The bombesin receptor subtype 3 (BRS-3) is considered an orphan receptor as it has a low affinity for bombesin-like peptides and no identified natural ligand. We have reported a novel form of gastrin-releasing peptide (GRP) present in high abundance in the pregnant uterus of women and sheep. As BRS-3 was originally cloned from guinea pig uterus, we postulated that the uterine GRP-like peptide may be its natural ligand. We have therefore cloned the gene for the sheep homologue of BRS-3 and determined its distribution. The sheep BRS-3 gene spans 4 kbp and comprises three exons with intron-exon borders at positions similar to those observed for the human and mouse BRS-3 genes. The predicted amino acid sequence of ovine BRS-3 has approximately 85% identity with the human, mouse and guinea pig receptors. Highly conserved amino acids important in mediating receptor G-protein coupling to second messengers and important in ligand binding were found to be conserved in ovine BRS-3. One potentially important deviation was noted: ovine BRS-3 possesses an arginine residue at position 294 instead of a histidine residue as found in all other BRS-3. His(294) was previously identified as important in ligand-receptor interactions while Arg(294) was implicated in high ligand affinity. Thus ovine BRS-3 may have binding characteristics different from those of the human, mouse and guinea pig BRS-3 receptors. In the ewe, BRS-3 mRNA expression was detected in pituitary and hypothalamus but not in tissues of the pregnant uterus (endometrium, myometrium, chorioallantois or amnion). Nor was BRS-3 expression detected in the non-pregnant uterus or in testis. This pattern of BRS-3 expression is similar to that observed in the mouse but different from that observed in the human, rat and guinea pig. We conclude that there is no local interaction between uterine GRP-like peptide and BRS-3. However, the high expression of BRS-3 in the pituitary coupled with elevated circulating levels of this GRP-like peptide during pregnancy suggests an alternate pathway. Cloning of the ovine BRS-3 gene will permit a detailed functional analysis of this receptor in the sheep and its role in the mediation of action of uterine GRP.
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JC Whitley, C Moore, AS Giraud, and A Shulkes
Eliza A Walthers, C Samuel Bradford, and Frank L Moore
We have cloned and characterized an opioid receptor-like (ORL1; also referred to as NOP) receptor from a urodele amphibian, the rough-skinned newt Taricha granulosa The cDNA clone encodes a protein of 368 amino acids that contains the seven hydrophobic domains characteristic of G-protein-coupled receptors, and has the highest sequence identity to the frog (Rana pipiens) nociceptin-like and human ORL1 opioid receptors (79.6 and 68.4%, respectively). Saturation binding assays on membranes from COS-7 cells transiently expressing the newt ORL1 (nORL) receptor revealed a single, high-affinity (estimated K d, 0.1974 nM) binding site for the ORL1-specific agonist [3H]orphanin FQ analog ([3H]oFQ). In competition binding assays, the [3H]oFQ-binding site, like the mammalian ORL1 receptor, had no affinity for the non-selective opioid receptor antagonist naloxone, the κ-selective agonists U69593 and U50488, or the μ- and δ-selective opioid receptor agonists DAMGO and DPDPE, respectively. However, the nORL receptor displayed higher affinities for the κ-selective agonists dynorphin A (1–13), dynorphin B, and dynorphin A (1–8) (K i values, 2.8, 151.8, and 183.0 nM, respectively) than its mammalian homologue. The tissue distribution of the nORL receptor, as determined by reverse transcriptase PCR, was also found to differ from reports on the mammalian ORL1 receptor, with mRNA detected in brain, spinal cord, and lung, but not detected in a number of other peripheral tissues reported to express the receptor in mammals. This is the first report describing the expression and characterization of an amphibian ORL1 receptor, and contributes to our understanding of the evolution of the opioid system.
C Samuel Bradford, Eliza A Walthers, Brian T Searcy, and Frank L Moore
A full-length cDNA that encodes a kappa (κ) opioid receptor has been isolated from the brain of a urodele amphibian, the rough-skinned newt Taricha granulosa. The deduced protein contains 385 amino acids and possesses features commonly attributed to G protein-coupled receptors, such as seven putative transmembrane domains. The newt κ receptor has 75% sequence identity to κ opioid receptors cloned from mammals, and 66% sequence identity to the κ opioid receptor reported for the zebrafish, with the greatest divergence in the extracellular N-terminus, the second and third extracellular loops and the intracellular C-terminus. Membranes isolated from COS-7 cells expressing the newt κ receptor possessed a single, high-affinity (Kd =1.5 nM) binding site for the κ-selective agonist U69593. In competition binding assays, the expressed newt κ receptor displayed high affinity for the κ-selective agonists GR89696, dynorphin A(1–13), U69593, U50488 and BRL52537, as well as the κ-selective antagonist nor-binaltorphimine and the non-selective antagonist naloxone. Rank order potencies and affinity constants were similar in competition binding studies that used either whole brain homogenates or membranes isolated from COS-7 cells expressing the newt κ receptor. The expressed receptor displayed essentially no affinity for the δ-selective agonist DPDPE ([d-penicillamine, d-penicillamine]enkephalin), but showed moderate affinity for the μ-selective agonist DAMGO ([d-Ala-MePhe, Gly-ol]enkephalin) and moderately high affinity for nociceptin (orphanin FQ), the endogenous ligand for the opioid receptor-like (ORL)1 receptor. These findings support the conclusions that a gene for the κ opioid receptor is expressed in amphibians and that the pharmacology of the newt κ receptor closely matches mammalian κ opioid receptors. However, the functional dichotomy between the classic opioid receptors (κ, δ, μ) and ORL1 appears less strict in amphibians than in mammals.
A Moore, C-L C Chen, J R E Davis, and I D Morris
IGF-I mRNA has been demonstrated in testicular tissue and, more recently, localized specifically to Leydig cells. This study investigated the expression of IGF-I and side-chain cleavage enzyme (SCC) mRNA in two preparations of rat interstitial testicular cells which were separated by buoyant density into Leydig cell-enriched and -depleted fractions. RNA was prepared from interstitial cells obtained from the testes of untreated adult and immature rats and adult rats treated with human chorionic gonadotrophin (hCG) or ethane dimethanesulphonate (EDS; to destroy Leydig cells). IGF-I mRNA was detected in all samples, with five major transcripts ranging from 7·5 to 0·6 kb. Leydig cells (3β-hydroxysteroid dehydrogenase-positive and sensitive to EDS) expressed abundant IGF-I and SCC mRNAs, and levels of both were increased following hCG treatment. However, in addition, IGF-I mRNA which was derived from non-Leydig interstitial cells was detected, in the complete absence of SCC message, either in the more buoyant interstitial cells or in both interstitial cell fractions following the destruction of Leydig cells by EDS treatment. IGF-I expression in the Leydig cell-depleted cell fraction was also increased by hCG treatment, and it is therefore suggested that at least part of this non-Leydig interstitial cell IGF-I mRNA originates in Leydig cell precursors. In conclusion, Leydig cells are not the sole origin of IGF-I mRNA in the testis, and the non-Leydig cell expression may be an important component of testicular IGF-I production.
K Boelaert, R Yu, L A Tannahill, A L Stratford, F L Khanim, M C Eggo, J S Moore, L S Young, N J L Gittoes, J A Franklyn, S Melmed, and C J McCabe
Human pituitary tumor-transforming gene (PTTG), known also as securin, is a multifunctional protein implicated in the control of mitosis and the pathogenesis of thyroid, colon, oesophageal and other tumour types. Critical to PTTG function is a C-terminal double PXXP motif, forming a putative SH3-interacting domain and housing the gene’s sole reported phosphorylation site. The exact role of phosphorylation and PXXP structure in the modulation of PTTG action in vitro remains poorly understood. We therefore examined the mitotic, transformation, proliferation and transactivation function of the C-terminal PXXP motifs of human PTTG. Live-cell imaging studies using an EGFP-PTTG construct indicated that PTTG’s regulation of mitosis is retained regardless of phosphorylation status. Colony-formation assays demonstrated that phosphorylation of PTTG may act as a potent inhibitor of cell transformation. In proliferation assays, NIH-3T3 cells stable transfected and overexpressing mutations preventing PTTG phosphorylation (Phos-) showed significantly increased [3H]thymidine incorporation compared with WT, whereas mutants mimicking constitutive phosphorylation of PTTG (Phos+) exhibited reduced cell proliferation. We demonstrated that PTTG transactivation of FGF-2 in primary thyroid and PTTG-null cell lines was not affected by PTTG phosphorylation but was prevented by a mutant disrupting the PXXP motifs (SH3-). Taken together, our data suggest that PXXP structure and phosphorylation are likely to exert independent and critical influences upon PTTG’s diverse actions in vitro.