Cloning and characterization of NE-dlg: a novel human homolog of the Drosophila discs large (dlg) tumor suppressor protein interacts with the APC protein

We have cloned a cDNA for a novel human homolog of the Drosophila discs large (dig) tumor suppressor protein, termed NE-dlg (neuronal and endocrine dig). Northern blot analysis revealed that the gene is highly expressed in neuronal and endocrine tissues. Fluorescence in situ hybridization (FISH) and radiation hybrid mapping studies localized the NE-dlg gene to chromosome Xq13. We also found that the NE-dlg gene encoded a 100 kDa protein. Immunolocalization studies using an NE-dlg antibody showed that the protein tended to be expressed in non-proliferating cells, such as neurons, cells in Langerhans islets of the pancreas, myocytes of the heart muscles, and the prickle and functional layer cells of the esophageal epithelium. Proliferative cells, including various cultured cancer cell lines and basal cells in the esophageal epithelium, showed little expression of the NE-dlg protein. In addition, yeast two-hybrid screening and in vitro binding assays revealed that the NE-dlg interacted with the carboxyl-terminal region of the APC tumor suppressor protein. These data suggest that NE-dlg negatively regulates cell proliferation through its interaction with the APC protein.     

Coupling of calcium receptors to inositol phosphate and cyclic AMP generation in mammalian cells and Xenopus laevis oocytes and immunodetection of receptor protein by region-specific antipeptide antisera

We hypothesized that manganese superoxide dismutase (MnSOD), known to be induced in rat mesothelial cells by asbestos fibers, cytokines, and hyperoxia, may also be induced in asbestos-related pleural diseases such as mesothelioma. MnSOD was assessed in healthy human pleural mesothelium (n = 6), in biopsy samples of human pleural mesothelioma (n = 7), in transformed nonmalignant human mesothelial cells (Met5A), and in two human mesothelioma cell lines (M14K and M38K) established from the tumor tissue of mesothelioma patients. There was no MnSOD immunoreactivity in five of the six samples of healthy pleural mesothelium, whereas MnSOD immunoreactivity was high in the tumor cells in all the mesothelioma samples. Northern blotting, immunohistochemistry, Western blotting, and specific activity measurements showed lower MnSOD in the nonmalignant Met5A mesothelial cells than in the M14K and M38K mesothelioma cells. In additional experiments the mesothelial and mesothelioma cells were exposed to menadione, which generates superoxide intracellularly, and to epirubicin, a cytotoxic drug commonly used to treat mesothelioma. The M38K mesothelioma cells were most resistant to menadione and epirubicin when assessed by LDH release or by adenine nucleotide (ATP, ADP, and AMP) depletion. These same cells showed not only the highest MnSOD levels, but also the highest mRNA levels and activities of catalase, whereas glutathione peroxidase and glutathione reductase levels did not differ significantly. We conclude that MnSOD expression is low in healthy human pleural mesothelium and high in human malignant mesothelioma. The most resistant mesothelioma cells contained coordinated induction of MnSOD and catalase.     

Reconstitution of receptors and GTP-binding regulatory proteins (G proteins) in Sf9 cells. A direct evaluation of selectivity in receptor.G protein coupling

The selectivity in coupling of various receptors to GTP-binding regulatory proteins (G proteins) was examined directly by a novel assay entailing the use of proteins overexpressed in Spodoptera frugiperda (Sf9) cells. Activation of G proteins was monitored in membranes prepared from Sf9 cells co-expressing selected pairs of receptors and G proteins (i.e. alpha, beta1, and gamma2 subunits). Membranes were incubated with [35S]guanosine 5'-(3-O-thio)triphosphate (GTPgammaS) +/- an agonist, and the amount of radiolabel bound to the alpha subunit was quantitated following immunoprecipitation. When expressed without receptor (but with beta1gamma2), the G protein subunits alphaz, alpha12, and alpha13 did not bind appreciable levels of [35S]GTPgammaS, consistent with a minimal level of GDP/[35S]GTPgammaS exchange. In contrast, the subunits alphas and alphaq bound measurable levels of the nucleotide. Co-expression of the 5-hydroxytryptamine1A (5-HT1A) receptor promoted binding of [35S]GTPgammaS to alphaz but not to alpha12, alpha13, or alphas. Binding to alphaz was enhanced by inclusion of serotonin in the assay. Agonist activation of both thrombin and neurokinin-1 receptors promoted a modest increase in [35S]GTPgammaS binding to alphaz and more robust increases in binding to alphaq, alpha12, and alpha13. Binding of [35S]GTPgammaS to alphas was strongly enhanced only by the activated beta1-adrenergic receptor. Our data identify interactions of receptors and G proteins directly, without resort to measurements of effector activity, confirm the coupling of the 5-HT1A receptor to Gz and extend the list of receptors that interact with this unique G protein to the receptors for thrombin and substance P, imply constitutive activity for the 5-HT1A receptor, and demonstrate for the first time that the cloned receptors for thrombin and substance P activate G12 and G13.     

Ras-dependent mitogen-activated protein kinase activation by G protein-coupled receptors. Convergence of Gi- and Gq-mediated pathways on calcium/calmodulin, Pyk2, and Src kinase

Many receptors that couple to heterotrimeric guanine-nucleotide binding proteins (G proteins) have been shown to mediate rapid activation of the mitogen-activated protein kinases Erk1 and Erk2. In different cell types, the signaling pathways employed appear to be a function of the available repertoire of receptors, G proteins, and effectors. In HEK-293 cells, stimulation of either alpha1B- or alpha2A-adrenergic receptors (ARs) leads to rapid 5-10-fold increases in Erk1/2 phosphorylation. Phosphorylation of Erk1/2 in response to stimulation of the alpha2A-AR is effectively attenuated by pretreatment with pertussis toxin or by coexpression of a Gbetagamma subunit complex sequestrant peptide (betaARK1ct) and dominant-negative mutants of Ras (N17-Ras), mSOS1 (SOS-Pro), and Raf (DeltaN-Raf). Erk1/2 phosphorylation in response to alpha1B-AR stimulation is also attenuated by coexpression of N17-Ras, SOS-Pro, or DeltaN-Raf, but not by coexpression of betaARK1ct or by pretreatment with pertussis toxin. The alpha1B- and alpha2A-AR signals are both blocked by phospholipase C inhibition, intracellular Ca2+ chelation, and inhibitors of protein-tyrosine kinases. Overexpression of a dominant-negative mutant of c-Src or of the negative regulator of c-Src function, Csk, results in attenuation of the alpha1B-AR- and alpha2A-AR-mediated Erk1/2 signals. Chemical inhibitors of calmodulin, but not of PKC, and overexpression of a dominant-negative mutant of the protein-tyrosine kinase Pyk2 also attenuate mitogen-activated protein kinase phosphorylation after both alpha1B- and alpha2A-AR stimulation. Erk1/2 activation, then, proceeds via a common Ras-, calcium-, and tyrosine kinase-dependent pathway for both Gi- and Gq/11-coupled receptors. These results indicate that in HEK-293 cells, the Gbetagamma subunit-mediated alpha2A-AR- and the Galphaq/11-mediated alpha1B-AR-coupled Erk1/2 activation pathways converge at the level of phospholipase C. These data suggest that calcium-calmodulin plays a central role in the calcium-dependent regulation of tyrosine phosphorylation by G protein-coupled receptors in some systems.     

Cloning and expression of Xenopus HGF-like protein (HLP) and Ron/HLP receptor implicate their involvement in early neural development

Endoglin was first identified on a cell line derived from pre-B acute lymphoblastic leukemia. This 180-kDa homodimeric glycoprotein was then shown to be primarily expressed on endothelial cells and to bind the beta 1 and beta 3 isoforms of TGF-beta with high affinity. We now demonstrate that pre-B leukemic cells express a functional TGF-beta 1 receptor complex. The levels of mRNA for these receptors and for TGF-beta 1 were quantitated by PCR. HOON, G2, and NALM-6 cell lines express similar levels of mRNA for TGF-beta 1 and for TGF-beta receptor I (R-I) and receptor II (R-II). HOON cells express ten times more endoglin than G2 and NALM-6 cells, whereas all three cell lines have low levels of betaglycan relative to other cell types. The receptors were identified by affinity labeling with 125I-labeled TGF-beta 1, chemical cross-linking, and specific immunoprecipitation. Endoglin, R-II, and R-I were co-precipitated by Abs to either endoglin or R-II, indicating that these proteins are forming a receptor complex on leukemic cells; no betaglycan could be immunoprecipitated. The receptor complex is functional, as demonstrated by inhibition of proliferation of HOON cells (80%) and NALM-6 cells (60%) with 25 pM TGF-beta 1. Furthermore, the motility of HOON and NALM-6 cells on immobilized fibronectin, which appears to be alpha 4 beta 1-integrin mediated, was stimulated two- to threefold by TGF-beta 1. These results suggest that active TGF-beta 1 produced in the bone marrow microenvironment might stimulate the motility of normal pre-B cells and the peripheral dissemination of leukemic pre-B cells.     

Multitissue circadian expression of rat period homolog (rPer2) mRNA is governed by the mammalian circadian clock, the suprachiasmatic nucleus in the brain

The period (per) gene, controlling circadian rhythms in Drosophila, is expressed throughout the body in a circadian manner. A homolog of Drosophila per was isolated from rat and designated as rPer2. The rPER2 protein showed 39 and 95% amino acid identity with mPER1 and mPER2 (mouse homologs of per) proteins, respectively. A robust circadian fluctuation of rPer2 mRNA expression was discovered not only in the suprachiasmatic nucleus (SCN) of the hypothalamus but also in other tissues including eye, brain, heart, lung, spleen, liver, and kidney. Furthermore, the peripheral circadian expression of rPer2 mRNA was abolished in SCN-lesioned rats that showed behavioral arrhythmicity. These findings suggest that the multitissue circadian expression of rPer2 mRNA was governed by the mammalian brain clock SCN and also suggest that the rPer2 gene was involved in the circadian rhythm of locomotor behavior in mammals.     

Cardiovascular characterization of pyrrolo[2,1-d][1,5]benzothiazepine derivatives binding selectively to the peripheral-type benzodiazepine receptor (PBR): from dual PBR affinity and calcium antagonist activity to novel and selective calcium entry blockers

The synthesis and cardiovascular characterization of a series of novel pyrrolo[2,1-d][1,5]-benzothiazepine derivatives (54-68) are described. Selective peripheral-type benzodiazepine receptor (PBR) ligands, such as PK 11195 and Ro 5-4864, have recently been found to possess low but significant inhibitory activity of L-type calcium channels, and this property is implicated in the cardiovascular effects observed with these compounds. In functional studies both PK 11195 (1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxa mide) and Ro 5-4864 (4'-chlorodiazepam) did not display selectivity between cardiac and vascular tissue. Therefore, several 7-(acyloxy)-6-arylpyrrolo[2,1-d][1,5]benzothiazepines, potent and selective peripheral-type benzodiazepine receptor ligands recently developed by us (3, 7-20), were subjected to calcium channel receptor binding assay. Some of these compounds showed an unexpected potency in displacing the binding of [3H]nitrendipine from L-type calcium channels, much higher than that reported for PK 11195 and Ro 5-4864 and equal to or higher than that of reference calcium antagonists such as verapamil and (+)-cis-diltiazem. Specifically, in rat cortex homogenate, our prototypic PBR ligand 7-acetoxy-6-(p-methoxyphenyl)pyrrolo[2,1-d][1,5]benzothiazepine (3) showed an IC50 equal to 0.13 nM for inhibition of [3H]nitrendipine binding. Furthermore, in functional studies this compound displayed a clear-cut selectivity for cardiac over vascular tissue. Comparison of calcium antagonist activity on guinea pig aorta strips with the negative inotropic activity, determined by using isolated guinea pig left atria, revealed that 3 displayed higher selectivity than the reference (+)-cis-diltiazem. Thus, the pyrrolobenzothiazepine 3 might represent a new tool for characterizing the relationship between the PBR and cardiac function. Furthermore, we have also investigated the structural dependence of binding to PBR and L-type calcium channels, and this study allowed us to identify a new class of potent calcium channel blockers selective for cardiac over vascular tissue, with no affinity for PBR. A number of structure-activity relationship trends have been identified, and a possible explanation is advanced in order to account for the observed differences in selectivity. Three structural features, namely, (i) the saturation of the C(6)-C(7) double bond, with a consequent higher molecular flexibility, (ii) the presence of a substituent in the benzofused ring, and (iii) a basic side chain at C-10 of the pyrrolobenzothiazepine ring system, were found to be responsible for potent L-type calcium channel antagonism and clear-cut selectivity for cardiac over vascular tissue. Among the synthesized compounds the pyrrolobenzothiazepine 62 was found to be the most promising selective calcium channel blocker. Additionally, the molecular structure determination of the key intermediate 48 by X-ray diffraction, molecular modeling, and NMR analysis is reported.     

Evidence for inhibition by protein kinase A of receptor/G alpha(q)/phospholipase C (PLC) coupling by a mechanism not involving PLCbeta2

The effects of cAMP on the oxytocin-stimulated increase in phosphatidylinositide turnover and the possible pathways involved were investigated in a human myometrial cell line (PHM1-41) and in COS-M6 cells overexpressing the oxytocin receptor. Preincubation with chlorophenylthio-cAMP (CPT-cAMP), forskolin, or relaxin inhibited oxytocin-stimulated phosphatidylinositide turnover in PHM1-41 cells, and the inhibition was reversed by H-89, a relatively specific protein kinase A inhibitor. Both CPT-cAMP and transiently expressed protein kinase A catalytic subunit inhibited stimulation by oxytocin and carbachol of [3H]inositol 1,3,4-trisphosphate formation in COS-M6 cells expressing oxytocin or muscarinic M1 receptors, respectively. CPT-cAMP also inhibited phosphatidylinositide turnover stimulation by endothelin-1 in PHM1-41 cells, further demonstrating the generality of the cAMP-inhibitory mechanism. Since G betagamma activation of phospholipase Cbeta2 (PLCbeta2) is a suggested target of protein kinase A, the possibility that the oxytocin receptor couples to PLCbeta2 via G alpha(i)G betagamma activation was explored. Western blot analysis of PHM1-41 cells and COS-M6 cells detected PLCbeta1 and PLCbeta3, but not PLCbeta2. In PHM1-41 cells, pertussis toxin reduced the oxytocin-stimulated increase in [3H]inositol 1,3,4-trisphosphate by 53%, and this was reversed completely by H-89. Thus, the inhibitory effect of pertussis toxin may result from an indirect effect of cAMP elevation. These data suggest that receptor/G alpha(q)-coupled stimulation of PLCbeta1 or PLCbeta3 can be inhibited by cAMP through a phosphorylation mechanism involving protein kinase A that does not involve PLCbeta2. In smooth muscle, this mechanism could constitute potentially important cross-talk between pathways regulating contraction and relaxation.     

Coexpression of ligand-gated P2X and G protein-coupled P2Y receptors in smooth muscle. Preferential activation of P2Y receptors coupled to phospholipase C (PLC)-beta1 via Galphaq/11 and to PLC-beta3 via Gbetagammai3

P2 receptor subtypes and their signaling mechanisms were characterized in dispersed smooth muscle cells. UTP and ATP stimulated inositol 1,4,5-triphosphate formation, Ca2+ release, and contraction that were abolished by U-73122 and guanosine 5'-O-(3-thio)diphosphate, and partly inhibited (50-60%) by pertussis toxin (PTX). ATP analogs (adenosine 5'-(alpha, beta-methylene)triphosphate, adenosine 5'-(beta, gamma-methylene)triphosphate, and 2-methylthio-ATP) stimulated Ca2+ influx and contraction that were abolished by nifedipine and in Ca2+-free medium. Micromolar concentrations of ATP stimulated both Ca2+ influx and Ca2+ release. ATP and UTP activated Gq/11 and Gi3 in gastric and aortic smooth muscle and heart membranes, Gq/11 and Gi1 and/or Gi2 in liver membranes, and Go and Gi1-3 in brain membranes. Phosphoinositide hydrolysis stimulated by ATP and UTP was mediated concurrently by Galphaq/11-dependent activation of phospholipase (PL) C-beta1 and Gbetagammai3-dependent activation of PLC-beta3. Phosphoinositide hydrolysis was partially inhibited by PTX or by antibodies to Galphaq/11, Gbeta, PLC-beta1, or PLC-beta3, and completely inhibited by the following combinations (PLC-beta1 and PLC-beta3 antibodies; Galphaq/11 and Gbeta antibodies; PLC-beta1 and Gbeta antibodies; PTX with either PLC-beta1 or Galphaq/11 antibody). The pattern of responses implied that P2Y2 receptors in visceral, and probably vascular, smooth muscle are coupled to PLC-beta1 via Galphaq/11 and to PLC-beta3 via Gbetagammai3. These receptors co-exist with ligand-gated P2X1 receptors activated by ATP analogs and high levels of ATP.     

The equine herpesvirus 1 (EHV-1) UL3 gene, an ICP27 homolog, is necessary for full activation of gene expression directed by an EHV-1 late promoter

We have previously reported that the equine herpesvirus 1 (EHV-1) XbaI G restriction fragment (nucleotides 1436 to 7943 relative to the left terminus of the EHV-1 genome [Kentucky A strain]) is required in combination with the EHV-1 immediate-early (IE) gene to achieve significant activation of two representative EHV-1 late promoter-chloramphenicol acetyltransferase (CAT) recombinants in transient expression assays. In this report, we demonstrate that the XbaI G-encoded UL3 gene (an ICP27 homolog) provides a trans-acting factor which acts (in combination with the EHV-1 IE gene product) to increase reporter gene expression directed by an EHV-1 late promoter-CAT recombinant plasmid. We show that cloned copies of UL3 can successfully substitute for the XbaI G fragment in CAT assays and that stop codon insertion within the UL3 open reading frame inhibits the ability of UL3 to activate reporter gene expression in trans.     

Molecular cloning, expression, and characterization of a novel human serine/threonine protein phosphatase, PP7, that is homologous to Drosophila retinal degeneration C gene product (rdgC)

A novel serine/threonine protein phosphatase (PPase) designated PP7 was identified from cDNA produced from human retina RNA. PP7 has a molecular mass of approximately 75 kDa, and the deduced amino acid sequence of PP7 contains a phosphatase catalytic core domain that possesses all of the invariant motifs of the PP1, PP2A, PP2B, PP4, PP5, and PP6 gene family. However, PP7 has unique N- and C-terminal regions and shares < 35% identity with the other known PPases. The unique C-terminal region of PP7 contains multiple Ca2+ binding sites (i.e. EF-hand motifs). This region of PP7 is similar to the Drosophila retinal degeneration C gene product (rdgC), and PP7 and rdgC share 42.1% identity. Unlike the other known PPases, the expression of PP7 is not ubiquitous; PP7 was only detected in retina and retinal-derived Y-79 retinoblastoma cells. Expression of recombinant human PP7 in baculovirus-infected SF21 insect cells produces an active soluble enzyme that is capable of utilizing phosphohistone and p-nitrophenyl phosphate as substrates. The activity of recombinant PP7 is dependent on Mg2+ and is activated by calcium (IC50 approximately equal to 250 microM). PP7 is not affected by calmodulin and is insensitive to inhibition by okadaic acid, microcystin-LR, calyculin A, and cantharidin.     

One class of growth hormone (GH) receptor and binding protein messenger ribonucleic acid in rat liver, GHR1, is sexually dimorphic and regulated by GH

In the rat, alternatively spliced messenger RNA (mRNA) species encode GH receptor (GHR) and GH-binding protein (GHBP). Additionally, these mRNAs are alternatively spliced in the 5'-untranslated region, resulting in at least two classes of GHR and GHBP mRNA with distinct first exons and identical coding regions. These alternative first exons define two unique classes of GHR and GHBP mRNA (called GHR1 and GHR2). The GHR1 class of RNA is expressed only in the liver, is far more abundant in females than males, and is particularly abundant during pregnancy. GHR1 RNA is induced later in development than is GHR2. Additional classes of GHR and GHBP RNA may also exist. The genomic structure of the GHR1 first exon reveals a putative promotor region with no TATA box, CAAT box, or other sequence elements suggesting specific responses. An in vivo approach was used to investigate the regulation of GHR1 expression. In female rats, gonadectomy was found to reduce the percentage of steady state GHR1 RNA levels in the liver, whereas male castration resulted in an induction of GHR1 RNA. However, short-term treatment with estrogen or testosterone had little effect, suggesting that direct regulation of GHR1 expression may occur through effector(s) other than gonadal steroids. Hypophysectomy abolished GHR1 RNA in females. Treatment of hypophysectomized females and castrated males with GH by single injection did not significantly induce GHR1 RNA, but treatment by continuous infusion of GH did. Little change in non-GHR1 RNA levels was observed for each of these treatments. The results suggest that: 1) the sexual dimorphism observed in total GHR and GHBP RNA in rat liver is attributable to the sexually dimorphic expression of the GHR1 class of RNA; 2) the sexually dimorphic pattern of GH release in rats regulates the GHR1 class of RNA; 3) changes in GHR and GHBP expression observed on gonadectomy, hypophysectomy, GH treatment, and pregnancy are best attributed to GHR1 regulation; and 4) since GHR1 is liver specific, the observed increases in serum GHBP concentration in response to sex steroids, GH pattern, and pregnancy are likely to originate from the liver.