The utilization of recombinant prostanoid receptors to determine the affinities and selectivities of prostaglandins and related analogs.

Stable cell lines that individually express the eight known human prostanoid receptors (EP(1), EP(2), EP(3), EP(4), DP, FP, IP and TP) have been established using human embryonic kidney (HEK) 293(EBNA) cells. These recombinant cell lines have been employed in radioligand binding assays to determine the equilibrium inhibitor constants of known prostanoid receptor ligands at these eight receptors. This has allowed, for the first time, an assessment of the affinity and selectivity of several novel compounds at the individual human prostanoid receptors. This information should facilitate interpretation of pharmacological studies that employ these ligands as tools to study human tissues and cell lines and should, therefore, result in a greater understanding of prostanoid receptor biology.

Genomic characterization of the human and mouse protein tyrosine phosphatase-1B genes.

PTP-1B is a ubiquitously expressed intracellular protein tyrosine phosphatase (PTP) that has been implicated in the negative regulation of insulin signaling. Mice deficient in PTP-1B were found to have an enhanced insulin sensitivity and a resistance to diet-induced obesity. Interestingly, the human PTP-1B gene maps to chromosome 20q13.1 in a region that has been associated with diabetes and obesity. Although there has been a partial characterization of the 3' end of the human PTP-1B gene, the complete gene organization has not been described. In order to further characterize the PTP-1B gene, we have cloned and determined the genomic organization for both the human and mouse PTP-1B genes including the promoter. The human gene spans >74 kb and features a large first intron of >54 kb; the mouse gene likewise contains a large first intron, although the exact size has not been determined. The organization of the human and mouse PTP-1B genes is identical except for an additional exon at the 3' end of the human that is absent in the mouse. The mouse PTP-1B gene maps to the distal arm of mouse chromosome 2 in the region H2-H3. This region is associated with a mouse obesity quantitative trait locus (QTL) and is syntenic with human chromosome 20. The promoter region of both the human and mouse genes contain no TATA box but multiple GC-rich sequences that contain a number of consensus SP-1 binding sites. The basal activity of the human PTP-1B promoter was characterized in Hep G2 cells using up to 8 kb of 5' flanking sequence. A 432 bp promoter construct immediately upstream of the ATG was able to confer maximal promoter activity. Within this sequence, there are at least three GC-rich sequences and one CCAAT box, and deletion of any of these elements results in decreased promoter activity. In addition, the promoter in a number of mouse strains contains, 3.5 kb upstream of the start codon, an insertion of an intracisternal a particle (IAP) element that possibly could alter the expression of PTP-1B mRNA in these strains.

Characterization of the cloned guinea pig leukotriene B4 receptor: comparison to its human orthologue.

A cDNA clone coding for the guinea pig leukotriene B4 (BLT) receptor has been isolated from a lung cDNA library. The guinea pig BLT receptor has an open reading frame corresponding to 348 amino acids and shares 73% and 70% identity with human and mouse BLT receptors, respectively. Scatchard analysis of membranes prepared from guinea pig and human BLT receptor-transfected human embryonic kidney (HEK) 293 EBNA (Epstein-Bar Virus Nuclear Antigen) cells showed that both receptors displayed high affinity for leukotriene B4 (Kd value of approximately 0.4 nM) and were expressed at high levels (Bmax values ranging from 9 to 12 pmol/mg protein). The rank order of potency for leukotrienes and related analogs in competition for [3H]leukotriene B4 specific binding at the recombinant guinea pig BLT receptor is leukotriene B4 > 20-OH-leukotriene B4 > 12(R)-HETE ((5Z,8Z,10E,12(R)14Z)-12-hydroxyeicosatetraen -1-oic acid) > 12(S)-HETE ((5Z,8Z,10E,12(S)14Z)-12-Hydroxyeicosatetraen -1-oic acid) > 20-COOH-leukotriene B4 > U75302 (6-(6-(3-hydroxy-1E,5Z-undecadienyl)-2-pyridinyl)-1,5-hexane diol) >> leukotriene C4 = leukotriene D4 = leukotriene E4. For the human receptor the rank order of 12(S)-HETE, 20-COOH-leukotriene B4 and U75302 was reversed. Xenopus melanophore and HEK aequorin-based reporter gene assays were used to demonstrate that the guinea pig and human BLT receptors can couple to both the cAMP inhibitory and intracellular Ca2+ mobilization signaling pathways. However, in the case of the aequorin-expressing HEK cells (designated AEQ17-293) transfected with either the guinea pig or human BLT receptor, expression of Galpha16 was required to achieve a robust Ca2+ driven response. Leukotriene B4 was a potent agonist in functional assays of both the guinea pig and human BLT receptors. U-75302 a leukotriene B4 analogue which possesses both agonistic and antagonistic properties behaved as a full agonist of the guinea pig and human BLT receptors in AEQ17-293 cells and not as an antagonist. The recombinant guinea pig BLT receptor will permit the comparison of the intrinsic potencies of leukotriene B4 receptor antagonists used in guinea pig in vivo models of allergic and inflammatory disorders.

Molecular cloning and characterization of the four rat prostaglandin E2 prostanoid receptor subtypes.

We have characterized the rat prostanoid EP1, EP2, EP3alpha and EP4 receptor subtypes cloned from spleen, hepatocyte and/or kidney cDNA libraries. Comparison of the deduced amino acid sequences of the rat EP receptors with their respective homologues from mouse and human showed 91% to 98% and 82% to 89% identity, respectively. Radioreceptor binding assays and functional assays were performed on EP receptor expressing human embryonic kidney (HEK) 293 cells. The KD values obtained with prostaglandin E2 for the prostanoid receptor subtypes EP1, EP2, EP3alpha and EP4 were approximately 24, 5, 1 and 1 nM, respectively. The rank order of affinities for various prostanoids at the prostanoid receptor subtypes EP2, EP3alpha and EP4 receptor subtypes was prostaglandin E2 = prostaglandin E1 > iloprost > prostaglandin F2alpha > prostaglandin D2 > U46619. The rank order at the prostanoid EP1 receptor was essentially the same except that iloprost had the highest affinity of the prostanoids tested. Of the selective ligands, butaprost was selective for prostanoid EP2, M&B28767 and sulprostone were selective for EP3alpha and enprostil displayed dual selectivity, interacting with both prostanoid receptor subtypes EP1 and EP3alpha. All four receptors coupled to their predominant signal transduction pathways in HEK 293 cells. Notably, using a novel aequorin luminescence assay to monitor prostanoid EP1 mediated increases in intracellular calcium, both iloprost and sulprostone were identified as partial agonists. Finally, by Northern blot analysis EP3 transcripts were most abundant in liver and kidney whereas prostanoid EP2 receptor mRNA was expressed in spleen, lung and testis and prostanoid EP1 receptor mRNA transcripts were predominantly expressed in the kidney. The rat prostanoid EP1 probes also detected additional and abundant transcripts present in all the tissues examined. These were found to be related to the expression of a novel protein kinase gene and not the prostanoid EP1 gene [Batshake, B., Sundelin, J., 1996. The mouse genes for the EP1 prostanoid receptor and the novel protein kinase overlap. Biochem. Biophys. Res. Commun. 227. 1329-1333].

Molecular cloning and characterization of the human prostanoid DP receptor.

A cDNA encoding a functional human prostanoid DP (hDP) receptor has been constructed from a genomic clone and a fragment cloned by 3'-rapid amplification of cDNA ends-polymerase chain reaction. The hDP receptor consists of 359 amino acid residues with a predicted molecular mass of 40,276 and has the putative heptahelical transmembrane domains characteristics of G-protein-coupled receptors. The deduced amino acid sequence of the hDP receptor, when compared with all other members of the prostanoid receptor family, shows the highest degree of identity with the hIP and hEP2 receptors, followed by the hEP4 receptor. Radioreceptor binding studies using membranes prepared from mammalian COS-M6 cells transiently transfected with an expression vector containing the DP receptor cDNA showed that the rank order of affinities for prostaglandins and prostaglandin analogs, in competition for [3H]prostaglandin D2 (PGD2) specific binding sites, was as predicted for the DP receptor, with PGD2 >> PGE2 > PGF2 alpha = iloprost > U46619. The signal transduction pathway of the cloned hDP receptor was studied by transfecting the hDP expression vector in HEK 293(EBNA) cells. Activation of the hDP receptor with PGD2 resulted in an elevation of intracellular cAMP and in mobilization of Ca2+, but did not lead to generation of inositol 1,4,5-trisphosphate. Northern blot analysis of human tissue showed that the hDP receptor was a very discrete tissue distribution and was detectable only in retina and small intestine. In summary, we have cloned and expressed a functional cDNA for the hDP receptor.

Detection of adenylate cyclase-coupled receptors in Xenopus oocytes by coexpression with cystic fibrosis transmembrane conductance regulator.

To detect heterologous expression of receptors coupled via G proteins to the stimulation of adenylate cyclase in Xenopus laevis oocytes, the receptor of interest is coexpressed with the cystic fibrosis transmembrane conductance regulator (CFTR)--a cAMP-dependent Cl- channel. The binding of an agonist to the expressed receptor stimulates adenylate cyclase resulting in intracellular cAMP elevation, which in turn activates the CFTR. The CFTR-mediated Cl- current response is then measured using the standard two-electrode voltage-clamp technique. This method has allowed us to detect functional expression in oocytes of the human EP2 and IP prostanoid receptors. This method should prove valuable for expression and identification of putative G protein-coupled receptors signaling through stimulation of adenylate cyclase, for structure/function studies, and for analysis of receptor antagonists and agonists.

Cloning and expression of a cDNA for the human prostanoid IP receptor.

A cDNA clone coding for a functional human prostanoid IP receptor has been isolated from a lung cDNA library. The human IP receptor consists of 386 amino acid residues with a predicted molecular mass of 40,961, and has the seven putative transmembrane domains characteristic of G-protein-coupled receptors. Challenge of Xenopus oocytes co-expressing the IP receptor and the cystic fibrosis transmembrane conductance regulator (cAMP-activated Cl- channel) with the stable prostacyclin analog iloprost resulted in specific inward Cl- currents, demonstrating that the cDNA encoded a functional IP prostanoid receptor coupled to elevation in cAMP. Radioreceptor binding studies using membranes prepared from mammalian COS cells transfected with the IP receptor cDNA showed that the rank order of potency for prostaglandins and prostaglandin analogs in competition for [3H]iloprost specific binding sites was as predicted for the IP receptor, with iloprost >> carbacyclin >> prostaglandin (PG) E2 > PGF 2 alpha = PGD2 = U46619. Northern blot analysis showed that IP mRNA was most abundantly expressed in kidney, with lesser amounts detected in lung and liver. In summary, we have cloned and expressed a cDNA for the human prostanoid IP receptor that is functionally coupled to a signaling pathway involving stimulation of intracellular cAMP production.

Cloning and expression of three isoforms of the human EP3 prostanoid receptor.

Functional cDNA clones coding for three isoforms of the human prostaglandin E receptor EP3 subtype have been isolated from kidney and uterus cDNA libraries. The three isoforms, designated hEP3-I, hEP3-II and hEP3-III, have open reading frames corresponding to 390, 388 and 365 amino acids, respectively. They differ only in the length and amino acid composition of their carboxy-terminal regions, beginning at position 360. The human EP3 receptor has seven predicted transmembrane spanning domains and therefore belongs to the G-protein-coupled receptor family. The rank order of potency for prostaglandins and related analogs in competition for [3H]PGE2 specific binding to membranes prepared from transfected COS cells was comparable for all three isoforms, and as predicted for the EP3 receptor, with PGE2 = PGE1 >> PGF2 alpha = iloprost > PGD2 >> U46619. In addition, the EP3-selective agonist MB28767 was a potent competing ligand with an IC50 value of 0.3 nM, whereas the EP1-selective antagonist AH6909 gave IC50 values of 2-7 microM and the EP2-selective agonist butaprost was inactive. In summary, we have cloned three isoforms of the human EP3 receptor having comparable ligand binding properties.

Cloning and expression of a cDNA for the human prostanoid FP receptor.

A cDNA clone coding for a functional human prostanoid FP receptor has been isolated from a uterus cDNA library. The human FP receptor consists of 359 amino acid residues with a predicted molecular mass of 40,060, and has the seven putative transmembrane domains characteristic of G-protein-coupled receptors. Challenge of Xenopus oocytes expressing the FP receptor with 10 nM of either prostaglandin (PG) F2 alpha or the selective FP-receptor agonist fluprostenol resulted in an elevation in intracellular Ca2+. Radioreceptor binding studies using membranes prepared from mammalian COS cells transfected with the FP receptor cDNA showed that the rank order of potency for prostaglandins and prostaglandin analogs in competition for [3H]PGF2 alpha specific binding sites was as predicted for the FP receptor, with PGF2 alpha approximately fluprostenol > PGD2 > PGE2 > U46619 > iloprost. In summary, we have cloned the human prostanoid FP receptor which is functionally coupled to the Ca2+ signalling pathway.

Enantioselective activation of the peroxisome proliferator-activated receptor.

A cell-based transactivation assay was established using the mouse full-length peroxisome proliferator-activated receptor (PPAR) cDNA sequence and the positive peroxisome proliferator-responsive regulatory element (-578 to -553) of the rat acyl-CoA oxidase gene promoter. Activation of the reporter plasmid was dependent on co-transfection of the full-length PPAR cDNA, and the response was greatly stimulated, up to 100-fold, by peroxisome proliferators such as Wy-14,643 ([4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio] acetic acid), nafenopin (2-methyl-2[p-(1,2,3,4-tetrahydro-1-naphthyl)phenoxy]-propionic acid), and clofibric acid (2-([p]-chlorophenoxy)-2-methylpropionic acid). Activation of the reporter plasmid promoter by the full-length PPAR cDNA also occurred at peroxisomal proliferator concentrations 40 times lower than that required for similar stimulation by a glucocorticoid-PPAR chimeric receptor. By using the stereoisomers of MK-571 ((+-)-3-(((3-(2-(7-chloro-2-quinolinyl)ethenyl)-phenyl)((3- (dimethylamino)-3-oxopropyl)-thio)methyl)-thio)propanoic acid), a potent leukotriene D4 receptor antagonist, we could show enantioselective activation of PPAR. The use of this compound in mice results in peroxisome proliferation; however, nearly all of the peroxisome proliferating activity can be attributed to the S enantiomer. Our results show a similar enantiomeric discrimination in PPAR activation of the reporter plasmid promoter, where again most of the activity can be attributed to the S enantiomer. The equivalent activities of these stereoisomers both in vivo and in the PPAR transactivation assay strongly implicate PPAR as a major component of the peroxisome proliferating mechanism in rodents.

The intracisternal A particle derived solo LTR promoter of the rat oncomodulin gene is not present in the mouse gene.

The rat gene encoding oncomodulin, a small calcium-binding protein related to parvalbumin, is under the control of a solo long terminal repeat (LTR) derived from an endogenous intracisternal A-particle (IAP). This gene was the first example of a mammalian gene regulated in normal cells by a promoter of retroviral origin (see also article by D. Robins and L. Samuelson in this volume). We show here that the oncomodulin LTR is a member of a small subset of sequence related solo LTR elements present in the rat genome and that a full length IAP genome containing LTRs of this type is no longer present in the rat genome. We have assayed the transcriptional activity of the oncomodulin LTR coupled to the human growth hormone gene as a reporter. Transfections in both Hela cells and 293 cells indicate the oncomodulin LTR promoter is sufficient to efficiently initiate transcription. In 293 cells (human embryo kidney cells transformed with human adenovirus type 5 DNA), the oncomodulin LTR is a strong promoter, capable of bidirectional transcription. Finally, we have determined the structure and the sequence of the mouse oncomodulin gene. Our results suggest that the integration of the IAP particle genome within the rat oncomodulin gene occurred after the rat and the mouse became distinct species.

Gene characterization and promoter analysis of the human 5-lipoxygenase-activating protein (FLAP).

The human gene for the recently identified 5-lipoxy-genase-activating protein (FLAP) has been cloned. The gene was isolated from two different genomic libraries and is contained within four overlapping bacteriophage clones. The gene spans greater than 31 kilobases and consists of five small exons and four large introns. Southern blot analysis of human genomic DNA suggests the presence of a single FLAP gene per haploid genome. A restriction site polymorphism was identified in intron II of the gene. This restriction fragment length polymorphism appears to be present in the normal population at a fairly high frequency. The transcription initiation site was located, at an adenine residue, 74 base pairs upstream of the ATG initiation codon. Examination of the sequence of the gene 5' to the mRNA start site revealed the presence of a possible TATA box (TGTAAT) 22 base pairs upstream and potential AP-2 and glucocorticoid receptor binding sites. Functional analysis of the FLAP gene promoter was assayed by transient transfection of mouse P388D1 cells (macrophage) and human HepG2 cells (hepatoma) with 5'-flanking sequences of the FLAP gene fused upstream of the chloramphenicol acetyltransferase reporter gene. Expression in the mouse macrophage cell line of the various FLAP gene promoter constructs revealed both tissue specificity and enhancer-like activities whereas in the hepatoma cell line only a minimum level of activity was obtained.

Retroviral long terminal repeat is the promoter of the gene encoding the tumor-associated calcium-binding protein oncomodulin in the rat.

Oncomodulin is a small calcium-binding protein normally found only in extra-embryonic tissues such as the placenta, but whose presence in a variety of tumors has been documented. We have isolated the oncomodulin gene from a Buffalo rat genomic library. The rat gene is approximately 9000 bases in length and consists of five exons and four introns. The introns interrupt the coding sequence of oncomodulin in positions identical with those previously reported for the parvalbumin gene, indicating that the two genes are derived from a common ancestor. Analysis of the promoter sequence of the oncomodulin gene revealed that the gene is under the control of a solo long terminal repeat element related to intracisternal-A particles, a family of endogenous retroviral elements. This represents a unique example of a mammalian gene transcribed in normal and tumor cells, from a promoter of viral origin.

Studies on emerging radiation leukemia virus variants in C57BL/Ka mice.

To analyze the emergence of radiation leukemia virus (RadLV) variants in primary X-ray-induced C57BL/Ka thymoma and to identify the virus responsible for the very high leukemogenic potential of passaged Kaplan strain BL/VL3 preparation, we cloned several primary and passaged ecotropic RadLV infectious genomes. By restriction analysis, we found that BL/VL3 cells harbor three related but different ecotropic RadLVs. Their restriction map differs significantly from those of primary RadLVs. Hybridization analysis also indicated that BL/VL3 and primary RadLVs differ in their p15E and long terminal repeat (LTR) regions. As compared with the LTR sequence of the putative parental endogenous ecotropic provirus, the LTR sequence of primary weakly leukemogenic RadLV has only one change, a C-rich sequence, generating a 6-base-pair direct repeat just in front of the promotor. The LTR of the primary nonleukemogenic RadLV only showed few base changes, mainly clustered in R and U5. The LTR from a moderately leukemogenic passaged BL/VL3 RadLV had conserved the C-rich sequence and acquired a 43-base-pair direct repeat in U3 and several other point mutations, small insertions, and deletions scattered in U3, R, and U5. All cloned primary RadLVs were fibrotropic, and some were weakly leukemogenic. All cloned BL/VL3 RadLVs were thymotropic and nonfibrotropic. The block of their replication was found to be after the synthesis of unintegrated linear and supercoiled viral DNA. Most of the BL/VL3 RadLVs were moderately leukemogenic, and one (V-13) was highly leukemogenic, being as virulent as the Moloney strain. We propose a model for the emergence of the RadLV variants and show that the virus responsible for the high leukemogenic potential of BL/VL3 preparation is a nondefective, ecotropic, lymphotropic, nonfibrotropic, unique retrovirus which most likely arose from a parental primary RadLV similar to those studied here.

Molecular analysis of emerging radiation leukemia virus variants of C57BL/Ka mice.

Molecular cloning of several primary or passaged RadLV variants and their biological characterization has allowed us to propose a model of their emergence following X-ray irradiation of C57BL/6 mouse.