The eel and perhaps other euryhaline teleost fish have the
The eel, and perhaps other euryhaline teleost fish, have the additional peptide, renoguanylin that exhibits high levels of expression within the kidney as well as the intestine. In terms of overall sequence, pre-prorenoguanylin is more homologous to pre-proguanylin than pre-prouroguanylin and a phenylalanine rather than an asparagine residue occupies position nine in the putative active peptide. This would suggest that any prorenoguanylin that is exposed to the renal tubular fluid would be hydrolysed and inactivated before it could bind and activate GC-C. Although having a higher amino Mouse Angiopoietin-2 / ANG2 / ANGPT2 Protein (His Tag) homology to guanylin, renoguanylin has a single amino acid (leucine) extension to the C-terminal of the peptide that is characteristic of uroguanylin peptides in mammals although in most other teleost species such as cod, flounder, plaice and salmon, the C-terminal of uroguanylin peptides are extended by two amino acids (Cramb et al., 2005, Takei and Yuge, 2007; unpublished observations). This C-terminal extension of uroguanylin, but not guanylin peptides is likely to have some functional significance and it is interesting that renoguanylin has this characteristic, especially if, as hypothesised above, that this peptide arose from a duplication of the guanylin rather than uroguanylin gene. The high levels of expression of renoguanylin within the kidney suggest that renoguanylin also has paracrine actions in this tissue, possibly acting on a baso-laterally located GC-C. However, further studies will be required to prove or disprove this hypothesis. Two isoforms of GC-C are expressed in intestine and renal tissues of the eel, and both have sequences and structural characteristics similar to all known mammalian membrane guanylate cyclases (Padayatti et al., 2004). Overall amino acid homologies are similar between the eel isoforms and hGC-C suggesting equivalent rates of divergence of all three genes indicating that the generation of the eel GC-C isoforms arose during the teleost-specific genome duplication event (Christoffels et al., 2004, Hoegg et al., 2004, Semon and Wolfe, 2007). Although amino acid homologies of the intracellular domain of the receptors are reasonably well conserved between each other (>75%) and to other species (>65%) the extracellular domains shows only limited homology between eel isoforms (<50%) or between other species (30%). This is perhaps strange considering the relatively high homologies between the active eel peptides (>75%) and the peptides of all other species (>50%). As the peptides are small (<18 aa.s) and are likely to form tightly coiled structures due to the disulphide bonds, the corresponding binding domain within the extracellular sequence of the GC-C receptor may also be relatively small in size. Indeed when comparing GC-C amino acid sequences across all species, a number of small (8–20 aa long) homologous regions appear to exist as clusters within the extracellular domain suggesting that some or all of these may be important for the peptide binding domain or for oligomerisation of the receptor (unpublished observations; Vaandrager, 2002). Conserved peptide regions present in the extracellular domain and the highly conserved sequences within the intracellular coiled-coil domain may be responsible for formation of the active trimeric structure proposed for GC-C isoforms (Vijayachandra et al., 2000). Like all known mammalian homologues, both eel GC-C isoforms contain a number of potential N-linked glycosylation sites (Fig. 2). Of the 10 potential glycosylation sites on GC-C1 and 11 potential sites on GC-C2 only four are conserved between isoforms. It is still uncertain whether any of these potential glycosylation sites do indeed harbour such post-translational modification and if so, if they have functional roles in peptide recognition and binding (Ghanekar et al., 2004, Hasegawa et al., 1999, Hirayama et al., 1992, Padayatti et al., 2004, Vaandrager et al., 1993). A potential C-terminal protein kinase C phosphorylation site, previously identified in various mammalian GC-C receptors (Vaandrager, 2002) is also present in both eel isoforms (Ser 1045 in GC-C1 and Ser 1049 in GC-C2). Previous reports suggest that phosphorylation of a serine residue near the C-terminal resulted in a synergistic potentiation of cyclase activity following peptide binding (Wada et al., 1996, Crane and Shanks, 1996).