Cloning and Functional Characterization of the Amphibian Mesotocin Receptor, a Member of the Oxytocin/Vasopressin Receptor Superfamily

Abstract

Mesotocin is the oxytocin-like hormone found in most terrestrial vertebrates from lungfishes to marsupials, which includes all non-mammalian tetrapods (amphibians, reptiles, and birds). It has the largest distribution in vertebrates after vasotocin found in all non-mammalian vertebrates and isotocin identified in bony fishes. In this study, we report the cloning and functional characterization of the cDNA for the mesotocin receptor (MTR) from the urinary bladder of the toad Bufo marinus. The cloned cDNA encodes a polypeptide of 389 amino acids that shows the greatest similarity to the teleost fish isotocin receptor and to mammalian oxytocin receptors with mutations in extracellular loops which are involved in ligand binding. When expressed in COSM6 cells, MTR exhibits the following relative order of ligand affinity: mesotocin > vasotocin = oxytocin > vasopressin > hydrin 1, isotocin, hydrin 2. Injection of MTR cRNA into Xenopus laevis oocytes induces membrane chloride currents in response to mesotocin, which indicates the coupling of the mesotocin receptor to the inositol phosphate/calcium pathway. This response is inhibited by an oxytocin antagonist, but not by a vasopressin antagonist specific for V, vasopressin receptors. MTR mRNA is not only found in toad urinary bladder, but also in kidney, muscle, and brain tissue of the toad as revealed by Northern blot analysis and reverse-transcriptase PCR. The results suggest a variety of functions for mesotocin and its receptor including, in particular, an involvement in the regulation of water and salt transport.Keywords: mesotocin receptor ; molecular evolution ; terrestrial amphibians ; water transport ; hormonebinding site.To date, thirteen vertebrate neurohypophysial hormones and five invertebrate neurohypophysial hormone-like peptides are known [1, 21. These neurohypophysial hormones are usually nonapeptides that exhibit several highly conserved residues such as the half-cystine residues at positions 1 and 6 that form a disulfide bridge.The neurohypophysial hormones used by man and most other mammals are [Arg']vasopressin and oxytocin. Vasopressin is the antidiuretic hormone that reabsorbs water from forming urine in the kidney and constricts vascular smooth muscle. Thus, vasopressin participates in the maintenance of normal water balance, blood volume, and blood pressure. Oxytocin influences mating behaviour, contracts uterine smooth muscle during labor, and is responsible for the milk let-down reflex.Most species have a vasopressin-like hormone with a polar residue in position 8 of the nonapeptide (i.e. arginine or lysine in mammals) and an oxytocin-like hormone with a nonpolar amino acid residue in position 8 (i.e. leucine in mammals [7, 81 sug-gests that mesotocin serves important neurotransmitter functions in addition to its hormonal role in water balance. Mesotocin, which was originally identified in amphibians [9], was evolutionary very stable, since it is present in all non-mammalian tetrapods investigated to date. It has been identified in lungfish, a...

Species, sex and individual differences in the vasotocin/vasopressin system: Relationship to neurochemical signaling in the social behavior neural network

“…In newts (i.e., C. pyrrhogaster ), three types of AVT receptors have been cloned and based on their structure they have been designated as V1a, V2 and V3/V1b receptors corresponding to the mammalian V1a, V2 and V1b receptors, respectively(Hasunuma and others, 2007). MT receptors have also been characterized and sequenced in amphibians(Akhundova and others, 1996; Kohno and others, 2003; Acharjee and others, 2004; Searcy and others, 2011). In birds, there are four nonapeptide receptors VT1-VT4(Tan and others, 2000; Gubrij and others, 2005; Leung and others, 2011).…”

Molecular cloning, pharmacological characterization, and histochemical distribution of frog vasotocin and mesotocin receptors

“…To date, the VT receptor (VTR) has been characterized in several non-mammalian species, including the white sucker Catostomus commersoni (Mahlmann et al 1994), the Pacific salmon Oncorhynchus kisutch (Mahlmann et al 1994), the cave-dwelling fish Astyanax fasciatus (Mahlmann et al 1994), the euryhaline flounder Platichthys flesus (Warne 2001), the anuran Xenopus laevis (Mahlmann et al 1994), and the domestic chicken Gallus gallus (Tan et al 2000). An MT receptor (MTR) has also been cloned in the giant toad Bufo marinus (Akhundova et al 1996). However, the sites of expression, the coupling mechanisms and the regulation of VTR and MTR have not been thoroughly investigated in non-mammalian vertebrates.…”

Vasotocin and isotocin regulate aquaporin 1 function in the sea bream

“…The Xenopus oocyte heterologous system has already been used for neuropeptide receptor characterization (Akhundova et al, 1996;Hausmann et al, 1995;Kimura et al, 1992;Mahlmann et al, 1994;Sugimoto et al, 1994). However, to our knowledge, characterization of the interaction between AVTR and ITR with AQP function has not been carried out, in spite of the conceivable co-regulation suggested by other authors in in vivo studies Hatakeyama et al, 2001;Konno et al, 2010).…”