al., 1999b). Interestingly, coexpression of the 1-subunit of sGC and the C-terminal catalytic domain of adenylyl cyclase (type I, II, or V), each of which is inactive when expressed alone, produced a catalytically active adenylyl cyclase (Weitmann et al., 1999). This chimeric enzyme was regulated by P-site inhibitors but was not stimulated by G_s or forskolin. These data support the suggestion that two catalytic domains are required for expression of nucleotide cyclase activity. Also, they support the suggestion that the catalytic domains of adenylyl and guanylyl cyclase are structurally and functionally homologous.

ii. Determinants of Purine Specificity. The primary structure of the catalytic domain is highly conserved in both pGCs and sGCs and closely related to the catalytic domain of adenylyl cyclases (Krupinski et al., 1989). Thus, insights into the function of guanylyl cyclase catalytic domains were obtained using the solution structure of the X-ray crystal of the rat type II adenylyl cyclase C₂ catalytic domain. This provided critical information about substrate binding to the catalytic domain (Tesmer et al., 1997). Three invariant residues (Lys, Asp, Gln) present in the active site of adenylyl cyclase interact with the purine ring, which determines substrate specificity. The catalytic subunits of rat sGC (₁₁) contain three invariant residues (Glu, Cys, Arg) in positions homologous to those of the three invariant residues in adenylyl cyclase (Fig. 2). Mutations of adenylyl and guanylyl cyclase, in which these three residues were exchanged, resulted in the exchange of nucleotide substrate specificity: the guanylyl cyclase mutant specifically utilized ATP as substrate, whereas the adenylyl cyclase mutant became a nonselective purine nucleotide cyclase (Sunahara et al., 1998). Both enzymes retained their ability to be regulated by activators that were specific for the parent nucleotide cyclase. Thus, cAMP production by the mutant guanylyl cyclase was regulated by SNP, whereas cyclic nucleotide production by the mutant adenylyl cyclase was activated by G_s. Similarly, mutation of retinal guanylyl cyclase (Ret GC 1) based on the structural model of adenylyl cyclase, in which Glu₉₂₅ was substituted with Lys and Cys₉₉₅ was substituted with Asp, reversed substrate specificity from GTP to ATP (Tucker et al., 1998). This mutant retained the characteristic ability of retinal guanylyl cyclases to be regulated by GCAP-1 and GCAP-2.