Characterization of the human cysteinyl leukotriene 2 receptor.

The contractile and inflammatory actions of the cysteinyl leukotrienes (CysLTs), LTC(4), LTD(4), and LTE(4), are thought to be mediated through at least two distinct but related CysLT G protein-coupled receptors. The human CysLT(1) receptor has been recently cloned and characterized. We describe here the cloning and characterization of the second cysteinyl leukotriene receptor, CysLT(2), a 346-amino acid protein with 38% amino acid identity to the CysLT(1) receptor. The recombinant human CysLT(2) receptor was expressed in Xenopus oocytes and HEK293T cells and shown to couple to elevation of intracellular calcium when activated by LTC(4), LTD(4), or LTE(4). Analyses of radiolabeled LTD(4) binding to the recombinant CysLT(2) receptor demonstrated high affinity binding and a rank order of potency for competition of LTC(4) = LTD(4) LTE(4). In contrast to the dual CysLT(1)/CysLT(2) antagonist, BAY u9773, the CysLT(1) receptor-selective antagonists MK-571, montelukast (Singulair(TM)), zafirlukast (Accolate(TM)), and pranlukast (Onon(TM)) exhibited low potency in competition for LTD(4) binding and as antagonists of CysLT(2) receptor signaling. CysLT(2) receptor mRNA was detected in lung macrophages and airway smooth muscle, cardiac Purkinje cells, adrenal medulla cells, peripheral blood leukocytes, and brain, and the receptor gene was mapped to chromosome 13q14, a region linked to atopic asthma.

Coexpression of full-length gamma-aminobutyric acid(B) (GABA(B)) receptors with truncated receptors and metabotropic glutamate receptor 4 supports the GABA(B) heterodimer as the functional receptor.

Direct evidence is lacking to show whether the gamma-aminobutyric acid (GABA)(B) gb1-gb2 heterodimer is the signaling form of the receptor. In this study, we tested whether gb1a or gb2 subunits when coexpressed with truncated receptors or metabotropic glutamate receptor mGluR4 could form functional GABA receptors. Coexpression of the ligand binding N-terminal domain of gb1a or the C-terminal portion of gb1a composing the seven-transmembrane segments and intracellular loops with gb2 could not reconstitute functional receptors. We next examined whether mGluR4, which forms homodimers and is structurally related to GABA(B), could act as a surrogate coreceptor for gb1 or gb2. The coexpression of mGluR4 and gb1a led to the expression of gb1a monomers on cell surface membranes as determined by immunoblot analysis and flow cytometry. However, mGluR4-gb1a heterodimers were not formed, and membrane-expressed gb1a monomers were not functionally coupled to adenylyl cyclase in human embryonic kidney 293 cells or activated inwardly rectifying potassium (Kir) channels in Xenopus oocytes. Similarly, the coexpression of mGluR4 and gb2 led to nonfunctional GABA receptors. GABA-activated distal signaling events resulted only after the coexpression and heterodimerization of gb1 and gb2. Taken together with the truncated receptor studies, the data suggest that a high degree of structural specificity is required to form the functional GABA(B) receptor that is a gb1-gb2 heterodimer.

Characterization of the human cysteinyl leukotriene CysLT1 receptor.

The cysteinyl leukotrienes-leukotriene C4(LTC4), leukotriene D4(LTD4) and leukotriene E4(LTE4)-are important mediators of human bronchial asthma. Pharmacological studies have determined that cysteinyl leukotrienes activate at least two receptors, designated CysLT1 and CysLT2. The CysLT1-selective antagonists, such as montelukast (Singulair), zafirlukast (Accolate) and pranlukast (Onon), are important in the treatment of asthma. Previous biochemical characterization of CysLT1 antagonists and the CysLT1 receptor has been in membrane preparations from tissues enriched for this receptor. Here we report the molecular and pharmacological characterization of the cloned human CysLT1 receptor. We describe the functional activation (calcium mobilization) of this receptor by LTD4 and LTC4, and competition for radiolabelled LTD4 binding to this receptor by the cysteinyl leukotrienes and three structurally distinct classes of CysLT1-receptor antagonists. We detected CysLT1-receptor messenger RNA in spleen, peripheral blood leukocytes and lung. In normal human lung, expression of the CysLT1-receptor mRNA was confined to smooth muscle cells and tissue macrophages. Finally, we mapped the human CysLT1-receptor gene to the X chromosome.

Discovery of a receptor related to the galanin receptors.

We report the isolation of a cDNA clone named GPR54, which encodes a novel G protein-coupled receptor (GPCR). A PCR search of rat brain cDNA retrieved a clone partially encoding a GPCR. In a library screening this clone was used to isolate a cDNA with an open reading frame (ORF) encoding a receptor of 396 amino acids long which shared significant identities in the transmembrane regions with rat galanin receptors GalR1 (45%), GalR3 (45%) and GalR2 (44%). Northern blot and in situ hybridization analyses revealed that GPR54 is expressed in brain regions (pons, midbrain, thalamus, hypothalamus, hippocampus, amygdala, cortex, frontal cortex, and striatum) as well as peripheral regions (liver and intestine). In COS cell expression of GPR54 no specific binding was observed for 125I-galanin. A recent BLAST search with the rat GPR54 ORF nucleotide sequence recovered the human orthologue of GPR54 in a 3.5 Mb contig localized to chromosome 19p13.3.

Cloning of a novel G-protein-coupled receptor GPR 51 resembling GABAB receptors expressed predominantly in nervous tissues and mapped proximal to the hereditary sensory neuropathy type 1 locus on chromosome 9.

Query of the expressed sequence tag database with the rat metabotropic GABABR1A receptor amino acid sequence using the TFASTA algorithm revealed two partial cDNA fragments whose sequence information was then used to isolate by PCR a novel full-length human cDNA encoding a putative G-protein-coupled receptor (GPCR), termed GPR 51. Sequence analysis revealed that it encoded a protein of 941 amino acids, similar in size and homology to GABAB receptors followed by metabotropic glutamate receptors but not other GPCRs. GPR 51 expressed in COS-1 cells showed no specific binding for [3H](+)baclofen and when expressed in Xenopus oocyte and Xenopus melanophore functional assays showed no activity to GABA, (-)baclofen, and glutamic acid. Northern blot analysis and in situ hybridization revealed that GPR 51 transcripts were predominantly expressed in the central nervous system with highest abundance in the cortex, thalamus, hippocampus, amygdala, cerebellum, and spinal cord. In contrast, GPR 51 receptor transcripts were almost not detected in the peripheral tissues. Gene GPR 51 was localized by radiation hybrid mapping to chromosome 9, 4.81 cR from the WI-8684 marker, and proximal to the hereditary sensory neuropathy type 1 locus.

Identification of a GABAB receptor subunit, gb2, required for functional GABAB receptor activity.

G protein-coupled receptors are commonly thought to bind their cognate ligands and elicit functional responses primarily as monomeric receptors. In studying the recombinant gamma-aminobutyric acid, type B (GABAB) receptor (gb1a) and a GABAB-like orphan receptor (gb2), we observed that both receptors are functionally inactive when expressed individually in multiple heterologous systems. Characterization of the tissue distribution of each of the receptors by in situ hybridization histochemistry in rat brain revealed co-localization of gb1 and gb2 transcripts in many brain regions, suggesting the hypothesis that gb1 and gb2 may interact in vivo. In three established functional systems (inwardly rectifying K+ channel currents in Xenopus oocytes, melanophore pigment aggregation, and direct cAMP measurements in HEK-293 cells), GABA mediated a functional response in cells coexpressing gb1a and gb2 but not in cells expressing either receptor individually. This GABA activity could be blocked with the GABAB receptor antagonist CGP71872. In COS-7 cells coexpressing gb1a and gb2 receptors, co-immunoprecipitation of gb1a and gb2 receptors was demonstrated, indicating that gb1a and gb2 act as subunits in the formation of a functional GABAB receptor.

Molecular characterization and expression of cloned human galanin receptors GALR2 and GALR3.

Galanin is a 29- or 30-amino acid peptide with wide-ranging effects on hormone release, feeding behavior, smooth muscle contractility, and somatosensory neuronal function. Three distinct galanin receptor (GALR) subtypes, designated GALR1, 2, and 3, have been cloned from the rat. We report here the cloning of the human GALR2 and GALR3 genes, an initial characterization of their pharmacology with respect to radioligand binding and signal transduction pathways, and a profile of their expression in brain and peripheral tissues. Human GALR2 and GALR3 show, respectively, 92 and 89% amino acid sequence identity with their rat homologues. Radioligand binding studies with 125I-galanin show that recombinant human GALR2 binds with high affinity to human galanin (K(D) = 0.3 nM). Human GALR3 binds galanin with less affinity (IC50 of 12 nM for porcine galanin and 75 nM for human galanin). Human GALR2 was shown to couple to phospholipase C and elevation of intracellular calcium levels as assessed by aequorin luminescence in HEK-293 cells and by Xenopus melanophore pigment aggregation and dispersion assays, in contrast to human GALR1 and human GALR3, which signal predominantly through inhibition of adenylate cyclase. GALR2 mRNA shows a wide distribution in the brain (mammillary nuclei, dentate gyrus, cingulate gyrus, and posterior hypothalamic, supraoptic, and arcuate nuclei), and restricted peripheral tissue distribution with highest mRNA levels detected in human small intestine. In comparison, whereas GALR3 mRNA was expressed in many areas of the rat brain, there was abundant expression in the primary olfactory cortex, olfactory tubercle, the islands of Calleja, the hippocampal CA regions of Ammon's horn, and the dentate gyrus. GALR3 mRNA was highly expressed in human testis and was detectable in adrenal gland and pancreas. The genes for human GALR2 and 3 were localized to chromosomes 17q25 and 22q12.2-13.1, respectively.

Platelet angiotensin II binding sites and early detection of preeclampsia.

OBJECTIVE: To determine if platelet angiotensin II binding density during the second or third trimester of pregnancy can be used as a marker for early detection of women who will develop preeclampsia. METHODS: We collected blood samples from 412 nulliparous pregnant women during their second or third trimesters. They were classified in four groups after delivery: normotensive (n=297), transient hypertensive (n=54), preeclamptic (n=39), and chronic hypertensive (n=22). We also studied 35 nonpregnant women and 122 women in the peripartum period. The binding capacity of platelet angiotensin II receptors was analyzed in each patient. RESULTS: In normotensive pregnancies, there was a significant decrease in mean (+/-standard error of the mean [SEM]) platelet binding in the second trimester (1.6+/-0.2 fmol/10(9) cells) compared with nonpregnant women (3.3+/-0.7 fmol/10[9] cells). No statistical differences were observed in the mean (+/-SEM) number of platelet angiotensin II binding sites between the groups studied in the third trimester (normal: 1.7+/-0.1 fmol/10(9) cells; transient hypertensive: 2.3+/-0.4 fmol/10(9) cells; preeclamptic: 1.6+/-0.4 fmol/10(9) cells, and chronic hypertensive: 1.6+/-0.6 fmol/10(9) cells), nor were any significant differences found in second-trimester values. At cutoff levels providing identical sensitivities, angiotensin II binding showed significantly lower positive predictive values than mean arterial pressure (P < .05). With this study's sample size, we could have demonstrated an improvement in positive predictive values of 20% with a statistical power (1-beta) of 90%. CONCLUSION: The measurement of platelet angiotensin II receptor density cannot be recommended for the early detection of preeclampsia.

Characterization of the hamster CYP11B2 gene encoding adrenal cytochrome P450 aldosterone synthase.

A CYP11B2 gene encoding cytochrome P450 aldosterone synthase (P450aldo) was isolated from a hamster genomic library. The gene, which contained 9 exons, was composed of 9,045 bp, of which 3,722 bp were located in the 5' untranslated region (5' UTR). A TATA box sequence (gataaa) and other putative cis elements, previously named Ad1 to Ad6, were identified in the 5' UTR of the hamster gene comparable to the CYP11B2 gene of other animal species. Footprint analysis showed protection by nuclear protein extracts from hamster adrenal zona glomerulosa (ZG) in the regions containing the above mentioned cis elements. In addition, a new protected cis element, between -143 and -161 bp, was demonstrated, and gel-shift assays revealed that the sequence of this new cis element was specifically retarded by factors in the nuclear extracts of hamster adrenal ZG. We then examined the transcriptional activity of the 5' UTR of the CYP11B2 gene, using chloramphenicol acyltransferase (CAT) as the reporter gene. Ten deletion plasmids were constructed using a modified pCAT vector. Transient transfections of the chimeric reporter constructs into Y1 cells showed that the highest basal promoter activity was obtained with the construct containing up to -134 bp. Increasing the length of the regulatory region of CYP11B2 gene to -167 bp resulted in less than two-thirds of the maximal activity, indicating the probability of putative inhibitory cis elements in this area of the gene. Forskolin stimulated the expression of the reporter gene of deletion plasmids excepting the construct containing only the TATA box, and the highest activity also occurred with the -134 bp construct. TPA had no stimulatory effects on any of the constructs, and interestingly it slightly inhibited CAT activity. In contrast to TPA, staurosporine, an inhibitor of the PKC pathway, stimulated CAT activity. To conclude, the promoter region of the hamster CYP11B2 gene transfected in Y1 cells is responsive to forskolin, indicating that the gene is controlled by the PKA signaling pathway. Paradoxically, staurosporine, but not TPA, stimulates the promoter activity of the CYP11B2 gene, indicating that PKC might, at least in Y1 cells, act as a negative regulator on the aldosterone synthase promoter. Moreover, a new cis element was shown to exert a negative effect on basal as well as on stimulated activities of the hamster promoter CYP11B2 gene.

Characterization of the hamster CYP11B2 gene regulatory regions.

We have isolated a hamster CYP11B2 gene encoding the cytochrome P450 aldosterone synthase. In comparison with the CYP11B2 gene of other species, cis-elements named Ad1, Ad2, Ad3, and Ad4, were identified in the 5'-untranslated region of the hamster gene. Mouse adrenal tumor cells were transiently transfected with chimaeric reporter constructs, fused to the bacterial chloramphenicol acyltransferase (CAT) reporter gene, to study the regulation of expression of the hamster CYP11B2 gene. The highest basal expression was obtained with the -130 bp construct. Decreasing the length of the regulatory region of the CYP11B2 gene beyond that of -130 bp, to exclude Ad2 and Ad1 elements, resulted in successive decreases in CAT activity. Increasing the length of the regulatory region beyond that of -130 bp also resulted in a reduction of CAT activity, indicating the presence of inhibitory cis-elements in this area of the gene. Forskolin stimulated the CAT activity of all constructs, the highest of which occurred with the -130 bp construct, indicating that the gene is controlled by the PKA signalling pathway. TPA, however, had no stimulatory effects on any of these constructs. Staurosporine, an inhibitor of the PKC pathway, stimulated cells transfected with the different constructs in a similar manner as forskolin, indicating that PKC might act, at least in Y-1 cells, as a negative regulator on the hamster CYP11B2 promoter.