INTRODUCTION

Cyclic GMP is a ubiquitous intracellular second messenger produced by an ever-expanding family of guanylyl cyclases (GCs), which are classified into two major forms, those found in the plasma membrane (membrane GC) and those in the cytoplasm (soluble GC) (Kusakabe and Suzuki, 2000; Loretz and Pollina, 2000; Wedel and Garbers, 2001). The soluble GC is a heme-containing heterodimer and is activated by nitric oxide or carbon monoxide (Wedel and Garbers, 2001). The membrane GC is a protein having a single membrane-spanning region and is activated by various endogenous and exogenous peptides. The membrane GC is further divided into three subfamilies such as the natriuretic peptide (NP) receptors, the sensory organ-specific membrane GCs, and the enterotoxin/guanylin receptors (Fig. 1) (Kusakabe and Suzuki, 2000).

Fig. 1.

Molecular phylogenetic relationship of membrane GCs between medaka fish and rat. The amino acid sequences of the catalytic domains of GCs were subjected to phylogenetic analysis as described previously (Yamagami et al., 2003). The following GenBank/EMBL/DDBJ accession numbers for the sequences were used: X14773 (rat GC-A); M26896 (rat GC-B); M55636 (rat GC-C); L37203 (rat GC-D); L36029 (rat GC-E); L36030 (rat GC-F); AF024622 (rat GC-G); AB004921 (OlGC1); AB030274 (OlGC2); AB000899 (OlGC3); AB000900 (OlGC4); AB000901 (OlGC5); AB007192 (OlGC6); AB023489 (OlGC7); AS054814 (OlGC8); AB162944 (OlGC9); AB015874 (OlGC-R2); U21101 (sea urchin sperm membrane GC).

Heat-stable enterotoxins (STa), which are small peptides secreted by some pathogenic strains of Escherichia coli, are the first identified natural compound to activate a membrane GC in mammals (Field et al., 1978; Hughes et al., 1978; Vaandrager, 2002). The receptor for STa has been shown to be located primarily on the apical or brush border membrane of mammalian intestinal epithelial cells which are known as a rich source of a membrane GC (De Jonge, 1975; Vaandrager, 2002). Earlier works have demonstrated that STa actually serves as an extracellular activator of a membrane GC and it causes severe secretory diarrhea in mammals (Field et al., 1978; Hughes et al., 1978; Field et al., 1989a,b). Subsequently, it has been shown that an intestinal membrane GC is different from the membrane GCs present in most other tissues which would be stimulated by NPs (Kuno et al., 1986; Waldman et al., 1986; Vaandrager, 2002). In contrast, several initial studies on the membrane GC in mammalian intestine suggested that there are several proteins which show both STa-binding and GC activity (Kuno et al., 1986; Waldman et al., 1986; Ivens et al., 1990; Thompson and Giannella, 1990). However, the cloning and expression experiments of an intestinal membrane GC (designated GC-C in mammals) revealed that the membrane GC itself is the STa receptor and GC-C showed a considerable homology to NP receptors such as GC-A and GC-B (Schulz et al., 1990). Later, it was demonstrated that the endogenous ligands (guanylin and uroguanylin) also activate GC-C (Currie et al., 1992; Hamra et al., 1993).

The STa/guanylin receptor type membrane GC has been identified from various vertebrates, including human (De Sauvage et al., 1991; Singh et al., 1991), rat (Schulz et al., 1990), pig (Wada et al., 1994), guinea pig (Kruhoeffer et al., EMBL/Genbank/DDDJ accession number Z74734), cow (Goraczniak et al., EMBL/Genbank/DDDJ accession number AF081464), opossum (London et al., 1999), birds (Krause et al., 1995), reptilians (Krause et al., 1997), amphibian (MacFarland, EMBL/Genbank/DDDJ accession number D49837), European eel (Comrie et al., 2001), and medaka fish (Mantoku et al., 1999; Iio et al., 2005), as shown in Table 1. In mammals, only a single membrane GC (GC-C) is known to be the STa/guanylin receptor (Schulz et al., 1990), although two membrane GCs are identified in the intestine of the European eel Anguilla anguilla (Comrie et al., 2001) and the medaka fish Oryzias latipes (Mantoku et al., 1999; Iio et al., 2005). Here, we describe recent progresses on the studies of the STa/guanylin receptor type membrane GC of the non-mammalian vertebrates, mainly on the medaka fish and discuss their potential contribution to understanding on the roles in vertebrates.

Table 1.

Enterotoxin/guanylin receptor type guanylyl cyclase in vertebrates

STRUCTURE OF THE ENTEROTOXIN/GUANYLIN RECEPTOR TYPE MEMBRANE GC

The STa/guanylin receptor type membrane GC, as well as other type of membrane GCs, is composed of an extra-cellular domain, a single transmembrane domain, and an intracellular region comprising a kinase-like domain and a catalytic domain.

The extracellular domain of the STa/guanylin receptor type membrane GC functions as the binding site for both (uro)guanylin and STa (Schulz et al., 1990; Lucas et al., 2000; Mantoku et al., 1999; Iio et al., 2005) (Fig. 2). The binding site for STa is thought to be located at the N-terminal region to the transmembrane domain (Hasegawa et al., 1999b; Wada et al., 1996a), which is also conserved in medaka fish STa/guanylin receptor type membrane GCs (OlGC6 and OlGC9) (Mantoku et al., 1999; Iio et al., 2005). The extracellular domain contains 8-10 N-glycosylation sites, depending on species, which are probably not absolutely required for binding to the ligand(s) but are important for proper folding of the domain for STa-binding (Hasegawa et al., 1999a; Nandi et al., 1996; Ghanekar et al., 2004; Vaandrager, 2002).

Fig. 2.

Schematic drawing of the structure of the mammalian enterotoxin/guanylin receptor type membrane GC. Lines in each domain represent the location of the residue or region. Conserved residue or region between OlGC6 and OlGC9 are underlined. STa, heat stable enterotoxin; PKC, protein kinase C; PDZ, Post-synaptic density-95/Dlg-1/ZO-1.

The kinase-like domain in the intracellular region is located between the transmembrane domain and the catalytic domain and has homology to the catalytic site of receptor tyrosine kinases (Schulz et al., 1990; Vaandrager, 2002). However, the domain is thought to have no protein kinase activity since it lacks a conserved Gly-rich region and an Asp residue requiring for positioning of γ-phosphate of ATP and transferring the phosphate group to the substrate, respectively (Schulz et al., 1990; Vaandrager, 2002). It has been reported that the GC-C lacking the kinase-like domain is constitutivelly fully active and could not longer be activated by STa, suggesting that the kinase-like domain plays a critical role in the signal transduction from the ligand-binding domain to the catalytic domain (Rudner et al., 1995; Dashmane et al., 1997; Bhandari et al., 2001; Vaandrager, 2002). The kinase-like domain is also thought to interact with adenine nucleotides based on the results of in vitro assays, although the activation by STa does not depend on the presence of ATP. Therefore, occupation of the domain by ATP might stabilize the activated membrane GC and protect it against rapid desensitization (Katwa et al., 1992; Gazzano et al., 1991; Vaandrager, 2002). The presence of Lys⁵¹⁶ residue in the domain seems to be critical for the possible proper orientation of ATP (Bhandari et al., 2001). The Lys residue is conserved in OlGC6 and OlGC9 (Iio et al., 2005).

The catalytic domain of the STa/guanylin receptor type membrane GC is highly conserved with those of the NP receptor type and sensory organ-specific type membrane GCs. The STa/guanylin receptor type membrane GC contains an approximately 60 amino acid long extension distal to the catalytic domain, similar to the sensory organ-specific type membrane GCs but not to the NP receptor type membrane GCs and deletion of this extended portion (tail) results in unresponsive to STa (Wada et al., 1996b; Vaandrager, 2002). In relation to this, it is important to mention that Ser¹⁰²⁹ residue in the tail of porcine GC-C is phosphorylated by PKC (Wada et al., 1996b; Crane and Shanks, 1996) (Fig. 2) and that phosphorylation of Ser¹⁰²⁹ residue plays a critical role in activating the cyclase, especially in synergy with STa (Wada et al., 1996b; Crane and Shanks, 1996). Ser¹⁰²⁹ is conserved in OlGC9, but not in OlGC6 (Iio et al., 2005). On the other hand, it is reported that a novel PDZ protein termed IKEPP interacts with the C-terminal 4 amino acid residues of the STa/guanylin receptor type membrane GC and is involved in the regulation of the cyclase activity (Scott et al., 2002) (Fig. 2). These C-terminal target residues recognized by PDZ protein are also conserved in OlGC6 and OlGC9 (Iio et al., 2005).

Based on the results of the studies by Vaandrager et al. (1994), it appears that the STa/guanylin receptor type membrane GC is a homomultimer without regard to the absence or presence of ligands. Recently, it was demonstrated that both the extracellular and intracellular domains exist as trimers and a ligand is required to generate or stabilize the trimeric extracellular domain (Hasegawa et al., 1999a; Vijayachandra et al., 2000). A region located in the linker between the kinase-like and catalytic domains is implied as the intracellular multimerization sequence (Vijayachandra et al., 2000) (Fig. 2) and the amino acid sequences of the region are also conserved in OlGC6 and OlGC9 (Iio et al., 2005). From the point of view of the catalytic mechanism, a membrane GC requires two catalytic subunits to convert GTP into cGMP (Hurley, 1998). Therefore, further studies, probably three-dimensional structural studies, are needed for describing the detailed catalytic mechanisms of this type membrane GCs.

ACTIVATION OF THE ENTEROTOXIN/GUANYLIN RECEPTOR TYPE MEMBRANE GC

Mammalian STa/guanylin receptor type membrane GC has been shown to be activated upon endogenous or exogenous ligands binding to the receptor domain and the studies in the in vivo and in vitro effects of the ligands on the GC demonstrated the following functions of the GC in the mammalian intestine (Fig. 3): (1) Regultion of the fluidity of the intestinal contents by stimulation of secretion of Cl⁻ and inhibition of absorption of NaCl. As a result, it prevents dehydration and consequently obstruction of the intestine on one hand and the loss of water (diarrhea) on the other hand (Vaandrager and de Jonge, 1994; Vaandrager, 2002). (2) Regulation of pH of the intestinal contents by stimulation of HCO₃⁻ secretion and inhibition of H⁺ extrusion mediated by a Na⁺/H⁺ exchanger. As a result, it stimulates the digestion and absorption of food components (Fawcus et al., 1997; Joo et al., 1998; Guba et al., 1996; Vaandrager, 2002). (3) Regulation of the Na⁺ homeostasis by limiting intestinal Na⁺ uptake and by stimulation of Na⁺ excretion by the kidney especially in the case of high salt intake. In this regard, it should be mentioned that uroguanylin secreted by the intestinal cells into the blood stream acts as an intestinal natriuretic hormone along a postulated endocrine intestine-kidney axis (Forte et al., 2000; Vaandrager, 2002). (4) Protection against carcinogenesis by activation of cyclic nucleotide-gated (CNG) channels and/or inhibition of Na⁺/Ca²⁺ exchange, which leads to alterations in the intracellular Ca²⁺ concentrations and thus inhibiting DNA synthesis in colon carcinoma cells (Pitari et al., 2001; Pitari et al., 2003). Despite the aforementioned potential physiological roles of mammalian STa/guanylin receptor type membrane GCs, targeted disruption of the GC-C gene induced no deleterious effects in mice and, in fact, confers resistance to STa-induced diarrhea (Schulz et al., 1997; Mann et al., 1997). However, all vertebrates have the STa/guanylin receptor type membrane GC gene, implying that it serves some more important and undefined physiological roles.

Fig. 3.

Schematic drawing of the function of the mammalian enterotoxin/guanylin receptor-type membrane GC in the intestinal cells. STa: heat-stable enterotoxin, CFTR: cystic fibrosis transmembrane regulator, HNE-3: Na⁺/H⁺ exchanger-3, CNG channel: cyclic nucleotide-gated channel, PKG: cGMP dependant protein kinase, PKA: cAMP dependant protein kinase, PDE: phosphodiesterase.

It has been reported that the receptors for STa are found throughout the intestinal tract of all birds examined (Krause et al., 1995). In certain kinds of birds, cellular cGMP-accumulating responses to both STa and rat guanylin have been observed in the proximal small intestine, suggesting that the functional receptors for endogenous peptides like guanylin and uroguanylin exist in the apical membranes of enterocytes throughout the avian intestine (Krause et al., 1995). The receptors for guanylin and uroguanylin have also been identified on the mucosal surface of the enterocytes lining in the intestine of reptiles (Krause et al., 1997). Furthermore, guanylin-like peptides that stimulate cGMP accumulation in human T84 intestinal cells have also been isolated from the intestinal mucosa of reptiles (Krause et al., 1997). These suggest that the functional receptor/membrane GC, probably a STa/guanylin receptor type membrane GC and it's ligand(s) exist in the intestinal tract of birds and reptiles as in the intestine of mammals. However, cDNA of the STa/guanylin receptor type membrane GC have yet been cloned neither from birds nor from reptiles.

Recently, two cDNAs for the STa/guanylin receptor type membrane GCs have been cloned from the European eel Anguilla anguilla (GC-C1 and GC-C2) (Comrie et al., 2001) and also from medaka fish (OlGC6 and OlGC9) (Mantoku et al., 1999; Iio et al., 2005). Considering that the intestine is an essential organ for fish osmoregulation, the STa/guanylin receptor type membrane GCs may play the major roles in the teleost osmoregulation. In eels, it has been demonstrated that the expression of the GC-C2 gene in the eel intestine was increased by 100% after transfer of fresh water-acclimated eels to sea water and developmental maturation of yellow eels into pre-migratory silver eels resulted in a significant increase in the intestinal expression of the GC-C2 gene (Comrie et al., 2001), although in the medaka fish such transcriptional change was found neither of the OlGC6 gene nor the OlGC9 gene upon changes in environmental salinity (Iio et al., 2005). In this regard, it should be mentioned that cDNAs for three distinct guanylin-like peptides are cloned from Japanese ell Anguilla japonica and that the expression of all of guanylin-like peptides expression was increased after adaptation of the eel to seawater (Yuge et al., 2000). These strongly suggest that these peptides play important roles in seawater adaptation and act on regulation of water and salt absorption.

Although it is not known whether the European eel GC-C1 and GC-C2 are activated by STa and/or endogenous peptides (Comrie et al., 2001), in medaka fish it is demonstrated that STa activates OlGC9 but not OlGC6 and on the contrary, the medaka fish intestine extract, in which endogenous ligands should be contained, activates OlGC6 but not OlGC9 (Fig. 4) (Iio et al., 2005). These results suggest that the structural differences between OlGC6 and OlGC9 are responsible for differential activation by endogenous ligand(s) and STa (Iio et al., 2005). In this regard, the following facts should be useful for explanation of the differential activation by endogenous ligand(s) and STa. The structure of STa has been demonstrated to be similar to that of guanylin or uroguanylin (Nakazato, 2001) and the activation mechanisms of mammalian GC-C by STa have been explained by various standpoints of view (Fig. 2): (1) Importance of glycosylation sites (Asn¹⁹⁵ and Asn⁴⁰²) in the extra-cellular domain for proper folding of the domain for STa-binding (Nandi et al., 1996; Ghanekar et al., 2004), (2) Phospholylation of Ser¹⁰⁵² in the intracellular domain by PKC for conformation change suitable for an active form upon binding of STa to the extracellular domain (Wada et al., 1996b), (3) Oligomerlization (Vaandrager et al., 1994; Vijayachandra et al., 2000), (4) Interaction of the carboxyl termini with PDZ protein (Scott et al., 2002), and (5) ATP-binding to the kinase-like domain (Bhandari et al., 2001). Among them, a region for mediating oligomerization, PDZ domain, and a Lys residue in the kinase-like domain to stabilize the ATP-binding site are conserved in OlGC6 and OlGC9 (Fig. 2). In addition to these, the residues SPTFIWK which are suggested to be involved in STa-binding in porcine GC-C (Hasegawa et al., 1999b) are also conserved in both OlGC6 and OlGC9 (Fig.2). Considering these, the STa-binding site identified in porcine GC-C may not be related to the differential activation of OlGC6 and OlGC9 by STa. On the other hand, two glycosylation sites (Asn¹⁹⁵ and Asn⁴⁰²) which is essential for proper folding of the extracellular domain to allow ligand-binding in porcine GC-C (Hasegawa et al., 1999a) are conserved in OlGC9, but not in OlGC6 and a RNNSFQK sequence (residues 1050-1056 in OlGC9) which corresponds to the consensus phosphorylation sequence (RXXS¹⁰⁵²XK) in porcine GC-C is found in OlGC9 but not in OlGC6. These structural differences between OlGC6 and OlGC9 may be responsible to differential binding and subsequent activation by possible endogenous ligand(s) and STa (Iio et al., 2005) and may be useful when medaka fish STa/guanylin receptor type membrane GCs works in the regulation of salt and water transport in the medaka fish intestine. Further study on the differential activation mechanisms of two medaka fish STa/guanylin receptor type membrane GCs by endogenous ligand(s) and STa may be advantageous for further understanding of the ligand-stimulating mechanism of the STa/guanylin receptor type membrane GCs.

Fig. 4.

GC activity in the COS-7 cells transfected with OlGC6 or OlGC9 in the presence or absence of medaka fish intestine extract (A) or STa (B). Shaded boxes indicate the cGMP concentrations after treatment of the cells by the intestine extract (26.56 μg protein/μl) or 1×10⁻⁶ M STa. Black boxes indicate cGMP concentrations without peptides. Transfection and cGMP assay were performed in four independent experiments and the values are expressed as mean±S.D.

TRANSCRIPTION OF THE ENTEROTOXIN/GUANYLIN RECEPTOR TYPE MEMBRANE GC GENE

Using mammalian cultured cell lines, it has been demonstrated that the intestinal transcriptional regulator Cdx2 mediates the intestinal epithelial cell-specific expression of the humam GC-C gene through the 5′-flanking region between −83 and −75 (Park et al., 2000; Di Guglielmo et al., 2001) and that hepatocyte nuclear factor-4 (HNF-4) is a key regulator of the intestine-specific expression of the human GC-C gene through the 5′-flanking region between −46 and −29 (Mann et al., 1996; Swenson et al., 1999) (Fig. 5). However, the consensus binding sequences for HNF-4 are not found in the 5′-flanking region of the medaka fish OlGC6 gene and those for Cdx2, which are found in the region, do not seem to be involved in the transcription of the OlGC6 gene (Nakauchi and Suzuki, 2003), suggesting that the transcriptional regulatory mechanism of the OlGC6 gene differs from that of the human GC-C gene. Using CACO-2 cells (human intestine-derived cell line) and COS1 cells (African green monkey kidney-derived cell line), it was demonstrated that the 5′-flanking region between −98 and −89 of the OlGC6 gene is important for the transcriptional regulation only in the intestine-derived CACO-2 cells and that the sequence for this region are not similar to the regulatory element of the GC-C gene (Nakauchi and Susuki, 2003).

Fig. 5.

Schematic drawing of several consensus sequences for transcriptional factors found in the 5′-flanking region of the OlGC6, OlGC9, and human GC-C genes. Open ellipses and closed boxes represent consensus sequence for hepatocyte nuclear factor-4 (HNF-4) and Cdx2, respectively. A TATA box is indicated as an open triangle. The numbers below the line indicate the length of the 5′-flanking sequence.

Foreign DNA transfer and expression experiments in medaka fish have been used to investigate transcriptional regulation of tissue-specific genes, suggesting that medaka fish is adapted as a model vertebrate to study transcriptional regulation (Kusakabe and Suzuki, 2000). To understand the regulatory mechanisms of the OlGC6 transcription in vivo, the transgenic medaka fish were generated by microinjection of the OlGC6-reporter fusion gene constructs into the medaka fish embryos (Fig. 6) (Nakauchi and Suzuki, 2003). Grown up transgenic founders were mated with non-transgenic littermates to establish a line, and the embryos of each F₁ were analyzed to examine the transmission of the transgene (Fig. 6). The experiments using these F₁ transgenic medaka fishes demonstrated that the AGACCTTTGC nucleotides in the 5′-flanking region of the OlGC6 gene (region between −90 and −81) play a critical role in the transcription of the gene in vivo (Nakauchi and Suzuki, 2003). Furthermore, it was shown that a medaka fish homologue (designated as OlPC4) of mammalian positive co-factor 4 (PC4) activates the transcription of the OlGC6 gene through the AGACCTTTGC element (Nakauchi et al., 2005). The recombinant OlPC4 has been demonstrated to exhibit certain preferences with respect to binding sequences. In this regard, it should be mentioned that binding of human PC4 to a random sequence is much less efficient than binding to a promoter-containing sequence, although there is no direct evidence for the sequence-specific binding of human PC4 to any human gene (Kretzschmar et al., 1994; Ge and Roeder, 1994; Kaiser et al., 1995). Therefore, PC4 may exert still unknown functions in the recognition of the binding sequence (Nakauchi et al., 2005). PC4 is known as a transcriptional coactivator and it is demonstrated that human PC4 activates the transcription of some genes by interacting with several types of activators and general transcriptional factors, and also with TATA-binding protein (TBP)-associated factors (TAFs), as well as with a specific type of coactivator, thereby acting as an adaptor that links upstream activators with the basal transcriptional machinery (Kretzschmar et al., 1994; Ge and Roeder, 1994) (Fig. 7). These results suggest that PC4 plays an important role in the regulation of the genes transcribed by RNA polymerase II, although its physiological roles are still largely unknown (Kretzschmar et al., 1994; Ge and Roeder, 1994). Considering that mammalian PC4 requires upstream activators and interacts with many factors (Kretzschmar et al., 1994; Ge and Roeder, 1994) and our recent results demonstrating that OlPC4 requires the additional factor(s) which would be expressed in medaka fish intestine, OlPC4 may also interact with some unknown factor(s) and/or activator(s) binding to the upstream region of the OlGC6 gene yet to be identified (Nakauchi et al., 2005).

Fig. 6.

The procedure used for the investigation of the transcriptional regulation in vivo using transgenic medaka fish. Using F1, the reporter gene expression can be analyzed by various methods, e. g. Reverse transcription-polymerase chain reaction (RT-PCR) analysis, in situ hybridization analysis using reporter gene RNA probe.

Fig. 7.

Schematic drawing of the eukaryotic transcriptional machinery containing PC4. PC4 regulates transcription by interacting with activators, general transcriptional factors, general co-factors, the TATA binding protein (TBP)-associated factors (TAFs), and the RNA polymerase II (pol II) holoenzyme-associated mediator complex that binds to the carboxy-terminal domain (CTD) of the largest subunit of RNA pol II.

Since the size of the human genome is almost four times larger than that of medaka fish (Tanaka, 1995), more dynamic changes could be occurred in the human genome during evolutional processes and such changes may lead to different transcriptional regulatory mechanisms of human genome. In fact, the 5′-flanking regions involved in the intestinal cell-specific transcriptional regulation of the human GC-C gene are different from the corresponding region in the medaka fish OlGC6 gene, and on the contrary, that of the OlGC6 gene is also different from the corresponding region in GC-C gene (Fig. 5). These potential transcriptional regulatory sequences are not found in the 5′-flanking region of the OlGC9 gene, suggesting that the transcriptional regulatory mechanism of the OlGC9 gene is different from those of the OlGC6 and human GC-C genes (Iio et al., 2005). Recently, it was demonstrated that the some parts in the 5′-flanking region of the OlGC9 gene are involved in the transcriptional regulation when CACO-2 cells and COS1 cells were used (Iio et al., 2005). Further investigations on determination of the detailed cis-regulatory region in the OlGC9 gene and on identification of the transcriptional factor(s) interacting with the region will reveal more details about the mechanism of the transcriptional regulation of the STa/guanylin receptor type guanylyl cyclase genes.

It has been reported that the human GC-C gene locates on the chromosome 12 and the size of the GC-C gene is 85 kbp consisting of 27 exons (Lucas et al., 2000; Vaandrager, 2002). In medaka fish, the OlGC6 gene is 16 kbp, much smaller than that of the human GC-C gene but the OlGC6 gene consists of 27 exons, the number of which is the same as that of the human GC-C gene (Mantoku et al., 1999). The OlGC6 gene was mapped to linkage group 19 (LG19) and the OlGC9 gene was mapped to linkage group 8 (LG8). The genes on LG8 and LG19 share the same ancestral chromosome (proto-chromosome 2), suggesting that the OlGC6 and OlGC9 genes were duplicated from the same ancestral gene (Naruse et al., 2004; Iio et al., 2005). However, the differential activation of OlGC6 and OlGC9 by possible endogenous ligand(s) and STa and actually no similarity in the 5′-flanking region between the OlGC6 and OlGC9 genes suggest that after gene duplication the nucleotide sequences of both genes and subsequent biological functions of both genes' translation products were altered independently during evolutional processes. Further studies to solve the differential activation mechanisms would be useful for understanding of the ligand-stimulating mechanism of the mammalian STa/guanylin receptor type membrane GC.