The homology screening approach has been used to clone a new member of the guanine-nucleotidebinding-protein-coupled receptor superfamily from guinea pig uterus. The cloned cDNA encodes a 399-amino-acid protein and shows the highest amino acid similarity to members of the bombesin receptor family; 52% and 47% similarity to the gastrin-releasing-peptide (GRP) receptor and the neuromedin-B receptor, respectively. Binding experiments with the stably transfected LLC-PK1 cell line expressing the new receptor protein confirmed the bombesin-like nature of the cloned receptor.The relative order of ligand affinity, GRP = neuromedin C & neuromedin B, suggests that the cloned cDNA represents the GRP subtype rather than the neuromedin-B subtype of bombesin receptors. Northern-blot analysis of mRNA species from several guinea-pig tissues showed that the mRNA for the new bombesin receptor subtype is expressed mainly in uteri of pregnant animals.Two related peptides, ranatensin and bombesin, found in frog skin (Nakajima et al., 1970;Anastasi et al., 1971) were the first members of a large family of regulatory peptides, named later bombesin-like peptides. To date, more than 20 peptides of this family are known to share a common amino acid sequence at their C-terminus. The peptides can be classified into three subfamilies according to their C-terminal tripeptide; the bombesin, the ranatensin/litorin and the phyllolitorin subfamilies (Erspamer, 1988). To date, there are only three bombesin-like peptides identified in mammalian tissues; gastrin-releasing peptide (McDonald et al., 1979), neuromedin B (Minamino et al., 1983) and neuromedin C (Minamino et al., 1984). Gastrin-releasing peptide (GRP) and neuromedin C (C-terminal decapeptide of GRP) belong to the bombesin subfamily and neuromedin B belongs to the ranatensin/litorin subfamily (Fig.

[Arg*]vasopressin and oxytocin are the two main members of the neurohypophysial hormone family found to be present in nearly all mammals.

The gene encoding the human homologue of the guinea pig uterine bombesm receptor [( 1992) Eur. J. Biochem. 208,405] was isolated from a genomic lambda library by the PCR/homology screening approach. The gene spans more than 4 kb and consists of 3 exons and 2 introns. The deduced amino acid sequence shows about 86% identity to that of guinea pig bombesin receptor. This subtype of bombesin receptor is expressed in the pregnant uterus and in two human tumour cell lines, T47D (ductal breast carcinoma) and A431 (epidermal carcinoma). PCR analysis of genomic DNA from human-mouse cell hybrids allows the cloned gene to be localized to the region q26q28 on chromosome X.

A cDNA encoding guinea-pig uterine substance P (SP) receptor has been isolated using the homology screening approach. Northern blot analysis reveals that the corresponding mRNA, of approx. 4.8 kb, is expressed in all tissues tested, but predominantly in the uteri of non-pregnant animals; during pregnancy its expression is reduced. The guinea-pig SP receptor was expressed in COS-7 cells and demonstrated relative Iigand affinity in the order: SP » neurokinin A > neurokinin B.Substance P (SP) belongs to the tachykinin family of peptides which includes, in addition to SP, the peptides substance K (neurokinin A) and neuromedin K (neurokinin B). The tachykinins share a common carboxylterminal sequence and give rise to similar spectra of biological activity. SP was the first of these neuropeptides tobe discovered, and is the best characterized. It causes smooth muscle contraction and vasodilation, and plays a role in neuron excitation, pain ttansmission and netve regeneration [1,2]. An important target organ for SP is the uterus. lrnmunocytochemical studies have shown that numerous SP-positive neurons innervate the myometrium and uterine arteries [3][4][5]. SP, in combination with other neuropeptides, plays an essential role in the regulation of myometrial activity and, consequently, in the maintenance of pregnancy and parturition.SP exerts its effect via the SP receptor (so called . NK-1 receptor ), a G-protein-coupled receptor which utilises the phosphatidyl inositol second messenger system.cDNAs corresponding to the rat [6,7] and human [8] SP receptors have recently been cloned and characterized. Here we report the molecular cloning of the guinea-pig SP receptor cDNA and its expression in COS cells. The aim of our study was the isolation of cDNAs corresponding to neuropepÜde receptors expressed in the guinea-pig uterus. These are G-protein-coupled receptors that share common structural features and have consetved amino acid sequences, particularly within the transmembrane domains. Therefore, we chose the homology screening approach to analyse guinea-pig uterine cDNA library constructed in ,\gtlO bacteriophage. A degenerate oligonucleotide (5'-GG2A~CCAGCAGA

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...