Foreign glycoproteins expressed from recombinant vesicular stomatitis viruses are incorporated efficiently into virus particles.

In a previous study we demonstrated that vesicular stomatitis virus (VSV) can be used as a vector to express a soluble protein in mammalian cells. Here we have generated VSV recombinants that express four different membrane proteins: the cellular CD4 protein, a CD4-G hybrid protein containing the ectodomain of CD4 and the transmembrane and cytoplasmic tail of the VSV glycoprotein (G), the measles virus hemagglutinin, or the measles virus fusion protein. The proteins were expressed at levels ranging from 23-62% that of VSV G protein and all were transported to the cell surface. In addition we found that all four proteins were incorporated into the membrane envelope of VSV along with the VSV G protein. The levels of incorporation of these proteins varied from 6-31% of that observed for VSV G. These results suggest that many different membrane proteins may be co-incorporated quite efficiently with VSV G protein into budding VSV virus particles and that specific signals are not required for this co-incorporation process. In fact, the CD4-G protein was incorporated with the same efficiency as wild type CD4. Electron microscopy of virions containing CD4 revealed that the CD4 molecules were dispersed throughout the virion envelope among the trimeric viral spike glycoproteins. The recombinant VSV-CD4 virus particles were about 18% longer than wild type virions, reflecting the additional length of the helical nucleocapsid containing the extra gene. Recombinant VSVs carrying foreign antigens on the surface of the virus particle may be useful for viral targeting, membrane protein purification, and for generation of immune responses.

Membrane fusion protein synexin (annexin VII) as a Ca2+/GTP sensor in exocytotic secretion.

Exocytotic membrane fusion and secretion are promoted by the concerted action of GTP and Ca2+, although the precise site(s) of action in the process are not presently known. However, the calcium-dependent membrane fusion reaction driven by synexin (annexin VII) is an in vitro model for this process, which we have now found to be further activated by GTP. The mechanism of fusion activation depends on the unique ability of synexin to bind and hydrolyze GTP in a calcium-dependent manner, both in vitro and in vivo in streptolysin O-permeabilized chromaffin cells. The required [Ca2+] for GTP binding by synexin is in the range of 50-200 microM, which is known to occur at exocytotic sites in chromaffin cells, neurons, and other cell types. Previous immunolocalization studies place synexin at exocytotic sites in chromaffin cells, and we conclude that synexin is an atypical G protein that may be responsible for both detecting and mediating the Ca2+/GTP signal for exocytotic membrane fusion.

The weaver mutation of GIRK2 results in a loss of inwardly rectifying K+ current in cerebellar granule cells.

The weaver mutation in mice results in a severe ataxia that is attributable to the degeneration of cerebellar granule cells and dopaminergic neurons in the substantia nigra. Recent genetic studies indicate that the GIRK2 gene is altered in weaver. This gene codes for a G-protein-activated, inwardly rectifying K+ channel protein (8). The mutation results in a single amino acid substitution (glycine-->serine) in the pore-forming H5 region of the channel. The functional consequences of this mutation appear to depend upon the co-expression of other GIRK subunits--leading to either a gain or loss of function. Here, we show that G-protein-activated inwardly rectifying K+ currents are significantly reduced in cerebellar granule cells from animals carrying the mutant allele. The reduction is most pronounced in homozygous neurons. These findings suggest that the death of neurons in weaver is attributable to the loss of GIRK2-mediated currents, not to the expression of a nonspecific cation current.

Primary structure and tissue distribution of the orphanin FQ precursor.

The heptadecapeptide orphanin FQ (OFQ) is a recently discovered neuropeptide that exhibits structural features reminiscent of the opioid peptides and that is an endogenous ligand to a G protein-coupled receptor sequentially related to the opioid receptors. We have cloned both the human and rat cDNAs encoding the OFQ precursor proteins, to investigate whether the sequence relationships existing between the opioid and OFQ systems are also found at the polypeptide precursor level, in particular whether the OFQ precursor would encode several bioactive peptides as do the opioid precursors, and to study the regional distribution of OFQ sites of synthesis. The entire precursor protein displays structural homology to the opioid peptide precursors, especially preprodynorphin and preproenkephalin. The predicted amino acid sequence of the OFQ precursor contains a putative signal peptide and one copy of the OFQ sequence flanked by pairs of basic amino acid residues. Carboxyl-terminal to the OFQ sequence, the human and rat precursors contain a stretch of 28 amino acids that is 100% conserved and thus may encode novel bioactive peptides. Two peptides derived from this stretch were synthesized but were found to be unable to activate the OFQ receptor, suggesting that if they are produced in vivo, these peptides would likely recognize receptors different from the OFQ receptor. To begin analyzing the sites of OFQ mRNA synthesis, Northern analysis of human and rat tissues were carried out and showed that the OFQ precursor mRNA is mainly expressed in the brain. In situ hybridization of rat brain slices demonstrated a regional distribution pattern of the OFQ precursor mRNA, which is distinct from that of the opioid peptide precursors. These data confirm that the OFQ system differs from the opioid system at the molecular level, although the OFQ and opioid precursors may have arisen from a common ancestral gene.

Activation of muscarinic acetylcholine receptors via their allosteric binding sites.

Ligands that bind to the allosteric-binding sites on muscarinic acetylcholine receptors alter the conformation of the classical-binding sites of these receptors and either diminish or increase their affinity for muscarinic agonists and classical antagonists. It is not known whether the resulting conformational change also affects the interaction between the receptors and the G proteins. We have now found that the muscarinic receptor allosteric modulators alcuronium, gallamine, and strychnine (acting in the absence of an agonist) alter the synthesis of cAMP in Chinese hamster ovary (CHO) cells expressing the M2 or the M4 subtype of muscarinic receptors in the same direction as the agonist carbachol. In addition, most of their effects on the production of inositol phosphates in CHO cells expressing the M1 or the M3 muscarinic receptor subtypes are also similar to (although much weaker than) those of carbachol. The agonist-like effects of the allosteric modulators are not observed in CHO cells that have not been transfected with the gene for any of the subtypes of muscarinic receptors. The effects of alcuronium on the formation of cAMP and inositol phosphates are not prevented by the classical muscarinic antagonist quinuclidinyl benzilate. These observations demonstrate for the first time that the G protein-mediated functional responses of muscarinic receptors can be evoked not only from their classical, but also from their allosteric, binding sites. This represents a new mechanism of receptor activation.

The yeast and mammalian isoforms of phosphatidylinositol transfer protein can all restore phospholipase C-mediated inositol lipid signaling in cytosol-depleted RBL-2H3 and HL-60 cells.

The mammalian phosphatidylinositol transfer proteins (PITP) and the yeast Saccharomyces cerevisiae PITP (SEC14p) that show no sequence homology both catalyze exchange of phosphatidylinositol (PI) between membranes compartments in vitro. In HL-60 cells where the cytosolic proteins are depleted by permeabilization, exogenously added PITPalpha is required to restore G protein-mediated phospholipase Cbeta (PLCbeta) signaling. Recently, a second mammalian PITPbeta form has been described that shows 77% identity to rat PITPalpha. We have examined the ability of the two mammalian PITPs and SEC14p to restore PLC-mediated signaling in cytosol-depleted HL-60 and RBL-2H3 cells. Both PITPalpha and PITPbeta isoforms as well as SEC14p restore G protein-mediated PLCbeta signaling with a similar potency. In RBL-2H3 cells, crosslinking of the IgE receptor by antigen stimulates inositol lipid hydrolysis by tyrosine phosphorylation of PLCgamma1. Permeabilization of RBL cells leads to loss of PLCgamma1 as well as PITP into the extracellular medium and this coincides with loss of antigen-stimulated lipid hydrolysis. Both PLCgamma1 and PITP were required to restore inositol lipid signaling. We conclude that (i) because the PI binding/transfer activities of PITP/SEC14p is the common feature shared by all three transfer proteins, it must be the relevant activity that determines their abilities to restore inositol lipid-mediated signaling and (ii) PITP is a general requirement for inositol lipid hydrolysis regardless of how and which isoform of PLC is activated by the appropriate agonist.

Ras2 signals via the Cdc42/Ste20/mitogen-activated protein kinase module to induce filamentous growth in Saccharomyces cerevisiae.

RAS2val19, a dominant activated form of Saccharomyces cerevisiae Ras2, stimulates both filamentous growth and expression of a transcriptional reporter FG(TyA)::lacZ but does not induce the mating pathway reporter FUS1::lacZ. This induction depends upon elements of the conserved mitogen-activated protein kinase (MAPK) pathway that is required for both filamentous growth and mating, two distinct morphogenetic events. Full induction requires Ste20 (homolog of mammalian p65PAK protein kinases), Ste11 [an MEK kinase (MEKK) or MAPK kinase (MEK) kinase], Ste7 (MEK or MAPK kinase), and the transcription factor Ste12. Moreover, the Rho family protein Cdc42, a conserved morphogenetic G protein, is also a potent regulator of filamentous growth and FG(TyA)::lacZ expression in S. cerevisiae. Stimulation of both filamentous growth and FG(TyA)::lacZ by Cdc42 depends upon Ste20. In addition, dominant negative CDC42Ala118 blocks RAS2val19 activation, placing Cdc42 downstream of Ras2. Our results suggest that filamentous growth in budding yeast is regulated by an evolutionarily conserved signaling pathway that controls cell morphology.

ARD1, a 64-kDa bifunctional protein containing an 18-kDa GTP-binding ADP-ribosylation factor domain and a 46-kDa GTPase-activating domain.

The alpha subunits of the heterotrimeric guanine nucleotide-binding proteins (G proteins) hydrolyze GTP at a rate significantly higher than do most members of the Ras family of approximatelly 20-kDa GTP-binding proteins, which depend on a GTPase-activating protein (GAP) for acceleration of GTP hydrolysis. It has been demonstrated that an inserted domain in the G-protein alpha subunit, not present in the much smaller Ras-like proteins, is responsible for this difference [Markby, D. W., Onrust, R. & Bourne, H. R. (1993) Science 262, 1895-1900]. We report here that ARD1, a 64-kDa protein with an 18-kDa carboxyl-terminal ADP-ribosylation factor (ARF) domain, exhibited significant GTPase activity, whereas the ARF domain, expressed as a recombinant protein in Escherichia coli, did not. Addition of the 46-kDa amino-terminal extension (similarly synthesized in E. coli) to the GTP-binding ARF-domain of ARD1 enhanced GTPase activity and inhibited GDP dissociation. The kinetic properties of mixtures of the ARF and non-ARF domains were similar to those of an intact recombinant ARD1. Physical association of the two proteins was demonstrated directly by gel filtration and by using the immobilized non-ARF domain. Thus, like the alpha subunits of heterotrimeric G proteins, ARD1 appears to consist of two domains that interact to regulate the biological activity of the protein.

Detection of membrane-bound HLA-G translated products with a specific monoclonal antibody.

A monomorphic anti-HLA-G monoclonal antibody (mAb) was obtained by immunization of HLA-B27/human beta 2-microglobulin double-transgenic mice with transfected murine L cells expressing both HLA-G and human beta 2-microglobulin. This mAb, designated BFL.1, specifically recognizes, by flow cytometry analysis, the immunizing HLA-G-expressing cells, whereas it does not bind to parental untransfected or to HLA-B7- and HLA-A3-transfected L cells, suggesting that it distinguishes between classical HLA-A and -B and nonclassical HLA-G class I molecules. This was further assessed by the absence of BFL.1 reactivity with a number of human cell lines known to express classical HLA class I proteins. In addition, we showed that the BFL.1 mAb also labels HLA-G-naturally-expressing JEG-3 and HLA-G-transfected JAR human choriocarcinoma cell lines as well as a subpopulation of first-trimester placental cytotrophoblast cells. Further biochemical studies were performed by immunoprecipitation of biotinylated membrane lysates: BFL.1, like the monomorphic W6/32 mAb, immunoprecipitated a 39-kDa protein in HLA-G-expressing cell lines, a size corresponding to the predicted full-length HLA-G1 isoform. However, in contrast to W6/32, which immunoprecipitates both classical and nonclassical HLA class I heavy chains, BFL.1 mAb does not recognize the class Ia products. Such a mAb should be a useful tool for analysis of HLA-G protein expression in various normal and pathological human tissues and for determination of the function(s) of translated HLA-G products.

Genetic dissection of Alzheimer disease, a heterogeneous disorder.

The genetics of Alzheimer disease (AD) are complex and not completely understood. Mutations in the amyloid precursor protein gene (APP) can cause early-onset autosomal dominant AD. In vitro studies indicate that cells expressing mutant APPs overproduce pathogenic forms of the A beta peptide, the major component of AD amyloid. However, mutations in the APP gene are responsible for 5% or less of all early-onset familial AD. A locus on chromosome 14 is responsible for AD in other early-onset AD families and represents the most severe form of the disease in terms of age of onset and rate of decline. Attempts to identify the AD3 gene by positional cloning methods are underway. At least one additional early-onset AD locus remains to be located. In late-onset AD, the apolipoprotein E gene allele epsilon 4 is a risk factor for AD. This allele appears to act as a dose-dependent age-of-onset modifier. The epsilon 2 allele of this gene may be protective. Other late-onset susceptibility factors remain to be identified.

Molecular characterization of a second melatonin receptor expressed in human retina and brain: the Mel1b melatonin receptor.

A G protein-coupled receptor for the pineal hormone melatonin was recently cloned from mammals and designated the Mel1a melatonin receptor. We now report the cloning of a second G protein-coupled melatonin receptor from humans and designate it the Mel1b melatonin receptor. The Mel1b receptor cDNA encodes a protein of 362 amino acids that is 60% identical at the amino acid level to the human Mel1a receptor. Transient expression of the Mel1b receptor in COS-1 cells results in high-affinity 2-[125I]iodomelatonin binding (Kd = 160 +/- 30 pM). In addition, the rank order of inhibition of specific 2-[125I]iodomelatonin binding by eight ligands is similar to that exhibited by the Mel1a melatonin receptor. Functional studies of NIH 3T3 cells stably expressing the Mel1b melatonin receptor indicate that it is coupled to inhibition of adenylyl cyclase. Comparative reverse transcription PCR shows that the Mel1b melatonin receptor is expressed in retina and, to a lesser extent, brain. PCR analysis of human-rodent somatic cell hybrids maps the Mel1b receptor gene (MTNR1B) to human chromosome 11q21-22. The Mel1b melatonin receptor may mediate the reported actions of melatonin in retina and participate in some of the neurobiological effects of melatonin in mammals.

In vitro autoradiography of receptor-activated G proteins in rat brain by agonist-stimulated guanylyl 5'-[gamma-[35S]thio]-triphosphate binding.

Agonists stimulate guanylyl 5'-[gamma-[35S]thio]-triphosphate (GTP[gamma-35S]) binding to receptor-coupled guanine nucleotide binding protein (G proteins) in cell membranes as revealed in the presence of excess GDP. We now report that this reaction can be used to neuroanatomically localize receptor-activated G proteins in brain sections by in vitro autoradiography of GTP[gamma-35S] binding. Using the mu opioid-selective peptide [D-Ala2,N-MePhe4,Gly5-ol]enkephalin (DAMGO) as an agonist in rat brain sections and isolated thalamic membranes, agonist stimulation of GTP[gamma-35S] binding required the presence of excess GDP (1-2 mM GDP in sections vs. 10-30 microM GDP in membranes) to decrease basal G-protein activity and reveal agonist-stimulated GTP[gamma-35S] binding. Similar concentrations of DAMGO were required to stimulate GTP[gamma-35S] binding in sections and membranes. To demonstrate the general applicability of the technique, agonist-stimulated GTP[gamma-35S] binding in tissue sections was assessed with agonists for the mu opioid (DAMGO), cannabinoid (WIN 55212-2), and gamma-aminobutyric acid type B (baclofen) receptors. For opioid and cannabinoid receptors, agonist stimulation of GTP[gamma-35S] binding was blocked by incubation with agonists in the presence of the appropriate antagonists (naloxone for mu opioid and SR-141716A for cannabinoid), thus demonstrating that the effect was specifically receptor mediated. The anatomical distribution of agonist-stimulated GTP[gamma-35S] binding qualitatively paralleled receptor distribution as determined by receptor binding autoradiography. However, quantitative differences suggest that variations in coupling efficiency may exist between different receptors in various brain regions. This technique provides a method of functional neuroanatomy that identifies changes in the activation of G proteins by specific receptors.

Physical mapping, cloning, and identification of genes within a 500-kb region containing BRCA1.

BRCA1 is a breast/ovarian cancer susceptibility gene on human chromosome 17q21. We describe a complete and detailed physical map of a 500-kb region of genomic DNA containing the BRCA1 gene and the partial cloning in phage P1 artificial chromosomes. Approximately 70 exons were isolated from this region, 11 of which were components of the BRCA1 gene. Analysis of the other exons revealed a rho-related G protein and the interferon-induced leucine-zipper protein IFP-35.

Micrornas 9a, 9b, 9c And 315 Regulate Expression Of A Reporter For The Neuronal Microtubule-Associated Protein Futsch/map1b

Fragile X syndrome (FXS) is the most common form of inherited mental retardation in humans. FXS is caused by loss of the Fragile X Mental Retardation Protein (FMRP), an important regulator of neuronal mRNA translation. Patients with FXS display cognitive deficits including memory problems. Protein synthesis-dependent long-term changes in synaptic plasticity are involved in the establishment and maintenance of long-term memory. One prevalent theory of FXS pathology predicts that FMRP is required to negatively regulate the translation of important mRNAs at the synapse. We are investigating microRNAs (miRNAs) as a potential regulator of synaptic FMRP-regulated mRNAs that have previously been described as being crucial to the process of synaptic plasticity. The general hypothesis underlying this thesis is that FMRP may negatively regulate the expression of futsch (the Drosophila homologue of the microtubule-associated protein gene MAP1B) via the miRNA pathway. The first step we took in testing this hypothesis was to confirm that futsch is subject to miRNA-mediated translational control. Using in silico target analysis, we predicted that several neuronally expressed miRNAs target the futsch mRNA 3'UTR and repress expression of Futsch protein. Then, using an in vitro luciferase reporter system, we showed that miR-315 and members of the miR-9 family selectively down-regulated futsch reporter translation. We have confirmed by site- directed mutagenesis that the miRNA interaction with the futsch 3'UTR is specific to the miRNA seed region binding site. Interestingly, reduction of FMRP levels by RNAi had no effect on futsch 3'UTR reporter expression. Together, these data suggest regulation of futsch expression by the miRNA pathway might be independent of FMRP activity. However, additional experiments need to be completed to confirm these preliminary results.

A synthetic biology-based device prevents liver injury in mice

BACKGROUND & AIMS The liver performs a panoply of complex activities coordinating metabolic, immunologic and detoxification processes. Despite the liver's robustness and unique self-regeneration capacity, viral infection, autoimmune disorders, fatty liver disease, alcohol abuse and drug-induced hepatotoxicity contribute to the increasing prevalence of liver failure. Liver injuries impair the clearance of bile acids from the hepatic portal vein which leads to their spill over into the peripheral circulation where they activate the G-protein-coupled bile acid receptor TGR5 to initiate a variety of hepatoprotective processes. METHODS By functionally linking activation of ectopically expressed TGR5 to an artificial promoter controlling transcription of the hepatocyte growth factor (HGF), we created a closed-loop synthetic signalling network that coordinated liver injury-associated serum bile acid levels to expression of HGF in a self-sufficient, reversible and dose-dependent manner. RESULTS After implantation of genetically engineered human cells inside auto-vascularizing, immunoprotective and clinically validated alginate-poly-(L-lysine)-alginate beads into mice, the liver-protection device detected pathologic serum bile acid levels and produced therapeutic HGF levels that protected the animals from acute drug-induced liver failure. CONCLUSIONS Genetically engineered cells containing theranostic gene circuits that dynamically interface with host metabolism may provide novel opportunities for preventive, acute and chronic healthcare. LAY SUMMARY Liver diseases leading to organ failure may go unnoticed as they do not trigger any symptoms or significant discomfort. We have designed a synthetic gene circuit that senses excessive bile acid levels associated with liver injuries and automatically produces a therapeutic protein in response. When integrated into mammalian cells and implanted into mice, the circuit detects the onset of liver injuries and coordinates the production of a protein pharmaceutical which prevents liver damage.