Enterotoxin/Guanylin Receptor Type Guanylyl Cyclases in Non-Mammalian Vertebrates

Abstract Cyclic GMP is a ubiquitous intracellular second messenger produced by guanylyl cyclases (GCs). The enterotoxin/guanylin receptor type membrane GC (designated as GC-C in mammals) is activated by exogenous ligands such as heat-stable enterotoxins (STa), small peptides secreted by some pathogenic strains of Escherichia coli which cause severe secretory diarrhea and also activated by endogenous ligands such as guanylin and uroguanylin. The STa/guanylin receptor type membrane GC, as well as other type membrane GCs, is composed of an extracellular domain, a single transmembrane domain, and an intracellular region comprising a kinase-like domain and a catalytic domain. The STa/guanylin receptor type membrane GC is identified in various vertebrates including fishes, amphibians, reptiles, and birds, implying that it serves some important and undefined physiological roles in the intestine of non-mammalian vertebrates, e.g. the regulation of water and salt absorption. In mammals, only a single membrane GC (GC-C) is known to be the STa/guanylin receptor. On the contrary, two membrane GC cDNAs are cloned from the intestine of the European eel Anguilla anguilla (GC-C1 and GC-C2) and the medaka fish Oryzias latipes (OlGC6 and OlGC9). OlGC6 and OlGC9 are structurally distinct and show different ligand responsibility. Accumulated evidences indicate that the transcriptional regulatory mechanism of the human GC-C gene is different from that of the corresponding medaka fish GC gene; the human GC-C gene is regulated by Cdx2 and/or HNF-4, and the medaka fish OlGC6 gene is regulated by OlPC4, which is a medaka fish homologue of the mammalian transcriptional positive co-factor 4 (PC4). Furthermore, the transcriptional regulatory mechanism of the OlGC9 gene is different from those of both the OlGC6 and human GC-C genes, indicating that the study on these two medaka fish GCs will be useful for further understanding of the STa/guanylin receptor type membrane GC in the vertebrates.


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).
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). 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). 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., 1989).
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). 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).

MEMBRANE GC
The STa/guanylin receptor type membrane GC, as well as other type of membrane GCs, is composed of an extracellular 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 proximal 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).
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). 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). 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). 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). The presence of Lys 516 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). In relation to this, it is important to mention that Ser 1029 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 Ser 1029 residue plays a critical role in activating the cyclase, especially in synergy with STa (Wada et al., 1996b;Crane and Shanks, 1996). Ser 1029 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 in PDZ proteins 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.
(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). (4)  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 A. 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 the extracellular domains of 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)  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) is 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 (Asn 195 and Asn 402 ) 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 (RXXS 1052 XK) 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.

MEMBRANE GC GENE
Using 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).
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.    Various of these elements interact with one another.