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The RNA-binding protein CsrA (carbon storage regulator) is a new kind of global regulator, which facilitates specific mRNA decay. A recombinant CsrA protein containing a metal-binding affinity tag (CsrA-H6) was purified to homogeneity and authenticated by N-terminal sequencing, matrix-assisted laser desorption/ionization time of flight mass spectrometry, and other studies. This protein was entirely contained within a globular complex of approximately 18 CsrA-H6 subunits and a single approximately 350-nucleotide RNA, CsrB. cDNA cloning and nucleotide sequencing revealed that the csrB gene is located downstream from syd in the 64-min region of the Escherichia coli K-12 genome and contains no open reading frames. The purified CsrA-CsrB ribonucleoprotein complex was active in regulating glg (glycogen biosynthesis) gene expression in vitro, as was the RNA-free form of the CsrA protein. Overexpression of csrB enhanced glycogen accumulation in E. coli, a stationary phase process that is repressed by CsrA. Thus, CsrB RNA is a second component of the Csr system, which binds to CsrA and antagonizes its effects on gene expression. A model for regulatory interactions in Csr is presented, which also explains previous observations on the homologous system in Erwinia carotovora. A highly repeated nucleotide sequence located within predicted stem-loops and other single-stranded regions of CsrB, CAGGA(U/A/C)G, is a plausible CsrA-binding element.

The carbon storage regulator gene, csrA, modulates the expression of genes in the glycogen biosynthesis and gluconeogenesis pathways in Escherichia coli and has been cloned, mapped and sequenced (T. Romeo, M. Gong, M.Y. Liu, and A.M. Brun-Zinkernagel, J. Bacteriol. 175:4744-4755, 1993; T. Romeo and M. Gong, J. Bacteriol. 175:5740-5741, 1993). We have now conducted experiments that begin to elucidate a unique mechanism for csrA-mediated regulation. Steady-state levels of glgC transcripts, encoding ADP-glucose pyrophosphorylase, were elevated by up to sixfold in a csrA::kanR mutant and were less than 6.5% of wild-type levels in a strain containing pCSR10 (csrA+), as shown by S1 nuclease protection analysis. The rate of chemical decay of these transcripts after adding rifampin to cultures was dramatically reduced by the csrA::kanR mutation. Deletion studies of a glgC'-'lacZ translational fusion demonstrated that the region surrounding the initiation codon was important for csrA-mediated regulation and indicated that neither csrA-mediated regulation nor stationary phase induction of glgC expression originates at the level of transcript initiation. Cell-free (S-200) extracts containing the CsrA gene product potently and specifically inhibited the in vitro transcription-translation of glg genes. The deduced amino acid sequence of CsrA was found to contain the KH motif, which characterizes a subset of diverse RNA-binding proteins. The results indicate that CsrA accelerates net 5'-to-3' degradation of glg transcripts, potentially through selective RNA binding.

Guanine nucleotide exchange factors (GEFs) catalyze exchange of GDP for GTP by stabilizing the nucleotide-free state of the small GTPases through their Dbl homology/pleckstrin homology (DH.PH) domains. Unconventionally, PDZ-RhoGEF (PRG), a member of the RGS-RhoGEFs, binds tightly to both nucleotide-free and activated RhoA (RhoA.GTP). We have characterized the interaction between PRG and activated RhoA and determined the structure of the PRG-DH.PH-RhoA.GTPgammaS (guanosine 5'-O-[gamma-thio]triphosphate) complex. The interface bears striking similarity to a GTPase-effector interface and involves the switch regions in RhoA and a hydrophobic patch in PRG-PH that is conserved among all Lbc RhoGEFs. The two surfaces that bind activated and nucleotide-free RhoA on PRG-DH.PH do not overlap, and a ternary complex of PRG-DH.PH bound to both forms of RhoA can be isolated by size-exclusion chromatography. This novel interaction between activated RhoA and PH could play a key role in regulation of RhoGEF activity in vivo.

The heterotrimeric G proteins, G(12) and G(13), mediate signaling between G protein-coupled receptors and the monomeric GTPase, RhoA. One pathway for this modulation is direct stimulation by Galpha(13) of p115 RhoGEF, an exchange factor for RhoA. The GTPase activity of both Galpha(12) and Galpha(13) is increased by the N terminus of p115 Rho guanine nucleotide exchange factor (GEF). This region has weak homology to the RGS box sequence of the classic regulators of G protein signaling (RGS), which act as GTPase-activating proteins (GAP) for G(i) and G(q). Here, the RGS region of p115 RhoGEF is shown to be distinctly different in that sequences flanking the predicted "RGS box" region are required for both stable expression and GAP activity. Deletions in the N terminus of the protein eliminate GAP activity but retain substantial binding to Galpha(13) and activation of RhoA exchange activity by Galpha(13). In contrast, GTRAP48, a homolog of p115 RhoGEF, bound to Galpha(13) but was not stimulated by the alpha subunit and had very poor GAP activity. Besides binding to the N-terminal RGS region, Galpha(13) also bound to a truncated protein consisting only of the Dbl homology (DH) and pleckstrin homology (PH) domains. However, Galpha(13) did not stimulate the exchange activity of this truncated protein. A chimeric protein, which contained the RGS region of GTRAP48 in place of the endogenous N terminus of p115 RhoGEF, was activated by Galpha(13). These results suggest a mechanism for activation of the nucleotide exchange activity of p115 RhoGEF that involves direct and coordinate interaction of Galpha(13) to both its RGS and DH domains.

Glutamate is the main excitatory neurotransmitter in the mammalian central nervous system and is removed from the synaptic cleft by sodium-dependent glutamate transporters. To date, five distinct glutamate transporters have been cloned from animal and human tissue: GLAST (EAAT1), GLT-1 (EAAT2), EAAC1 (EAAT3), EAAT4, and EAAT5 (refs 1,2,3,4,5). GLAST and GLT-1 are localized primarily in astrocytes6,7, whereas EAAC1 (refs 8, 9), EAAT4 (refs 9,10,11) and EAAT5 (ref. 5) are neuronal. Studies of EAAT4 and EAAC1 indicate an extrasynaptic localization on perisynaptic membranes that are near release sites8,9,10. This localization facilitates rapid glutamate binding, and may have a role in shaping the amplitude of postsynaptic responses in densely packed cerebellar terminals12,13,14,15. We have used a yeast two-hybrid screen to identify interacting proteins that may be involved in regulating EAAT4—the glutamate transporter expressed predominately in the cerebellum—or in targeting and/or anchoring or clustering the transporter to the target site. Here we report the identification and characterization of two proteins, GTRAP41 and GTRAP48 (for glutamate transporter EAAT4 associated protein) that specifically interact with the intracellular carboxy-terminal domain of EAAT4 and modulate its glutamate transport activity.

The activity of phospholipase D (PLD) is regulated by a variety of hormonal stimuli and provides a mechanistic pathway for response of cells to extracellular stimuli. The two identified mammalian PLD enzymes possess highly homologous C termini, which are required for catalytic activity. Mutational analysis of PLD1 and PLD2 reveals that modification of as little as the C-terminal threonine or the addition of a single alanine attenuates activity of the enzyme. Protein folding appears to be intact because mutant enzymes express to similar levels in Sf9 cells and addition of peptides representing the C-terminal amino acids, including the simple hexamer PMEVWT, restores partial activity to several of the mutants. Analysis of several mutants suggests a requirement for the hydrophobic reside at the -2-position but not an absolute requirement for the hydroxyl side chain of threonine at the C terminus. The inability of peptides amidated at their C termini to effect restoration of activity indicates the involvement of the C-terminal alpha carboxyl group in functional activity of these enzymes. The ability of peptides to restore activity to PLD enzymes mutated at the C terminus suggests a flexible interaction of this portion of the molecule with a catalytic core constructed on conserved HKD motifs. Participation of these C termini residues in either stabilization of the catalytic site or the enzymatic reaction itself remains to be determined. This requirement for the C terminus provides an excellent potential site for interaction with regulatory proteins that may either enhance or down-regulate the activity of these enzymes in vitro.

Mutations of the geneLpsselectively impede lipopolysaccharide (LPS) signal transduction in C3H/HeJ and C57BL/10ScCr mice, rendering them resistant to endotoxin yet highly susceptible to Gram-negative infection. The codominantLpsdallele of C3H/HeJ mice was shown to correspond to a missense mutation in the third exon of the Toll-like receptor-4 gene (Tlr4), predicted to replace proline with histidine at position 712 of the polypeptide chain. C57BL/10ScCr mice are homozygous for a null mutation ofTlr4. Thus, the mammalian Tlr4 protein has been adapted primarily to subserve the recognition of LPS and presumably transduces the LPS signal across the plasma membrane. Destructive mutations ofTlr4predispose to the development of Gram-negative sepsis, leaving most aspects of immune function intact.

The csrA gene encodes a global regulatory protein which facilitates glgC mRNA decay in vivo. A purified recombinant CsrA protein was found to inhibit in vitro glg (glycogen biosynthesis) gene expression posttranscriptionally and bind specifically to a glgC runoff transcript without causing its decay. Our results provide further insight into the mechanism by which CsrA functions as an mRNA decay factor.

The carbon storage regulator gene, csrA, encodes a factor which negatively modulates the expression of the glycogen biosynthetic gene glgC by enhancing the decay of its mRNA (M. Y. Liu, H. Yang, and T. Romeo, J. Bacteriol. 177:2663-2672, 1995). When endogenous glycogen levels in isogenic csrA+ and csrA::kanR strains were quantified during the growth curve, both the rate of glycogen accumulation during late exponential or early stationary phase and its subsequent rate of degradation were found to be greatly accelerated by the csrA::kanR mutation. The expression of the biosynthetic genes glgA (glycogen synthase) and glgS was observed to be negatively modulated via csrA. Thus, csrA is now known to control all of the known glycogen biosynthetic genes (glg), which are located in three different operons. Similarly, the expression of the degradative enzyme glycogen phosphorylase, which is encoded by glgY, was found to be negatively regulated via csrA in vivo. The in vitro transcription-translation of glgY was also specifically inhibited by the purified CsrA gene product. These results demonstrate that localization of glycogen biosynthetic and degradative genes within the Escherichia coli glgCAY operon facilitates their coordinate genetic regulation, as previously hypothesized (T. Romeo, A. Kumar, and J. Preiss, Gene 70:363-376, 1988). The csrA gene did not affect glycogen debranching enzyme, which is now shown to be encoded by the gene glgX.

Current evidence suggests that a few global regulatory factors mediate many of the extensive changes in gene expression that occur as Escherichia coli enters the stationary phase. One of the metabolic pathways that is transcriptionally activated in the stationary phase is the pathway for biosynthesis of glycogen. To identify factors that regulate glycogen biosynthesis in trans, a collection of transposon mutants was generated and screened for mutations which independently increase or decrease glycogen levels and the expression of a plasmid-encoded glgC'-lacZ fusion. The glycogen excess mutation TR1-5 was found to be pleiotropic. It led to increased expression of the genes glgC (ADPglucose pyrophosphorylase) and glgB (glycogen branching enzyme), which are representative of two glycogen synthesis operons, and the gluconeogenic gene pckA (phosphoenolpyruvate carboxykinase), and it exhibited effects on cell size and surface (adherence) properties. The mutated gene was designated csrA for carbon storage regulator. Its effect on glycogen biosynthesis was mediated independently of cyclic AMP (cAMP), the cAMP receptor protein, and guanosine 3'-bisphosphate 5'-bisphosphate (ppGpp), which are positive regulators of glgC expression. A plasmid clone of the native csrA gene strongly inhibited glycogen accumulation and affected the ability of cells to utilize certain carbon sources for growth. Nucleotide sequence analysis, complementation experiments, and in vitro expression studies indicated that csrA encodes a 61-amino-acid polypeptide that inhibits glycogen biosynthesis. Computer-assisted data base searches failed to identify genes or proteins that are homologous with csrA or its gene product.

Interleukin-1 (IL-1), which plays an important role in the inflammatory response, was found to induce colony-stimulating factor-1 (CSF-1) expression in the MIA PaCa-2 cells. IL-1-induced CSF-1 production was markedly suppressed (70%) by pertussis toxin. This inhibition by pertussis toxin was reversed by benzamide, an inhibitor of ADP-ribosylation reactions. Similarly, IL-1-induced CSF-1 production was inhibited by cholera toxin and this inhibition was reversed by an arginine analog, p -methoxybenzylaminodecamethylene guanidine sulfate. Dibutyryl-cAMP as well as other cAMP elevating agents such as theophylline and forskolin also suppressed IL-1-induced CSF-1 production, suggesting that cAMP concentrations inversely regulate the biosynthesis of CSF-1. Measurement of cAMP concentration indicated that IL-1 treatment of MIA PaCa-2 cells did not change the cAMP level. IL-1-induced CSF-1 production was not suppressed by the protein kinase C (PKC) inhibitor, H7, under conditions in which 12- O -tetradecanoylphorbol-13-acetate-induced CSF-1 production was completely abolished. These data suggest that IL-1-induced CSF-1 production is not mediated via the activation of PKC. Analysis of oncogene c-fos and c-jun expression has shown the enhancement of expression of both protooncogenes prior to CSF-1, suggesting that the expression of these two oncogenes may be the mechanism which triggers CSF-1 gene expression.