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Vol. 52, Issue 3, 375-414, September 2000

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.)

Abstract
I. Introduction
II. Guanylyl Cyclases
    A. Molecular Biology
        1. Identification of the Members of the Guanylyl Cyclase Family.
        2. Structure and Location of Guanylyl Cyclase Genes.
        3. Genetic Disorders Associated with Guanylyl Cyclases.
    B. Membrane-Bound Guanylyl Cyclases
        1. Introduction.
        2. Isotypes of Particulate Guanylyl Cyclases.
            a. Natriuretic Peptide Receptors.
            b. Intestinal Peptide Receptor Guanylyl Cyclase.
            c. Orphan Receptor Guanylyl Cyclases.
        3. Structure of Particulate Guanylyl Cyclases.
            a. Extracellular Domain.
               i. Glycosylation of Receptors.
               ii. Cysteines and Oligomerization of Receptors.
            b. Transmembrane Domain.
            c. Juxtamembrane domain.
            d. Kinase Homology Domain.
               i. Structure.
               ii. Kinase Activity.
               iii. Phosphorylation.
            e. Hinge Region.
            f. Catalytic Domain.
               i. Dimerization of Catalytic Domains Is Required for Enzymatic Activity.
               ii. Determinants of Purine Specificity.
               iii. Configuration of the Catalytic Site.
            g. Carboxyl Terminal Tail.
        4. Receptor-Effector Coupling and Particulate Guanylyl Cyclase Function.
            a. Interaction of Ligand and Receptor.
            b. Oligomerization of Receptors.
            c. Regulation by Adenine Nucleotides.
               i. Allosteric Activation of Guanylyl Cyclases by Nucleotides.
               ii. Allosteric Inhibition of Guanylyl Cyclases by Nucleotides.
            d. Kinase Homology Domain.
            e. Phosphorylation and Homologous and Heterologous Desensitization.
            f. Accessory Protein Regulation.
            g. Model for Coupling of Particulate Guanylyl Cyclase Receptor and Effector.
    C. Soluble Guanylyl Cyclase
        1. Subunit Structure and Isotypes of Soluble Guanylyl Cyclase.
        2. Domain Structure.
        3. Regulation of Soluble Guanylyl Cyclase by Ligands.
            a. Nitric Oxide.
            b. Protoporphyrin IX.
            c. Catalytic Mechanism.
            d. Divalent Cations.
III. Cyclic GMP and Cell Signaling
    A. Introduction
    B. Protein Kinases
        1. Cyclic GMP-Dependent Protein Kinases.
        2. Cyclic AMP-Dependent Protein Kinases and Cyclic GMP Signaling.
    C. Cyclic Nucleotide-Gated Channels
    D. Cyclic GMP-Regulated Phosphodiesterases
    E. Cyclic GMP and Cell Physiology
        1. Motility of Vascular Smooth Muscle.
        2. Intestinal Fluid and Electrolyte Homeostasis.
        3. Phototransduction.
IV. Conclusions
Acknowledgments
References


    Abstract
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Guanylyl cyclases are a family of enzymes that catalyze the conversion of GTP to cGMP. The family comprises both membrane-bound and soluble isoforms that are expressed in nearly all cell types. They are regulated by diverse extracellular agonists 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-dependent protein kinases, cGMP-regulated phosphodiesterases, and cyclic nucleotide-gated ion channels. Guanylyl cyclases and cGMP-mediated signaling cascades play a central role in the regulation of diverse (patho)physiological processes, including vascular smooth muscle motility, intestinal fluid and electrolyte homeostasis, and retinal phototransduction. Topics 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 regulating enzymatic activity, and molecular sequences coupling ligand binding to catalytic activity. A brief overview is presented of the downstream events controlled 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
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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 alpha - and beta -subunits. The beta -subunit had a molecular mass of ~70 kDa, whereas the alpha -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 alpha 1- and beta 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 alpha 1 and beta 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.


                              
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TABLE 1
Soluble guanylyl cyclase isoform chromosomal localization and tissue distribution


                              
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TABLE 2
Particulate guanylyl cyclase isoforms, ligand and cofactor specificities, chromosomal localization, and tissue distribution

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, radiolabeled egg peptides could be chemically cross-linked to a sperm cell surface protein of the same size as that recognized by antiserum against guanylyl cyclase (Shimomura 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 activate guanylyl cyclase and to increase [cGMP]i in mammalian tissues (Hamet et al., 1984; Waldman et al., 1984; Winquist et al., 1984). Subsequently, ANP binding and guanylyl cyclase activity were copurified, strongly suggesting the two activities reside in a single molecule (Kuno et al., 1986; Paul et al., 1987; Shimonaka et al., 1987; Meloche 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 peptide 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 extracellular 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 regulatory domain has sequence similarity with protein kinases, 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 alpha - and beta -subunits of sGC and have limited identity with the two catalytic domains 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 conserved 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 alpha - and a second beta -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 alpha 1/beta 1 (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) is expressed in the olfactory neuroepithelium in a zonal pattern resembling that of the seven-transmembrane domain odorant receptors (Fülle et al., 1995). Two other members of the sensory tissue subfamily of guanylyl cyclases, guanylyl cyclase E (GC-E, retGC-1) and guanylyl 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 residues 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 [Ca2+]i through guanylyl cyclase-activating proteins (GCAPs). The recently cloned GC-G most closely resembles the natriuretic peptide receptors, although it is not activated by natriuretic peptides (Schulz et al., 1998b). Apparently, the family of mammalian guanylyl cyclases is relatively small because low stringency library screening and degenerate PCR have not yielded an abundance of unique cDNAs. In contrast, Caenorhabditis elegans has approximately 30 genes encoding 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 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).

The genes encoding human sGC subunits alpha 3 (equivalent to alpha 1) and beta 3 (equivalent to beta 1) 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 coordinated regulation of gene expression. The genes encoding alpha 1 and beta 1 sGC subunits in the medaka fish are organized in tandem within a 34-kb span (Mikami et al., 1999). The activity of the 5'-upstream region of each of the medaka fish genes was analyzed using green fluorescent protein reporter constructs expressed in medaka embryos (Mikami et al., 1999). Although the alpha 1 upstream region promoted expression of green fluorescent protein, the beta 1 5' region was insufficient, suggesting expression of the alpha 1- and beta 1-genes is coordinated. However, the alpha 2-subunit, which also can form an active dimer in vitro with beta 1, is encoded by a gene on chromosome 11 (Yu et al., 1996). That alpha 2- and beta 1-subunits dimerize under physiological conditions argues against the requirement for coordinated regulation of expression of alpha - and beta -subunits (Russwurm et al., 1998).

The structure of several genes for pGC has been determined, and the organization of their domains is reflected in the conservation of the intron/exon arrangement. This arrangement is most highly conserved in the portion of the gene encoding the catalytic and kinase homology domains. The extracellular domains of the guanylyl cyclases are conserved among, but not between, 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). However, 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 cyclases 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 5' regulatory regions 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 general transcription factors are present, the elements controlling tissue-specific expression are only now beginning to be explored. The GC-A gene promoter has at least three consensus binding sites for Sp1, a transcription 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 epithelium and primary and metastatic colorectal cancers (Carrithers et al., 1996). In the marsupial North American 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; London et al., 1997).

An initial characterization of the 5' flanking region of the gene for GC-C, using reporter gene constructs, suggested 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 factor-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). Mutation 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 transcription 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 binding site in the proximal GC-C gene promoter reduced the activity of a reporter gene construct expressed in intestinal cells to the level observed in extraintestinal cells (Park et al., 2000).

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.

Genetic alterations in other members of the guanylyl cyclase family are not associated with any described disease phenotype in humans. Several of the genes encoding guanylyl cyclases have been functionally eliminated 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 (Kishimoto et al., 1996). Null mice developed cardiac hypertrophy. 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 null mice exhibit many of the features of human essential hypertension and may prove to be a valuable model in which to study and develop treatment for this disease. Alterations in the sGC signaling pathway also may contribute to the development of hypertension. In the spontaneously hypertensive rat, an animal model for hypertension, the expression of both alpha 1- and beta 1-subunits of sGC and the expression of cGMP-dependent protein kinase (PKG) I are reduced in the aorta (Ruetten et al., 1999). The reduction in expression was observed even in young (normotensive) spontaneously hypertensive rats, suggesting this is an early event in the pathogenesis of the disease.

The gene encoding GC-C has also been subjected to targeted disruption (Mann et al., 1997; Schulz et al., 1997). Null mice were viable and healthy and were resistant to infection with the enterotoxigenic bacteria that cause diarrhea and death in wild-type mice. The normal physiological role of GC-C therefore remains undefined.

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.



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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 beta -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.

Based on their ligand specificities, pGCs have been classified as (1) natriuretic peptide receptors, (2) intestinal peptide-binding receptors, and (3) orphan receptors (Table 2). GC-A and GC-B bind to and are activated by natriuretic peptides, including ANP, BNP, and C-type natriuretic peptide (CNP). GC-C originally was characterized as the receptor for the family of homologous STs that produce secretory diarrhea. Recently, endogenous mammalian peptides, including guanylin, uroguanylin, and lymphoguanylin, were demonstrated to bind to and activate GC-C (Currie et al., 1992; Hamra et al., 1993; Forte et al., 1999). GC-D, -E, -F, and -G are orphan receptors for which ligands remain to be identified.

PGCs are expressed in almost all tissues in placental mammals (Table 2). GC-A mRNA is highly expressed in kidney, adrenal, and adipose tissue, and at lower levels in terminal ileum and human placenta (Lowe et al., 1989). GC-B mRNA is abundant in brain, lung, and kidney (Schulz et al., 1989). The mRNA for GC-C has been identified not only in intestinal mucosal cells in adult placental mammals, but also in many epithelia in marsupials (Forte et al., 1988; Schulz et al., 1990; Carrithers et al., 1996). In the central nervous system, GC-D is expressed in a subpopulation of olfactory sensory neurons, GC-E is expressed in retinal rod and cone cells, whereas GC-F is expressed only in retinal rod cells (Fülle et al., 1995; Yang et al., 1995). GC-G is predominantly expressed in the lung, the intestine, and skeletal muscle (Schulz et al., 1998b). Thus, the natriuretic peptide receptor-like cyclases, including GC-A, -B, and -G, are broadly expressed in many tissues. In contrast, GC-C and the sensory organ cyclases, GC-D, -E, and -F, all possess a C-terminal tail and are expressed in a tissue-specific fashion.

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).

The principal signaling mechanism by which natriuretic peptides induce their physiological effects involves activation of guanylyl cyclase-coupled receptors and accumulation of [cGMP]i. ANP has a selective affinity for GC-A, compared with GC-B, with a concentration for half-maximal response that is 1,000 times lower for the former than the latter (Schulz et al., 1989). ANP binding to GC-A has been demonstrated by both ligand binding analysis and affinity cross-linking studies (Leitman et al., 1988; Chinkers et al., 1989; Lowe et al., 1989; Jewett et al., 1993). ANP increases [cGMP]i in a concentration- and time-dependent fashion in a variety of cells and tissues (Chinkers et al., 1989; Schulz et al., 1989). Similarly, ANP activates guanylyl cyclase in cell-free membranes prepared from various cells and tissues (Hamet et al., 1984; Waldman et al., 1984; Leitman et al., 1987). The concentration-dependence of guanylyl cyclase activation and [cGMP]i accumulation induced by ANP compares favorably with that of receptor binding (Wong et al., 1995). Heterologous expression of GC-A in cells lacking endogenous expression of this protein results in the development of the ability of ANP to specifically bind to those cells, increase their pGC activity, and induce intracellular accumulation of [cGMP]i (Chinkers et al., 1989). ANP does not bind to tissues from animals that are homozygous null mutants for GC-A. These animals do not undergo natriuresis or diuresis in response to ANP and their aortic rings do not relax in response to that peptide (Kishimoto et al., 1996; Lopez et al., 1997). Furthermore, ANP directly activates GC-A purified from mammalian tissues (Kuno et al., 1986; Inagami et al., 1991; Waldman et al., 1991).

The endogenous receptor for BNP also appears to be GC-A, but BNP is 10-fold less potent than ANP in stimulating that receptor (Goeddel, 1991). BNP increases guanylyl cyclase activity and accumulation of [cGMP]i in cells and tissues in a pattern that mimics that of ANP (Goeddel, 1991). Similarly, BNP binds to heterologously expressed GC-A and increases guanylyl cyclase activity and [cGMP]i accumulation in those cells (Schulz et al., 1989). Aortic rings from animals that are homozygous null mutants for GC-A do not relax in response to BNP (Lopez et al., 1997).

The primary ligand for GC-B is CNP. Although ANP and BNP bind to this receptor with low affinity, CNP has a 50- to 500-fold greater affinity for GC-B than the other natriuretic peptides (Koller et al., 1991). CNP stimulates guanylyl cyclase activity and increases [cGMP]i in cells and tissues that express GC-B (Moriwaki et al., 1998; Chrisman and Garbers, 1999; Tao et al., 1999). Also, CNP binds to cells in which GC-B is heterologously expressed and increases activity of guanylyl cyclase and accumulation of [cGMP]i in those cells (Koller et al., 1991; Chrisman et al., 1993). CNP induces physiological effects in animals that are homozygous null mutants for GC-A (Lopez et al., 1997).

b. Intestinal Peptide Receptor Guanylyl Cyclase. GC-Coriginally was identified and cloned from mRNA extracted from intestinal mucosal cells (Schulz et al., 1990). Although GC-C possesses the conserved domain structure characteristic of the guanylyl cyclase family (Fig. 1), it does not serve as a receptor for natriuretic peptides. The first ligand identified for GC-C was ST, and binding of ST to GC-C activates guanylyl cyclase and increases [cGMP]i in intestinal cells (Schulz et al., 1990). ST is produced by bacteria that colonize the intestine, including Escherichia coli, Enterobacter sp., Klebsiella sp., and Yersinia enterocolitica (Rao et al., 1979; Thorne et al., 1979). This peptide contains six cysteines that form three intrachain disulfide bridges which stabilize the tertiary structure and confer the heat-stability characteristic of ST (Guerrant et al., 1980; Gariépy et al., 1987). Reduction of the disulfide bridges eliminates the ability of ST to bind to receptors, stimulate transmembrane signaling, and induce intestinal secretion and diarrhea (Staples et al., 1980). GC-C appears to be the only identified receptor for ST that is expressed in adult placental mammals (Guerrant et al., 1980). ST binds to GC-C in a concentration- and time-dependent manner (Guarino et al., 1987). Similarly, GC-C is activated by ST in a concentration-dependent manner and is not influenced by natriuretic peptides (Schulz et al., 1990; Krause et al., 1994a). Specific association of ST and GC-C has been demonstrated by ligand binding analysis and affinity cross-linking (Guarino et al., 1987; Schulz et al., 1990; Hugues et al., 1992). Interaction of ST and GC-C activates guanylyl cyclase in cell-free membranes prepared from intestinal cells (Gazzano et al., 1991a; Hugues et al., 1991). Similarly, ST induces accumulation of [cGMP]i in cells derived from intestine (Guerrant et al., 1980; Rao et al., 1980). Heterologous expression of GC-C confers specific ST binding to cells (Schulz et al., 1990; de Sauvage et al., 1991; Deshmane et al., 1995). Similarly, ST activates guanylyl cyclase and induces [cGMP]i accumulation in cells heterologously expressing GC-C (Schulz et al., 1990; de Sauvage et al., 1991). Disruption of the gene encoding GC-C results in mice that are resistant to ST-induced secretory diarrhea (Mann et al., 1997; Schulz et al., 1997). Homogenates of intestinal membranes from GC-C null animals specifically bind ST at very low levels, potentially identifying a binding protein other than GC-C (Mann et al., 1997). However, that novel binding protein has not yet been identified or characterized. Thus, GC-C remains the only identified receptor for ST and directly mediates the pathophysiological consequences of ligand-receptor interaction, including secretory diarrhea. A detailed discussion of the postreceptor mechanisms mediating ST-induced intestinal secretion is presented in a later section.

Unlike natriuretic peptide receptors, significant expression of GC-C appears limited to intestinal mucosal cells found from the duodenum to the rectum in adult placental mammals (Krause et al., 1994a). Expression of GC-C in intestinal, but not extraintestinal, cells has been identified by specific ST binding, stimulation of guanylyl cyclase, accumulation of cGMP, and detection of specific mRNA (Guerrant et al., 1980; Guarino et al., 1987; Schulz et al., 1990; Hugues et al., 1992; Krause et al., 1994a). Similarly, intestinal mucosal cells continue to express functional GC-C after undergoing neoplastic transformation to colorectal adenocarcinomas. Detection of this protein or its specific mRNA in extraintestinal sites appears to be a sensitive and specific marker for detecting metastatic colorectal cancer cells (Carrithers et al., 1996; Waldman et al., 1998; Cagir et al., 1999). Functional GC-C also has been detected in regenerating rat liver after surgical or chemical hepatectomy, but the functional significance of this observation remains to be elucidated (Laney et al., 1994). In contrast to placental mammals, GC-C in marsupials is expressed in the epithelia of the gut, gall bladder, lung, renal, and testis (Forte et al., 1988, 1989; White et al., 1989).

ST-like peptides that bind to and stimulate GC-C have been isolated from mammals. Guanylin and uroguanylin were isolated from intestinal mucosa; uroguanylin also has been isolated from urine (Currie et al., 1992; Hamra et al., 1993). These peptides share significant homology with ST, and their tertiary structure stabilized by two intrachain disulfide bonds is essential for biological activity (Currie et al., 1992; Hamra et al., 1993). The precise physiological importance of guanylin and uroguanylin remains to be elucidated. They may play a role in the regulation of fluid and electrolytes in the intestine (Hamra et al., 1993). However, mice that are homozygous null mutants for GC-C appear to develop normally, with normal intestinal function (Mann et al., 1997; Schulz et al., 1997). Uroguanylin, guanylin, and ST appear to induce natriuresis, diuresis, and kaliuresis in rodent kidney. These peptides may play a role in regulating fluid and electrolytes in the intestinal-renal axis (Greenberg et al., 1997; Fonteles et al., 1998). However, it is notable that analysis by sensitive nested reverse transcription-PCR does not detect expression of mRNA for GC-C in the kidney (Carrithers et al., 1996). Recently, a novel mRNA transcript encoding lymphoguanylin, a polypeptide related to guanylin and uroguanylin, was identified in spleen and lymphoid tissues from opossum (Forte et al., 1999). In these studies, the cDNA of lymphoguanylin was produced from isolated RNA and used to synthesize a putative endogenous peptide. Although ST, guanylin, and uroguanylin possess multiple disulfide bonds, synthetic lymphoguanylin contains only a single intrachain disulfide bond yet is biologically active (Forte et al., 1999). Because these studies did not isolate an endogenous peptide from tissues, the significance of this finding remains to be established.

c. Orphan Receptor Guanylyl Cyclases. PGCs for which endogenous ligands have not yet been identified are categorized as orphan receptors. Ligands known to activate other guanylyl cyclases do not activate GC-D, -E, -F, or -G (Fülle et al., 1995; Yang et al., 1995; Schulz et al., 1998b). The primary structures of GC-D, -E, and -F extracellular domains are homologous, and the expression of these pGCs is restricted to sensory tissues (Yang et al., 1995). The ligand of GC-G may resemble the natriuretic peptides because the extracellular domain of this pGC is homologous to that of the natriuretic peptide receptors (Schulz et al., 1998b).

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).

Glycosylation appears to play a role in the binding of ligand to the extracellular domain of pGCs. In a study performed in human embryonic kidney cells that were stably transfected with GC-A, fully glycoslyated GC-A specifically bound ANP, but removal of carbohydrate residues with endoglycosidase prevented binding of ANP to this receptor (Lowe and Fendly, 1992). In other studies performed in rat glioma cells that were stably transfected with GC-A, inhibition of N-glycosylation of GC-A by tunicamycin did not alter the ability of ANP to bind to cells and activate guanylyl cyclase (Heim et al., 1996). However, tunicamycin pretreatment inhibited the response of GC-A to ANP analogs, such as urodilatin [ANP (95-126)] (Heim et al., 1996). The different results obtained in these two studies might reflect differences in the type of ANP (native or analog) used. Similarly, when membranes prepared from cells that heterologously express GC-B were treated with endoglycosidase to remove carbohydrate from glycosylated sites in the protein, binding of CNP to the membranes was lost (Fenrick et al., 1996). Also, the activity of GC-B that had been deglycosylated was lower under stimulated conditions compared with the activity of fully glycosylated receptor (Fenrick et al., 1996). In close agreement, deglycosylation with endoglycosidase eliminated ST binding from the extracellular domain of GC-C (Hasegawa et al., 1999b). Studies in which each of the N-linked glycosylation sites was replaced with Ala demonstrated that Asn379, which is close to the transmembrane domain, was required for ligand binding and catalytic activity (Hasegawa et al., 1999a). Although glycosylation is important for binding of ligand, it does not appear to be required for distribution of receptors to the cell surface (Lowe and Fendly, 1992; Fenrick et al., 1996; Hasegawa et al., 1999b).

GC-D contains two potential N-linked glycosylation sites, whereas GC-E only contains one (Fülle et al., 1995; Yang et al., 1995). The extracellular domain of GC-G contains five potential N-linked glycosylation sites and is about 40% homologous with the extracellular domain of GC-A (Schulz et al., 1998b). Whether glycosylation of these orphan receptors is essential for expression on the cell surface or for transducing a signal has not yet been determined.

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.

Cysteine residues in the extracellular domain appear to mediate ligand-independent oligomerization of receptor monomers (Chinkers and Wilson, 1992). In ANPCR, the fifth cysteine in the extracellular domain (Cys469) is proximal to the membrane-spanning domain and forms an interchain disulfide bridge that stabilizes a dimeric structure (Itakura et al., 1994). Heterologously expressed human GC-A migrates as high molecular weight oligomeric complexes when subjected to SDS-PAGE under nonreducing conditions. These oligomeric complexes can be converted to monomers after SDS-PAGE under reducing conditions (Lowe, 1992). Truncated mutants of GC-A possessing only the extracellular domain also form oligomeric complexes, supporting the suggestion that the extracellular domain contributes the cysteines that form the interchain disulfide bonds mediating oligomerization (Lowe, 1992). GC-A from bovine adrenal gland migrates as a tetrameric 550-kDa complex when subjected to SDS-PAGE under nonreducing conditions but as a 140-kDa monomer when exposed to reducing conditions (Iwata et al., 1991). GC-C migrates as complexes of high molecular weight when subjected to SDS-PAGE under nonreducing conditions, but these complexes were converted to monomers on exposure to reducing conditions (Ivens et al., 1990). Heterologously expressed GC-C also forms higher order complexes that are converted to monomers on exposure to reducing conditions (Vaandrager et al., 1993a,b).

These data support the suggestion that, in the absence of ligands, pGCs spontaneously form complexes that are stabilized by disulfide bonds in the extracellular domain. Those cysteines that contribute to the formation of interchain disulfide bridges remain to be defined for each isoform of guanylyl cyclase. GC-A mutants, in which Cys423 near the membrane-spanning domain was replaced, spontaneously formed interchain disulfide bonds and underwent dimerization, presumably mediated by Cys432 that was unpaired in the mutant protein (Labrecque et al., 1999). However, the relevance of this observation to native receptors is unclear because dimerization in this study was associated with constitutive activity (Labrecque et al., 1999). Furthermore, GC-C, which also undergoes ligand-independent oligomerization, does not possess these conserved cysteines proximal to the membrane-spanning domain (Hasegawa et al., 1999b).

b. Transmembrane Domain. All pGCs have a single transmembrane domain similar to that in other members of the superfamily of growth factor receptors. The alpha -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). In contrast, deletions in the transmembrane domain of the transforming growth factor-beta receptor alter its ability to mediate transmembrane signaling (Zhu and Sizeland, 1999). In addition to membrane insertion, transmembrane domains may facilitate receptor oligomerization through alpha  helix-helix interactions (Lemmon and Engelman, 1994; Lemmon et al., 1994). The precise role of the transmembrane domain in pGCs beyond membrane localization remains to be defined. However, it is notable that truncated mutants of GC-A and GC-C, which contain the extracellular domain but lack the transmembrane domain, are capable of forming dimers and binding ligand (Chinkers and Wilson, 1992; Hasegawa et al., 1999b).

c. Juxtamembrane domain. The juxtamembrane domain is a short region of approximately 25 amino acids distal to the transmembrane domain in the cytoplasmic region of the protein. Although a precise function has not been ascribed to this domain, it may mediate alternate signaling mechanisms involving pGCs. This region in pGCs contains a consensus sequence that exists in other single transmembrane domain receptors and is important for their coupling to heterotrimeric G proteins (G proteins) and their downstream effectors.

Traditionally, G proteins are activated by the heptahelical family of receptors (Gudermann et al., 1995
). However, members of the single transmembrane domain growth factor receptor superfamily, of which pGCs are members, also activate G proteins and signal through their downstream effectors. These receptors include EGFR, the insulin-like growth factor receptor, and the insulin receptor (Okamoto et al., 1990; Okamoto and Nishimoto, 1991; Ramirez et al., 1995; Krieger-Brauer et al., 1997). All the G protein-coupled single transmembrane domain receptors contain a consensus sequence in their juxtamembrane domains. This consensus sequence interacts with and activates G proteins. The consensus sequence ranges in length from 14 to 20 amino acids, contains two basic residues in the N-terminal end and a BBXXB motif in the C-terminal end (B is a basic residue and X is a nonbasic, nonaromatic residue). Interestingly, GC-A, -B, and -C contain this consensus sequence in a homologous position in their juxtamembrane domains.

Similarly, ANPCR possesses the above consensus sequence in its juxtamembrane domain (Fuller et al., 1988; Lowe et al., 1990a). This truncated guanylyl cyclase has a short cytoplasmic domain of 37 amino acids (Lowe et al., 1990a) (Fig. 1). ANPCR binds ANP, BNP, and CNP, and its primary function appears to be clearance of natriuretic peptides from the circulation through constitutive ligand-independent endocytosis (Nussenzveig et al., 1990). However, this receptor also regulates a variety of physiological processes (Anand-Srivastava and Trachte, 1993), including inhibition of adenylyl cyclase in rat and human platelets (Anand-Srivastava et al., 1991; Marcil et al., 1996), atrial and ventricular cardiocytes (Anand-Srivastava and Cantin, 1986), rat heart (Anand-Srivastava et al., 1996), and pheochromocytoma cells (Drewett et al., 1992). ANPCR also regulates cellular growth in hepatoblastoma cells (Rashed et al., 1993), proliferation and invasion of matrix by endothelial cells (Pedram et al., 1997), [Ca2+]i in adrenal glomerulosa cells (Isales et al., 1992), activation of endothelial NO synthase (eNOS) in gastric SMCs (Murthy et al., 1998), activation of phospholipase C-beta 3 in tenia coli SMCs (Murthy and Makhlouf, 1999), and inhibition of MAP kinase in astrocytes (Prins et al., 1996).

Of significance, the juxtamembrane domain of ANPCR is required for receptor signaling (Murthy and Makhlouf, 1999). Antibodies directed against the cytoplasmic domain of ANPCR prevent signaling by this domain (Anand-Srivastava et al., 1996). Also, the juxtamembrane domain interacts with Go in membranes isolated from PC12 cells, presumably mediating inhibition of catecholamine secretion by ANP (Takida et al., 1999). Similarly, heterologously expressed ANPCR was coupled to activation of coexpressed eNOS through Gi in gastric SMCs (Murthy et al., 1998). In tenia coli SMCs, interaction of ANPCR with Gi and activation of phospholipase C-beta 3 by the beta gamma -subunit of Gi was mediated by the juxtamembrane domain consensus sequence. Mutation of the sequence eliminated coupling between the clearance receptor and activation of phospholipase C-beta 3 (Murthy and Makhlouf, 1999).

These studies demonstrate that the G protein-regulating consensus sequence within the context of the juxtamembrane domain of a member of the guanylyl cyclase family can couple with G proteins and regulate downstream effectors in a ligand-dependent manner. This consensus sequence mediates alternate transmembrane signaling in other single transmembrane receptors including EGFR, insulin-like growth factor receptor, and insulin receptor. These observations suggest the intriguing possibility that pGCs may signal through different pathways, including synthesis of cGMP through activation of the catalytic domain and regulation of G protein-coupled effectors through the consensus sequence in the juxtamembrane domain.

d. Kinase Homology Domain.

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 Km for ATP is 81 µM, and the kinase activity is unaffected by cyclic nucleotides, phorbol 12-myristate 13-acetate (PMA), L-alpha -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.

e. Hinge Region. The hinge region is a 43-residue domain in GC-A between the KHD and catalytic domain. This region mediates dimerization of catalytic subunits and is required for the expression of enzymatic activity. Truncated mutants of GC-A possessing only the catalytic domain migrate on SDS-PAGE as monomers that are devoid of enzymatic activity (Wilson and Chinkers, 1995). However, truncated mutants inclusive of the hinge region migrate as homodimers on SDS-PAGE and possess guanylyl cyclase catalytic activity (Thompson and Garbers, 1995; Wilson and Chinkers, 1995). The primary structure of the hinge domain is consistent with a coiled coil configuration that favors specific protein-protein interactions (Thompson and Garbers, 1995; Wilson and Chinkers, 1995). Indeed, incorporating this sequence into a yeast two-hybrid system demonstrated the hinge domain mediates spontaneous formation of protein multimers (Wilson and Chinkers, 1995). The hinge region mediates dimerization of catalytic subunits. Dimerization is generally required for catalytic activity of sGC, pGCs, and adenylyl cyclases (Thompson and Garbers, 1995; Wilson and Chinkers, 1995). In addition, this region may play a larger role in mediating holoreceptor dimerization because truncation mutants of GC-A lacking cytoplasmic domains failed to form dimers or higher-order structures in one study (Lowe, 1992). Whether this domain is necessary for dimerization of catalytic subunits or plays a role in mediating receptor oligomerization in guanylyl cyclases other than GC-A remains to be elucidated.

f. Catalytic Domain.

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 alpha - and beta -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 alpha  and beta  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 alpha 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 Galpha 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 (alpha 1beta 1) 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 Galpha 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.