The yeast KRE5 gene encodes a probable endoplasmic reticulum protein required for (1----6)-beta-D-glucan synthesis and normal cell growth.

Yeast kre mutants define a pathway of cell wall (1----6)-beta-D-glucan synthesis, and mutants in genes KRE5 and KRE6 appear to interact early in such a pathway. We have cloned KRE5, and the sequence predicts the product to be a large, hydrophilic, secretory glycoprotein which contains the COOH-terminal endoplasmic reticulum retention signal, HDEL. Deletion of the KRE5 gene resulted in cells with aberrant morphology and extremely compromised growth. Suppressors to the KRE5 deletions arose at a frequency of 1 in 10(7) to 1 in 10(8) and permitted an analysis of deletions which were found to contain no alkali-insoluble (1----6)-beta-D-glucan. These results indicate a role for (1----6)-beta-D-glucan in normal cell growth and suggest a model for sequential assembly of (1----6)-beta-D-glucan in the yeast secretory pathway.

Sequestration and phosphorylation of the prostaglandin E2 EP4 receptor: dependence on the C-terminal tail.

The prostaglandin E2 (PGE2) EP4 subtype is one of four prostanoid receptors that use PGE2 as the preferred ligand. We have investigated the agonist-mediated regulation of EP4 using a multifaceted approach. Short-term (30 min) agonist challenge of recombinant EP4 expressed in human embryonic kidney 293 cells (EP4-HEK293 cells) with PGE2 (1 microM) resulted in the desensitization of intracellular cyclic AMP (cAMP) accumulation and a reduction in cell surface [3H]PGE2 specific binding sites. These events correlated with sequestration of EP4, as visualized by immunofluorescence confocal microscopy and phosphorylation, as shown by [32P]orthophosphate labeling of the receptor. Stimulation of protein kinase A activity in EP4-HEK293 cells (10 microM forskolin or 1 mM 8-bromo-cAMP) did not induce EP4 desensitization, sequestration, or phosphorylation. In contrast, stimulation of protein kinase C activity (100 nM phorbol 12-myristate 13-acetate) attenuated PGE2-induced adenylyl cyclase activity and increased EP4 phosphorylation, but did not induce sequestration or a reduction in [3H]PGE2 specific binding sites. EP4 receptors containing a third intracellular loop deletion [EP4 (del. 215-263)] or a carboxyl-terminal tail truncation [EP4 (del. 355)] of EP4 were used to demonstrate that the C-terminal tail governs sequestration as well as phosphorylation of the receptor.

Photoaffinity labelling and radiation inactivation of the leukotriene B4 receptor in human myeloid cells.

The leukotriene (LT) B4 receptor has been characterized in the human monocyte leukemia THP-1 cell line. Scatchard analysis of [3H]LTB4 specific binding to THP-1 cell membranes revealed a single population of high affinity (KD = 56 pM) and saturable (2000 receptors/cell) binding sites. [3H]LTB4 specific binding was enhanced by divalent cations, but inhibited by both monovalent cations and a non-hydrolysable GTP analogue. Treatment with GTP analogue resulted in a concentration-dependent reduction in the number of high affinity binding sites, accompanied by the appearance of an equal number of binding sites of lower affinity (KD = 1250 pM). In contrast, Scatchard analysis with human polymorphonuclear leukocyte (PMN) membranes consistently revealed two populations of LTB4 receptors (KD = 48 pM and 270 pM). Treatment with GTP analogue, however, converted all these detectable binding sites to the lower affinity state. These data suggest that the LTB4 receptor in both THP-1 cell and PMN membranes exists in interconverting affinity states modulated by G-protein coupling. The similarity between the LTB4 receptors present in these two cell types was also substantiated by target-size analysis by radiation inactivation, which estimated a comparable molecular mass of 56.5 kDa and 52.8 kDa for the THP-1 cell and PMN LTB4 receptors, respectively. Finally, the presence of a single LTB4 receptor in PMN was demonstrated by direct photolabelling. Irradiation of frozen [3H]LTB4 equilibrium binding assay incubations resulted in complete photolysis of [3H]LTB4. Subsequent resolution of the tritiated PMN proteins by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) revealed one major radioactive peak migrating with an apparent molecular weight of 61,000. This peak was identified as the LTB4 receptor since radiolabelling could be completely inhibited by the presence of excess unlabelled LTB4 or the LTB4-receptor antagonist, L-662,328. Photolabelling was also partially inhibited by pretreatment with GTP analogue, consistent with G-protein uncoupling reagents reducing receptor affinity without complete inhibition. In summary, the LTB4 receptor identified in human myeloid cells is a G-protein coupled receptor with interconvertible high and low affinity states, having a molecular mass of 53-61 kDa.

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].

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.

Congenital deficiency of a 20-kDa subunit of mitochondrial complex I in fibroblasts.

The first component of the mitochondrial electron-transport chain is especially complex, consisting of 19 nuclear and seven mitochondrion-encoded subunits. Accordingly, a wide range of clinical manifestations are produced by the various mutations occurring in human populations. In this study, we analyze the subunit structure of complex I in fibroblasts from two patients who have distinct clinical phenotypes associated with complex I deficiency. The first patient died in the second week of life from overwhelming lactic acidosis. Severe complex I deficiency was evident in her fibroblasts, since alanine oxidation was markedly reduced whereas succinate oxidation was normal. Absence of a 20-kDa subunit was demonstrable when newly synthesized proteins were immunoprecipitated from pulse-labeled fibroblasts by anti-complex I antibody. Disordered assembly or decreased stability of the complex was suggested by deficiency of multiple subunits on Western immunoblots. The second patient exhibited a milder clinical phenotype, characterized by moderate lactic acidosis and developmental delay in childhood and by onset of seizures at 8 years of age. Oxidation studies demonstrated expression of the complex I deficiency in fibroblasts, but no subunit abnormalities were detected by immunoprecipitation or Western immunoblotting. This report demonstrates the utility of cultured fibroblasts in studying mutations affecting synthesis and assembly of complex I.

Deficiency of complex III of the mitochondrial respiratory chain in a patient with facioscapulohumeral disease.

Facioscapulohumeral disease (FSHD), an inherited neuromuscular disorder, is characterized by progressive wasting of specific muscle groups, particularly the proximal musculature of the upper limbs; the primary defect in this disorder is unknown. We studied a patient with FSHD to determine whether the mitochondrial respiratory chain was functionally abnormal. Muscle biopsy revealed fiber atrophy with patchy staining for oxidative enzymes. Electron microscopy of a liver section showed many enlarged mitochondria with paracrystalline inclusions. Decreased oxidation of the respiratory substrates-alanine and succinate-in skin fibroblasts suggested a deficiency of complex III of the electron-transport chain; cytochrome c oxidase activity (complex IV) was in the normal range. Biochemical analysis of liver supported the fibroblast data, since succinate oxidase activity (electron-transport activity through complexes II-IV) was reduced, whereas complex IV activity was normal. Furthermore, analysis of the cytochrome spectrum in liver revealed typical peaks for cytochromes cc1 and aa3, whereas cytochrome b (a component of complex III) was undetectable. Southern blot analysis of fibroblast mtDNA revealed no major deletions or rearrangements. Our study provides the first documentation of a specific enzyme-complex deficiency associated with FSHD.

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.

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 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.

Activation of the human peripheral cannabinoid receptor results in inhibition of adenylyl cyclase.

The human peripheral cannabinoid (CB2) receptor has been cloned by reverse transcription-polymerase chain reaction from human spleen RNA and expressed, to study both ligand binding characteristics and signal transduction pathways. Receptor binding assays used the aminoalkylindole [3H]Win 55212-2 and membranes from transiently transfected COS-M6 cells. Saturation analysis showed that [3H]Win 55212-2 specific binding to the CB2 receptor was of high affinity, with a Kd of 2.1 +/- 0.2 nM (four experiments), and a high level of expression was attained, with a maximal number of saturable binding sites of 24.1 +/- 4.4 pmol/mg of protein (four experiments). The rates of association and dissociation for [3H]Win 55212-2 specific binding were both rapid when measured at 30 degrees. [3H]Win 55212-2 specific binding to the CB2 receptor was moderately enhanced by divalent and monovalent cations but was only slightly inhibited by guanosine-5'-O-(3-thio)-triphosphate. Competition for [3H]Win 55212-2 specific binding to the CB2 receptor was stereoselective, with the following rank order of potency for the more active stereoisomers: HU-210 > (-)-CP-55940 approximately Win 55212-2 >> (-)delta 9-THC > anandamide. The signaling pathway of the human CB2 receptor was investigated in a CB2-CHO-K1 stable cell line. CB2 receptor activation by cannabinoid agonists inhibited forskolin-induced cAMP production in a concentration-dependent and stereoselective manner but did not increase either cAMP production or Ca2+ mobilization in fura-2/acetoxymethyl ester-loaded CB2-CHO-K1 cells. The CB2 receptor-mediated inhibition of forskolin-induced cAMP production was abolished by pretreatment of the cells with 10 ng/ml pertussis toxin. These results demonstrate that the CB2 receptor is functionally coupled to inhibition of adenylyl cyclase activity via a pertussis toxin-sensitive G protein.

Prostaglandin receptor EP(4) mediates the bone anabolic effects of PGE(2).

Prostaglandin (PG) E(2) is a potent inducer of cortical and trabecular bone formation in humans and animals. Although the bone anabolic action of PGE(2) is well documented, the cellular and molecular mechanisms that mediate this effect remain unclear. This study was undertaken to examine the effect of pharmacological inactivation of the prostanoid receptor EP(4), one of the PGE(2) receptors, on PGE(2)-induced bone formation in vivo. We first determined the ability of EP(4)A, an EP(4)-selective ligand, to act as an antagonist. PGE(2) increases intracellular cAMP and suppresses apoptosis in the RP-1 periosteal cell line. Both effects were reversed by EP(4)A, suggesting that EP(4)A acts as an EP(4) antagonist in the cells at concentrations consistent with its in vitro binding to EP(4). We then examined the effect of EP(4) on bone formation induced by PGE(2) in young rats. Five- to 6-week-old rats were treated with PGE(2) (6 mg/kg/day) in the presence or absence of EP(4)A (10 mg/kg/day) for 12 days. We found that treatment with EP(4)A suppresses the increase in trabecular bone volume induced by PGE(2). This effect is accompanied by a suppression of bone formation indices: serum osteocalcin, extent of labeled surface, and extent of trabecular number, suggesting that the reduction in bone volume is due most likely to decreased bone formation. The pharmacological evidence presented here provides strong support for the hypothesis that the bone anabolic effect of PGE(2) in rats is mediated by the EP(4) receptor.

Structure-activity relationship of biaryl acylsulfonamide analogues on the human EP(3) prostanoid receptor.

Potent and selective ligands for the human EP3 prostanoid receptor are described. Biaryl compounds bearing a tethered ortho substituted acidic moiety were identified as potent EP3 antagonists based on the SAR described herein. The binding affinity of key compounds on all eight human prostanoid receptors is reported.