Nucleotide sequence and structural organization of the human vasopressin pituitary receptor (V3) gene
Introduction
The hypothalamic nonapeptide arginine vasopressin (AVP) modulates body fluid osmolality, blood volume, liver metabolism, vascular constriction, hormonal response to stress and cell proliferation. These multiple actions are mediated by a family of GTP-binding proteins (G protein) coupled receptors. Three subtypes of vasopressin receptors have been identified (Jard, 1985): the V1a subtype is positively coupled to phospholipase C (PLC) and transcribed in many organs such as liver, brain, blood vessels and adrenals; the V2 subtype, which stimulates adenylate cyclase through the activation of Gs and is expressed in the kidney, and the V3 (or V1b) subtype, coupled to PLC like the V1a receptor. V3 receptors have been located by radioligand binding and pharmacological in-situ studies to the anterior pituitary corticotrophs in man and rat (Antoni, 1984, Antoni, 1988, DuPasquier et al., 1991). It is also expressed at lower levels in discrete brain areas and some endocrine cells (Burbach et al., 1995, Grazzini et al., 1996, Lolait et al., 1995, Richardson et al., 1995). This family also includes the receptors for the structurally related hormones oxytocin in mammals (Kimura et al., 1992, Rozen et al., 1995) and their analogs in lower vertebrates like vasotocin, mesotocin and conopressin (Akhundova et al., 1996, Mahlmann et al., 1994, Vankesteren et al., 1995).
The cDNAs coding for these receptors have been cloned in rat and man (Birnbaumer et al., 1992, de Keyzer et al., 1994, Lolait et al., 1995, Morel et al., 1992, Sugimoto et al., 1994). They all belong to the large superfamily of seven transmembrane domains receptors, and hV3 presents 37–38% sequence identity at the amino-acid level with the V2 receptors and 44–46% with the V1a and oxytocin receptors, particularly in the transmembrane domains 2, 3, 6 and 7 (de Keyzer et al., 1994).
We previously described the cloning, sequencing and functional expression of the human V3 receptor cDNA isolated from a pituitary corticotroph adenoma library (de Keyzer et al., 1994). We showed that its expression is tightly associated with expression of the proopiomelanocortin (POMC) gene in the pituitary as well as in many corticotroph tumors, suggesting that V3 belongs to the features of a well-established corticotroph phenotype. One of the hypotheses to explain this associated expression is that the V3 gene and the POMC gene share some aspects of their transcription mechanisms.
We report here the structure and sequence of the human (h) V3 gene and characterize its transcripts by S1 nuclease protection and RACE-PCR experiments.
Section snippets
Cloning of the human V3 receptor gene
The hV3 gene was isolated from a human liver genomic λEMBL4 library. Clones (1.2×106) were screened by standard plaque hybridization methods (Ausubel et al., 1994) with a 1.2 kb hV3 cDNA probe (de Keyzer et al., 1994) labeled with 32P by random priming. Positive clones, selected to span the whole gene, were subcloned into pBluescript (Stratagene) and sequenced on both strands (except for short intron regions) by the dideoxychain termination method. A database sequence comparison was made using
Isolation and characterization of the V3 gene
The human DNA library was initially screened with a human 1.2 kb cDNA probe including ca 400 nucleotides (nt) of 5′ UTR sequence. Eight clones were isolated from 1.2×106 pfu and further characterized by hybridization with fragments of the 5′ and 3′ non-coding regions of the cDNA. One of the clones encompassing the whole gene was selected after restriction mapping and subcloned as BamHI/EcoRI fragments in pBluescript for DNA sequencing. The gene spans ca 10.2 kb and is composed of two exons
Conclusions
We have established hV3gene structure and sequence and showed it to be considerably conserved among the human vasopressin/oxytocin receptors. We have mapped the transcription initiation site and provide promoter sequence information that will help to understand the mechanisms of hV3 gene transcription. In this view, we have located several putative transcription binding sites that may participate in hV3 transcription. In addition, an antisense transcript partially complementary to hV3 mRNA was
Acknowledgements
This work was supported in part by the Association pour la Recherche contre le Cancer (contract 6501 to Y.dK. and a fellowship to P.R.).
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