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M I Simon

Publications and source records attributed to M I Simon.

At least 91 records · Page 5Linked to original sources
PubMed1996

Mutations in a C. elegans Gqalpha gene disrupt movement, egg laying, and viability.

We find that C. elegans egl-30 encodes a heterotrimeric G protein a subunit more than 80% identical to mammalian Gqalpha family proteins, and which can function as a Gqalpha subunit in COS-7 cells. We have identified new egl-30 alleles in a selection for genes involved in the C. elegans acetylcholine response. Two egl-30 alleles specify premature termination of Gqalpha and are essentially lethal in homozygotes. Animals homozygous for six other egl-30 alleles are viable and fertile, but exhibit delayed egg laying and leave flattened tracks. Overexpression of the wild-type egl-30 gene produces the opposite behavior. Analysis of these mutants suggest that their phenotypes reflect defects in the muscle or neuromuscular junction.

Amino Acid Sequence↗
PubMed1996

Thermostable chemotaxis proteins from the hyperthermophilic bacterium Thermotoga maritima.

An expressed sequence tag homologous to cheA was previously isolated by random sequencing of Thermotoga maritima cDNA clones (C. W. Kim, P. Markiewicz, J. J. Lee, C. F. Schierle, and J. H. Miller, J. Mol. Biol. 231: 960-981, 1993). Oligonucleotides complementary to this sequence tag were synthesized and used to identify a clone from a T. maritima lambda library by using PCR. Two partially overlapping restriction fragments were subcloned from the lambda clone and sequenced. The resulting 5,251-bp sequence contained five open reading frames, including cheA, cheW, and cheY. In addition to the chemotaxis genes, the fragment also encodes a putative protein isoaspartyl methyltransferase and an open reading frame of unknown function. Both the cheW and cheY genes were individually cloned into inducible Escherichia coli expression vectors. Upon induction, both proteins were synthesized at high levels. T. maritima CheW and CheY were both soluble and were easily purified from the bulk of the endogenous E. coli protein by heat treatment at 80 degrees C for 10 min. CheY prepared in this way was shown to be active by the demonstration of Mg(2+)-dependent autophosphorylation with [32P]acetyl phosphate. In E. coli, CheW mediates the physical coupling of the receptors to the kinase CheA. The availability of a thermostable homolog of CheW opens the possibility of structural characterization of this small coupling protein, which is among the least well characterized proteins in the bacterial chemotaxis signal transduction pathway.

Amino Acid Sequence↗
PubMed1996

In vivo and in vitro characterization of Escherichia coli protein CheZ gain- and loss-of-function mutants.

Bacterial chemotaxis results from the ability of flagellated bacteria to control the frequency of switching between smooth-swimming and tumbling episodes in response to changes in concentration of extracellular substances. High levels of phosphorylated CheY protein are the intracellular signal for inducing the tumbling mode of swimming. The CheZ protein has been shown to control the level of phosphorylated CheY by regulating its rate of dephosphorylation. To identify functional domains in the CheZ protein, we made mutants by random mutagenesis of the cheZ gene and constructed a series of deletions. The map position and the in vivo and in vitro activity of the resulting gain- or loss-of-function mutant proteins define separate functional domains of the CheZ protein.

Bacterial Proteins↗
PubMed1996

A third P-domain peptide gene (TFF3), human intestinal trefoil factor, maps to 21q22.3.

Small peptides displaying a cysteine-rich module (termed P-domain or trefoil motif) form a recently increasing group of peptides abundantly expressed at mucosal surfaces of specific tissues and are associated with the maintenance of surface integrity. The estrogen-inducible pS2 gene (BCEI) and the human homolog to the porcine spasmolytic peptide (hsP) gene (SML1) appear synchronously expressed in healthy stomach mucosa and several carcinomas of the gastrointestinal tract. Both genes were shown to be located at 21q22.3. A new trefoil peptide from human intestinal mucosa (hITF/hP1.B) and its gene (TFF3) were described recently. By PCR analysis of a somatic cell hybrid panel and FISH using two large genomic recombinants (110 kb, 210 kb) cloned in the Bacterial Artificial Chromosome (BAC) system, we show that this gene coding for the new member of human P-domain/trefoil peptides also maps to chromosome region 21q22.3 suggesting a physical linkage of all three trefoil peptide genes.

Amino Acid Sequence↗
PubMed1996

G alpha q family members couple parathyroid hormone (PTH)/PTH-related peptide and calcitonin receptors to phospholipase C in COS-7 cells.

The PTH/PTH-related peptide (PTHrP) receptor and the calcitonin receptor mediate the action of their physiological ligands by activating two different effectors, adenylyl cyclase and phospholipase C. Whereas regulation of adenylyl cyclase via both receptors is thought to involve the G protein G(s), it is not known whether activation of phospholipase C results from coupling of the receptors to G(q) family members or whether beta gamma-subunit released from receptor-activated G(s) lead to phospholipase C activation. To elucidate the mechanism of this type of dual signaling, we reconstituted the signal transduction of the PTH/PTHrP and the calcitonin receptor in COS-7 and HEK293 cells. In COS-7 cells expressing the receptor alone, addition of the respective ligands resulted in the accumulation of cAMP and inositol phosphates. When cells were cotransfected with the cDNAs of receptor and different alpha-subunits of the Gq family (G alpha q, G alpha 11, G alpha 14, G alpha 15, and G alpha 16, a severalfold increase in the ligand-dependent inositol phosphate production could be observed, indicating that the receptors functionally interacted with all alpha-subunits of the G alpha q family. Additionally, whereas PTH treatment of HEK293 cells coexpressing both the PTH/PTHrP receptor and G alpha q increased both second messengers, the same treatment in cells expressing the PTH/PTHrP receptor alone increased only cAMP. Under all conditions tested, activation of phospholipase C via the PTH/PTHrP and calcitonin receptor required higher ligand concentrations than receptor-mediated adenylyl cyclase activation. Our data strongly support the idea that dual signaling of the PTH/PTHrP and calcitonin receptors is due to the a activation of different G proteins belonging to the G(s) and G(q) families.

Adenylyl Cyclases↗
PubMed1996

Organization of transmembrane signalling by heterotrimeric G proteins.

Heterotrimeric G proteins are key players in a transmembrane signalling system that is used by every cell to regulate its basal functions as well as to integrate its specific functions into the whole organism. The complexity of this task is reflected by the diversity of molecular components involved therein and the variety of their potential interactions that have been described thus far. G protein mediated cellular signalling obviously represents a network of interacting pathways that are highly dynamic and are subject to short and long term regulatory processes that adapt the system to changing conditions. The identification of signalling molecules and the description of their functions have provided the foundation for understanding signal transduction processes involving G proteins. Much work, however, is still required to provide an understanding of how these molecular events are orchestrated in time and in space in a living cell.

Animals↗
PubMed1995

The response regulators CheB and CheY exhibit competitive binding to the kinase CheA.

The autophosphorylating kinase CheA of the bacterial chemosensory signaling pathway donates a phosphoryl group to either of two regulator proteins, CheY or the receptor methylesterase (CheB). With isothermal titration calorimetry, it was demonstrated that CheA and CheA fragment composed of amino acid residues 1-233 (CheA1-233) bound to CheY with similar dissociation constants of 2.0 and 1.2 microM at 298 K, respectively, indicating that the CheY binding site is wholly within the 1-233 amino acid locus. CheB bound to CheA1-233 with a KD of 3.2 microM, and also bound to intact CheA with the same affinity. CheY was found to complete with CheB for binding to CheA1-233, in spite of the low level of sequence identity between CheY and the regulatory domain of CheB. The competitive nature of CheY and CheB binding was determined in two independent sets of experiments: titration experiments in which either a CheB-CheA1-233 complex was titrated with CheY or CheB was titrated with a CheY-CheA1-233 complex, and competitive affinity chromatography experiments that used a Ni-NTA-chelating resin as an affinity matrix for complexes of the histidine-tagged CheA1-233 fragment and CheY or CheB. The effects of phosphorylation, binding-site mutations, and active-site mutations were also studied to probe the influence of conformational changes in CheY as a regulatory mechanism of CheY-CheA Interactions. Phosphorylated CheY, in the presence of excess EDTA, was found to have a 2-fold lower affinity for CheA1-233, and 6 mM Mg2+ further reduced the affinity of phosphorylated CheY for CheA1-233 (ca. 3-fold), although Mg2+ on its own had no effect on the interactions of either CheB or CheY with CheA1-233. The data thus indicate that phosphorylated CheY has a significantly lower affinity for CheA under physiological conditions. The idea that phosphorylation may induce a significant conformational change, reducing the strength of the CheY-CheA interaction, is supported by the relative values of the association constants measured for CheY active-site and binding-site mutants. A binding-site mutation (A103V) in CheY, which is remote from the site of phosphorylation produced a 10-fold reduction in Ka, whereas active-site mutations produced a modest (2-fold) reduction.

Bacterial Proteins↗
PubMed1995

NMR studies of the phosphotransfer domain of the histidine kinase CheA from Escherichia coli: assignments, secondary structure, general fold, and backbone dynamics.

Multidimensional heteronuclear NMR techniques were applied to study the phosphotransfer domain, residues 1-134, of the histidine kinase CheA, from Escherichia coli, which contains the site of autophosphorylation, His48. Assignments of the backbone amide groups and side chain protons are nearly complete. Our studies show that this protein fragment consists of five alpha-helices (A-E) connected by turns. Analysis of NOE distance restraints provided by two-dimensional (2D) 1H-1H and three-dimensional (3D) 15N-edited NOESY spectra using model building and structure calculations indicates that the five helices form an antiparallel helix bundle with near-neighbor connectivity. The amino-terminal four helices are proposed to be arranged in a right-handed manner with helix E packing against helices C and D. From ideal hydrophobic helical packing and structure calculations, the site of autophosphorylation, His48, is nearly fully exposed to the solvent. We measured the NMR relaxation properties of the backbone 15N nuclei using inverse detected two-dimensional NMR spectroscopy. The protein backbone dynamics studies show that CheA1-134 is formed into a tight and compact structure with very limited flexibilities both in helices and turns. Structural implications of titration and phosphorylation experiments are briefly discussed.

Amino Acid Sequence↗
PubMed1995

G12 requirement for thrombin-stimulated gene expression and DNA synthesis in 1321N1 astrocytoma cells.

Thrombin stimulation of 1321N1 astrocytoma cells leads to Ras-dependent AP-1-mediated transcriptional activation and to DNA replication. In contrast to what has been observed in most cell systems, in 1321N1 cells these responses are pertussis toxin-insensitive. The pertussis toxin-insensitive G-protein G12 has been implicated in cell growth and transformation in different cell systems. We have examined the potential role of this protein in AP-1-mediated transcriptional activation and DNA synthesis in 1321N1 cells. Transient expression of an activated (GTPase-deficient) mutant of G alpha 12 increased AP-1-dependent gene expression. This response was inhibited by co-expression of a dominant negative Ala-15 Ras protein. To determine whether the pertussis toxin-insensitive G12 protein is involved in the thrombin-stimulated DNA synthesis, an inhibitory antibody against the C-terminal sequence of G alpha 12 subunit was microinjected into 1321N1 cells. Microinjection of the anti-G alpha 12 resulted in a concentration-dependent inhibition of thrombin-stimulated DNA synthesis. In contrast, microinjection of nonimmune IgG or an antibody directed against the C terminus of G alpha o did not reduce the mitogenic response to thrombin. Furthermore, microinjection of the anti-G alpha 12 antibody had no effect on fibroblast growth factor-stimulated DNA synthesis. These results demonstrate a specific role for G alpha 12 in the mitogenic response to thrombin in human astroglial cells.

Amino Acid Sequence↗
PubMed1995

G alpha 15 and G alpha 16 couple a wide variety of receptors to phospholipase C.

The murine G-protein alpha-subunit G alpha 15 and its human counterpart G alpha 16 are expressed in a subset of hematopoietic cells, and they have been shown to regulate beta-isoforms of inositide-specific phospholipase C. We studied the ability of a variety of receptors to interact with G alpha 15 and G alpha 16 by cotransfecting receptors and G-protein alpha-subunits in COS-7 cells. Activation of beta 2 adrenergic and muscarinic M2 receptors in cells expressing the receptors alone or together with G alpha q, G alpha 11, or G alpha 14 led to a very small stimulation of endogenous phospholipase C. However, when the receptors were coexpressed with G alpha 15 and G alpha 16, addition of appropriate ligands caused a severalfold increase in inositol phosphate production which was time- and dose-dependent. A similar activation of phospholipase C was observed when several other receptors which were previously shown to couple to members of the Gi and Gs family were coexpressed with G alpha 15/16. In addition, stimulation of inositol phosphate formation via receptors naturally coupled to phospholipase C was enhanced by cotransfection of G alpha 15 and G alpha 16. These data demonstrate that G alpha 15 and G alpha 16 are unique in that they can be activated by a wide variety of G-protein-coupled receptors. The ability of G alpha 15 and G alpha 16 to bypass the selectivity of receptor G-protein interaction can be a useful tool to understand the mechanism of receptor-induced G-protein activation. In addition, the promiscuous behavior of G alpha 15 and G alpha 16 toward receptors may be helpful in finding ligands corresponding to orphan receptors whose signaling properties are unknown.

Animals↗
PubMed1995

Different alpha 1-adrenergic receptor sequences required for activating different G alpha subunits of Gq class of G proteins.

In order to understand the specific interactions between receptors and guanine nucleotide-binding regulatory protein (G proteins), we attempted to delineate the alpha 1 B-adrenergic receptor sequences involved in activation of the alpha subunits of the Gq class of G proteins. A number of specific mutations were introduced into the third inner loop of the receptor, and the mutants were tested for their abilities to activate different G alpha subunits of the Gq class. Our results indicate that the receptor sequences required for activating G alpha q/11, G alpha 14, or G alpha 16 are different. The sequence extending from residues Lys240 to His252 is required for activation of G alpha q/11, but not for activation of G alpha 14 or G alpha 16. Two segments in the third loop of the receptor are required for activation of G alpha 14: one is located at the N terminus of the loop ending at residue Asn226, and the other is located at the C terminus of the loop starting from residue Ser278. The latter contains a BBXXB motif, which is apparently critical for G alpha 14 coupling, but not for G alpha 16 or G alpha q/11 coupling. Furthermore, the three amino acids stretch (Tyr217 to Val219) included in the N-terminal segment is not only required for G alpha 14 coupling, but also for G alpha q/11 coupling. It may be involved to some extent in G alpha 16 coupling as well.

Amino Acid Sequence↗
PubMed1995

Participation of the protein Go in multiple aspects of behavior in C. elegans.

The goa-1 gene encoding the alpha subunit of the heterotrimeric guanosine triphosphate-binding protein (G protein) Go from Caenorhabditis elegans is expressed in most neurons, and in the muscles involved in egg laying and male mating. Reduction-of-function mutations in goa-1 caused a variety of behavioral defects including hyperactive movement, premature egg laying, and male impotence. Expression of the activated Go alpha subunit (G alpha o) in transgenic nematodes resulted in lethargic movement, delayed egg laying, and reduced mating efficiency. Induced expression of activated G alpha o in adults was sufficient to cause these phenotypes, indicating that G alpha o mediates behavior through its role in neuronal function and the functioning of specialized muscles.

Amino Acid Sequence↗
PubMed1995

A segment of the C-terminal half of the G-protein beta 1 subunit specifies its interaction with the gamma 1 subunit.

The beta and gamma subunits of the heterotrimeric guanine nucleotide binding (G protein) act as a dimer and directly regulate various signal transduction pathways. By using cotransfection assays, we tested the ability of several beta gamma combinations to activate inositol phospholipid-specific phospholipase C (PI-PLC)-beta 2. Our findings indicate that only beta gamma combinations that form dimers will activate PI-PLC-beta 2. Since G beta 1 interacts with G gamma 1, while G beta 2 cannot, chimeras between G beta 1 and G beta 2 were used to identify the regions in beta 1 that determine its specific association with gamma 1. Our evidence demonstrates that a chimera between beta 2 and beta 1 that contains the C-terminal 173 amino acids of beta 1 can interact and activate PI-PLC-beta 2 with gamma 1. Chimeras that contain portions of the beta 1 C-terminal region display a weaker association with gamma 1. Furthermore, the contribution of each of these regions depends on the sequence context of each chimeric protein. However, the segment between residues 210 and 293 of beta 1 consistently plays a critical role in specifying association with gamma 1.

Amino Acid Sequence↗
PubMed1995

The N terminus of phosducin is involved in binding of beta gamma subunits of G protein.

Phosducin is a soluble phosphoprotein found in retinal photoreceptor cells and in the pineal gland. It binds to the beta gamma subunits of guanine nucleotide-binding proteins (G proteins) (G beta gamma) and may regulate G-protein function. In this study, the ability of specific regions of phosducin to bind G beta gamma was characterized. A series of deletion mutants were made in bovine phosducin. They were tested in cotransfection assays for their ability to inhibit G beta gamma-mediated phospholipase C beta 2 isoform activation. Overexpression of the N-terminal half of phosducin showed inhibition, whereas overexpression of the C-terminal half did not. The first 63 amino acid residues were required for inhibition. A tryptophan-to-valine substitution at residue 29, which is part of a well conserved 11-amino acid sequence, severely impaired phosducin inhibitory function. Glutathione S-transferase-phosducin fusion proteins were expressed in Escherichia coli to study phosducin-G beta gamma interaction in vitro. The N-terminal 63-amino acid fragment was able to bind to G beta gamma. In contrast, the C-terminal half failed to bind to G beta gamma. The substitution mutants showed little or no binding. Furthermore, direct measurements of interaction between G beta gamma and fragments of phosducin, using surface plasmon resonance technology, confirmed the assignment of binding activity to the 63-amino acid fragment and the importance of the tryptophan residue.

Amino Acid Sequence↗
PubMed1995

The three-dimensional structure of the aspartate receptor from Escherichia coli.

The crystal structure of the periplasmic domain of the aspartate receptor from Escherichia coli has been solved and refined to an R-factor of 0.203 at 2.3 A, resolution. The dimeric protein is largely helical, with four helices from each monomer forming a four-helix bundle. The dimer interface is constructed from four helices, two from each subunit, also packed together in a four-helix bundle arrangement. A sulfate ion occupies the aspartate-binding site. All hydrogen bonds made to aspartate are substituted by direct or water-mediated hydrogen bonds to the sulfate. Comparison of the Escherichia coli aspartate-receptor structure with that of Salmonella typhimurium [Milburn, Prive, Milligan, Scott, Yeh, Jancarik, Koshland & Kim (1991). Science, 254, 1342-1347; Scott, Milligan, Milburn, Prive, Yeh, Koshland & Kim (1993). J. Mol. Biol. 232, 555-573] reveals strong conservation in the structure of the monomer, but more divergence in the orientation of the subunits with respect to one another. Mutations that render the Escherichia coli receptor incapable of responding to maltose are either located in spatially conserved sites or in regions of the structures that have high temperature factors and are therefore likely to be quite flexible. The inability of the receptor from Salmonella typhimurium to respond to maltose may, therefore, be because of differences in amino acids located on the binding surface rather than structural differences.

Journal Article↗
PubMed1995

Functional analysis of a dominant negative mutant of G alpha i2.

The key event in receptor-catalyzed activation of heterotrimer G proteins is binding of GTP, which leads to subunit dissociation generating GTP-bound alpha subunits and free beta gamma complexes. We have previously identified a mutation that abolished GTP binding in G alpha o (S47C) and demonstrated that the mutant retained the ability to bind beta gamma and could act in a dominant negative fashion when expressed in Xenopus oocytes (Slepak, V.Z., Quick, M.W., Aragay, A.M., Davidson, N., Lester, H.A., and Simon, M.I. (1993) J. Biol. Chem. 268, 21889-21894). In the current work, we investigated the effects of the homologous mutant of G alpha i2 (S48C) upon signaling pathways reconstituted in transiently transfected COS-7 cells. We found that expression of the G alpha i2 S48C mutant prevented stimulation of phospholipase C (PLC) beta 2 by free beta gamma subunit complexes. This effect of G alpha i S48C was not readily reversible in contrast to the inhibitory effect of wild-type G alpha i2, which could be reversed upon activation of the cotransfected muscarinic M2 receptor, presumably by release of beta gamma from the G protein heterotrimer. Coexpression of G alpha i S48C or the wild-type G alpha i2 also dramatically decreased G16-mediated stimulation of PLC by C5a in the cells transfected with cDNAs encoding C5a receptor and G alpha 16. Activation of PLC via endogenous Gq or G11 in the presence of alpha 1C adrenergic receptors was similarly attenuated by coexpression of G alpha i or G alpha i S48C. Pertussis toxin treatment of the transfected cells enhanced the inhibition of the receptor-stimulated PLC by wild-type G alpha i subunits but did not influence the effects of the dominant negative mutant. The enhancement of the wild-type G alpha i inhibitory effect by pertussis toxin can be explained by stabilization of G alpha i binding to beta gamma as a result of ADP-ribosylation, while G alpha i S48C mutant binds beta gamma irreversibly even without pertussis toxin treatment. Therefore, a feasible mechanism to rationalize the attenuation of the G alpha 16 and Gq/11-mediated activation of PLC by cotransfected G alpha i is the competition between G alpha i and G alpha 16 or Gq/11 for the beta gamma complexes, which are necessary for the G protein coupling with receptors. These experiments provide new evidence for the role of beta gamma in the integration of signals controlling phosphoinositide release through different G alpha families.

Adenosine Diphosphate Ribose↗
PubMed1995

Mechanisms of rhodopsin inactivation in vivo as revealed by a COOH-terminal truncation mutant.

Although biochemical experiments suggest that rhodopsin and other receptors coupled to heterotrimeric guanosine triphosphate-binding proteins (G proteins) are inactivated by phosphorylation near the carboxyl (COOH)-terminus and the subsequent binding of a capping protein, little is known about the quenching process in vivo. Flash responses were recorded from rods of transgenic mice in which a fraction of the rhodopsin molecules lacked the COOH-terminal phosphorylation sites. In the single photon regime, abnormally prolonged responses, attributed to activation of individual truncated rhodopsins, occurred interspersed with normal responses. The occurrence of the prolonged responses suggests that phosphorylation is required for normal shutoff. Comparison of normal and prolonged single photon responses indicated that rhodopsin begins to be quenched before the peak of the electrical response and that quenching limits the response amplitude.

Animals↗