Cloning and functional pharmacology of two corticotropin-releasing factor receptors from a teleost fish

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Abstract

Although it is well established that fish possess corticotropin-releasing factor (CRF) and a CRF-like peptide, urotensin I, comparatively little is known about the pharmacology of their cognate receptors. Here we report the isolation and functional expression of two complementary DNAs (cDNAs), from the chum salmon Oncorhynchus keta, which encode orthologues of the mammalian and amphibian CRF type 1 (CRF1) and type 2 (CRF2) receptors. Radioligand competition binding experiments have revealed that the salmon CRF1 and CRF2 receptors bind urotensin I with ∼8-fold higher affinity than rat/human CRF. These two peptides together with two related CRF-like peptides, namely, sauvagine and urocortin, were also tested in cAMP assays; for cells expressing the salmon CRF1 receptor, EC50 values for the stimulation of cAMP production were between 4.5±1.8 and 15.3±3.1 nM. For the salmon CRF2 receptor, the corresponding values were: rat/human CRF, 9.4±0.4 nM; urotensin I, 21.2±2.1 nM; sauvagine, 0.7±0.1 nM; and urocortin, 2.2±0.7 nM. We have also functionally coupled the O. keta CRF1 receptor, in Xenopus laevis oocytes, to the endogenous Ca2+-activated chloride conductance by co-expression with the G-protein α subunit, Gα16. The EC50 value for channel activation by rat/human CRF (11.2±2.6 nM) agrees well with that obtained in cAMP assays (15.3±3.1 nM). We conclude that although sauvagine is 13- and 30-fold more potent than rat/human CRF and urotensin I, respectively, in activating the salmon CRF2 receptor, neither receptor appears able to discriminate between the native ligands CRF and urotensin I.

Introduction

Corticotropin-releasing factor (CRF) is a 41-residue neuropeptide that was originally isolated from ovine hypothalamus by virtue of its ability to stimulate the secretion of adrenocorticotropic hormone (ACTH) and β-endorphin from anterior pituitary cells Spiess et al., 1981, Vale et al., 1981. This molecule, which coordinates the body's response to stressors (see Ito and Miyata, 1999, Koob and Heinrichs, 1999), binds to two main receptor subtypes, CRF1 and CRF2, that are members of the G-protein-coupled receptor superfamily Chalmers et al., 1996, Radulovic et al., 1999. The latter receptor exists, in several species, in two forms (CRF and CRF) that arise by alternative splicing and which have different amino-terminal sequences (Chalmers et al., 1996). In addition, a third CRF2 receptor isoform (CRF) has been described in man (Kostich et al., 1998), and the amino terminus of this is quite distinct from those of the CRF and CRF receptors. Binding of CRF to either the CRF1 or CRF2 receptor increases intracellular cAMP levels by the stimulation of adenylate cyclase activity Chalmers et al., 1996, Dieterich and DeSouza, 1996.

Several years ago, a mammalian peptide that exhibits 44% identity to CRF was identified and named urocortin (Vaughan et al., 1995). Although this was demonstrated to bind with nanomolar affinity to both the CRF1 and the CRF2 receptor, immunocytochemical data suggested that urocortin, rather than CRF, might be an endogenous ligand for the latter receptor. However, a subsequent study shed doubt on this notion by showing that most of the major sites of expression of the CRF2 receptor gene are poorly innervated by urocortin-containing projections (Bittencourt et al., 1999). Very recently, two mammalian urocortin-like peptides have been identified and characterised. One of these has been named urocortin II by Reyes et al. (2001) and stresscopin-related peptide by Hsu and Hsueh (2001); the other has been called urocortin III (Lewis et al., 2001) and stresscopin (Hsu and Hsueh, 2001). Both of these neuropeptides appear to be highly selective for the CRF2 receptor and they, as well as urocortin, are able to induce an increase in intracellular cAMP levels.

Two other peptides also bind to the mammalian CRF1 and CRF2 receptors, namely, sauvagine and urotensin I. The former was first characterised from the skin of Phyllomedusa sauvagei, a frog native to Central and South America (Montecucchi and Henschen, 1981), while the latter was originally sequenced from two teleost fish, the white sucker Catostomus commersoni (Lederis et al., 1982) and the carp Cyprinus carpio (Ichikawa et al., 1982). CRF is also found in non-mammalian species such as fish and frogs (see Lovejoy and Balment, 1999), and two urocortin-like peptides have very recently been identified, by database searches, in the pufferfish Fugu rubripes and Tetraodon nigroviridis (Lewis et al., 2001).

The presence of multiple ligands for two CRF receptor subtypes in a given species raises the question as to how the corresponding genes co-evolved (see Darlison and Richter, 1999). Although there exists a significant body of information on the sequences of CRF and CRF-like peptides in different chordate species (see Lovejoy and Balment, 1999, Lewis et al., 2001), much less is known about the sequences and pharmacologies of CRF receptors in lower vertebrates. One of the best studied non-mammalian species is Xenopus laevis, for which two CRF receptors have been identified by complementary DNA (cDNA) cloning (Dautzenberg et al., 1997). Interestingly, while the two amphibian receptors have a similar affinity for Xenopus CRF (KD=7.8±1.6 and 9.4±2.1 nM for the CRF1 and CRF2 receptors, respectively), the Xenopus CRF2 receptor has an almost 60-fold higher affinity for sauvagine than the Xenopus CRF1 receptor (KD=0.9±0.1 and 51.4±6.6 nM, respectively). During the preparation of this manuscript, the cloning of cDNAs for three distinct CRF receptors from the brown bullhead catfish, Ameiurus nebulosus, was reported (Arai et al., 2001). While two of the catfish receptors appear to be orthologous to the mammalian and amphibian CRF1 and CRF2 receptors, the evolutionary origin of the catfish CRF3 receptor is unclear. Furthermore, in functional assays, neither CRF nor urotensin I appeared to be selective for any of the three receptors. To gain further insight into the pharmacology and phylogeny of CRF receptors, we have cloned cDNAs from the chum salmon, Oncorhynchus keta, and expressed these in human embryonic kidney 293 (HEK 293) cells and X. laevis oocytes.

Section snippets

Cloning of O. keta full-length CRF receptor cDNAs

Total RNA was isolated from O. keta brain and heart using RNAClean™ (AGS, Heidelberg, Germany), digested with RNase-free DNase (Promega, Mannheim, Germany), and used as template for first-strand cDNA synthesis with Moloney murine leukemia virus (M-MLV) reverse transcriptase (Promega) and random nonamer primers (Stratagene, Amsterdam Zuidoost, The Netherlands). This cDNA was amplified, in the polymerase chain reaction (PCR), using Taq DNA polymerase (Promega) and two degenerate primers: 5′-TGC

Results

Using degenerate oligonucleotide primers in the PCR, followed by a combination of conventional library screening and the RACE technique, we have isolated two O. keta full-length cDNAs that encode putative G-protein-coupled receptors. The deduced amino-acid sequences each contain seven putative membrane-spanning domains and exhibit strong similarity to those of previously identified vertebrate CRF receptors (Fig. 1). Thus, for example, the salmon CRF1 receptor (430 amino acids; Mr=49,595 Da)

Discussion

We have described here the sequences of two members of the G-protein-coupled receptor superfamily from the teleost fish Oncorhynchus keta. These receptors are the orthologues of previously identified vertebrate CRF1 and CRF2 receptors. The salmon CRF1 and CRF2 receptors have been pharmacologically characterised using competition ligand-binding and cAMP assays; the latter experiments have also demonstrated that both receptors couple to the stimulation of adenylate cyclase activity, a property of

Acknowledgements

We thank Drs. Rainer Reinscheid, Robert J. Harvey and Hans-Jürgen Kreienkamp for advice and help with some of the experiments, Sönke Harder for oligonucleotide synthesis and Agata Blaszcyk-Wewer for DNA sequencing. We also gratefully acknowledge Dr. Emily Liman (Boston, USA) for pGEMHE, Drs. T. Liepold and J. Spiess (Göttingen, Germany) for the rat CRF1 receptor cDNA, and Prof. Dr. Volker Höllt (Magdeburg, Germany) for the rat GIRK1 cDNA. This work was supported by a Stipendium from the

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