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Biomedical subjects

M I Simon

Publications and source records attributed to M I Simon.

At least 127 records · Page 7Linked to original sources
PubMed1993

Purification and characterization of recombinant G16 alpha from Sf9 cells: activation of purified phospholipase C isozymes by G-protein alpha subunits.

A cDNA encoding G16 alpha, the alpha subunit of a heterotrimeric guanine nucleotide-binding protein, was expressed in Sf9 cells using recombinant baculovirus. G16 alpha in membrane extracts of Sf9 cells activated phospholipase C-beta 1 (PLC-beta 1) in the presence of guanosine 5'-[gamma-thio]triphosphate; the system could not be activated by Al3+, Mg2+, and F-. The G16 alpha in the cytosolic fraction of Sf9 cells did not stimulate PLC-beta 1. Concurrent expression of the G-protein beta gamma subunit complex increased the amount of G16 alpha in Sf9 cell membranes. The guanosine 5'-[gamma-thio]triphosphate-activated form of G16 alpha was purified from cholate extracts of membranes from cells expressing G16 alpha, and the G-protein beta 2 and gamma 2 subunits. G16 alpha activated PLC-beta 1, PLC-beta 2, and PLC-beta 3 in a manner essentially indistinguishable from that of Gq alpha. G16 alpha-mediated activation of PLC-beta 1 and PLC-beta 3 greatly exceeded that of PLC-beta 2. G16 alpha did not activate PLC-gamma 1 or PLC-delta 1. Thus, two distantly related members of the Gq alpha family, Gq alpha and G16 alpha, have the same ability to activate the known isoforms of PLC-beta.

Aluminum↗
PubMed1993

Assembly and function of a quaternary signal transduction complex monitored by surface plasmon resonance.

We have used surface plasmon resonance biosensor technology to monitor the assembly and dynamics of a signal transduction complex which controls chemotaxis in Escherichia coli. A quaternary complex formed which consisted of the response regulator CheY, the histidine protein kinase CheA, a coupling protein CheW and a membrane-bound chemoreceptor Tar. Using various experimental conditions and mutant proteins, we have shown that the complex dissociates under conditions that favour phosphorylation of CheY. Direct physical analysis of interactions among proteins in this signal transduction pathway provides evidence for a previously unrecognized binding interaction between the kinase and its substrate. This interaction may be important for enhancing substrate specificity and preventing 'crosstalk' with other systems. The approach is generally applicable to furthering our understanding of how signalling complexes transduce intracellular messages.

Bacterial Proteins↗
PubMed1993

The transforming activity of activated G alpha 12.

Heterotrimeric GTP-binding proteins transduce receptor-mediated extracellular signals to their effectors. Several activated mutations of Gs alpha and Gi alpha have been shown to be associated with endocrine tumors. In this report, we have evaluated the transforming activity of an putative activated form of G alpha 12 subunit. We found that transient expression in NIH3T3 cells of the G alpha 12 mutant with substitution of glutamine-229 for leucine could lead to focus formation and that stably transfected NIH3T3 cells could form colonies in soft agar and tumors in nude mice.

3T3 Cells↗
PubMed1993

Expression of CheA fragments which define domains encoding kinase, phosphotransfer, and CheY binding activities.

The histidine protein kinase CheA is a central component of the Escherichia coli chemotaxis system. The autophosphorylation activity of CheA is controlled by membrane-bound chemoreceptors and by the CheW coupling protein. CheA phosphorylates the CheY and CheB proteins which respectively control the direction of flagellar rotation and the level of receptor adaptation, thereby regulating the cells' chemotactic response. Genes encoding three polypeptide fragments of CheA were constructed and expressed in order to better define the functional organization of the wild-type protein. These fragments allowed the identification of regions of the protein responsible for CheY binding, phosphotransfer, and kinase activity. The kinase domain was expressed as a 30-kDa polypeptide corresponding to the central portion of the wild-type protein which contains sequences homologous to other histidine kinases. It was able to phosphorylate a 15-kDa amino-terminal phosphotransfer domain which was separately expressed and purified. This latter domain is capable of phosphotransfer to CheY despite the fact that it lacks the ability to stably bind CheY. CheY was immobilized to a dextran matrix through a single cysteine residue which was introduced into the protein at a position far removed from the active site. A stable binding site for CheY was mapped to a segment between the site of autophosphorylation and the kinase domain by using surface plasmon resonance to detect binding to the immobilized CheY. The region of the kinase which tightly binds the unphosphorylated substrate may play an important role in regulating the specificity of the signal transducing system.

Bacterial Proteins↗
PubMed1993

Purification from Sf9 cells and characterization of recombinant Gq alpha and G11 alpha. Activation of purified phospholipase C isozymes by G alpha subunits.

Members of the Gq alpha subfamily of heterotrimeric guanine nucleotide-binding proteins (G proteins) activate phospholipase C (PLC). The complementary DNAs (cDNAs) for the G protein alpha subunits Gq alpha and G11 alpha were expressed in insect (Sf9) cells using recombinant baculovirus. Active, nonaggregated, and membrane-associated protein was generated only when the alpha subunit cDNA was expressed together with cDNAs encoding G protein beta and gamma subunits. Recombinant alpha subunits (rGq alpha and rG11 alpha) were purified by three-step procedures, as was a PLC-activating alpha subunit(s) endogenous to Sf9 cells. Guanosine 5'-3-(thio)triphosphate (GTP gamma S) activated rGq alpha and rG11 alpha with an apparent K0.5 of 30 microM; similarly high concentrations of the nucleotide were required to observe [35S]GTP gamma S binding to rGq alpha. Activated rGq alpha and rG11 alpha each stimulated all three isoforms of purified PLC-beta with the rank order of potency PLC-beta 1 = PLC-beta 3 > or = PLC-beta 2; both alpha subunits also stimulated PLC-beta 1 and PLC-beta 3 to a much greater extent (10-fold) than they did PLC-beta 2. In contrast, activated rGq alpha and rG11 alpha failed to stimulate either PLC-delta 1 or PLC-gamma 1. Recombinant Gi alpha 1, Gi alpha 2, Gi alpha 3, Go alpha (A), Gs alpha, and Gz alpha all failed to stimulate any of the isoforms of PLC. The apparent affinities of rGq alpha and rG11 alpha for PLC-beta 1 and their capacities to activate the enzyme were similar to values observed for purified brain Gq alpha/11 alpha. Purified brain beta gamma subunits also stimulated the three isoforms of PLC-beta. The capacities of rGq alpha and rG11 alpha to activate PLC-beta 1 and PLC-beta 3 greatly exceeded those of beta gamma, whereas Gq alpha, G11 alpha and beta gamma were roughly equiefficacious with PLC-beta 2; the alpha subunits were more potent than beta gamma in all cases. The effects of alpha and beta gamma together were nonadditive for both PLC-beta 1 and PLC-beta 2. These results demonstrate that Gq alpha and G11 alpha specifically and selectively stimulate beta isoforms of PLC and confirm the idea that these members of the Gq alpha subfamily of G proteins are physiological regulators of this signaling pathway.

Animals↗
PubMed1993

G protein-coupled signal transduction pathways for interleukin-8.

Interleukin-8 (IL-8) is one of the major mediators of the inflammatory response. The pathways by which IL-8 activates inositide-specific phospholipase C (PLC) were investigated by co-expression of different components of the guanosine triphosphate binding protein (G protein) pathway in COS-7 cells. Two distinct IL-8 receptors reconstituted ligand-dependent activation of endogenous PLC when transfected together with the G protein alpha subunits G alpha 14, G alpha 15, or G alpha 16. However, reconstitution was not observed with cells that overexpressed G alpha q or G alpha 11. Furthermore, IL-8 receptors interacted with endogenous pertussis toxin-sensitive G proteins or with the recombinant G protein Gi to release free beta gamma subunits that could then specifically activate the beta 2 isoform of PLC. These findings suggest that IL-8 acts through signal-transducing pathways that are limited to specific heterotrimeric G proteins and effectors. These may provide suitable targets for the development of anti-inflammatory agents.

Animals↗
PubMed1993

Activation of the phosphosignaling protein CheY. I. Analysis of the phosphorylated conformation by 19F NMR and protein engineering.

CheY, the 14-kDa response regulator protein of the Escherichia coli chemotaxis pathway, is activated by phosphorylation of Asp57. In order to probe the structural changes associated with activation, an approach which combines 19F NMR, protein engineering, and the known crystal structure of one conformer has been utilized. This first of two papers examines the effects of Mg(II) binding and phosphorylation on the conformation of CheY. The molecule was selectively labeled at its six phenylalanine positions by incorporation of 4-fluorophenylalanine, which yielded no significant effect on activity. One of these 19F probe positions monitored the vicinity of Lys109, which forms a salt bridge to Asp57 in the apoprotein and has been proposed to act as a structural "switch" in activation. 19F NMR chemical shift studies of the labeled protein revealed that the binding of the cofactor Mg(II) triggered local structural changes in the activation site, but did not perturb the probe of the Lys109 region. The structural changes associated with phosphorylation were then examined, utilizing acetyl phosphate to chemically generate phsopho-CheY during NMR acquisition. Phosphorylation triggered a long-range conformational change extending from the activation site to a cluster of 4 phenylalanine residues at the other end of the molecule. However, phosphorylation did not perturb the probe of Lys109. The observed phosphorylated conformer is proposed to be the first step in the activation of CheY; later steps appear to perturb Lys109, as evidenced in the following paper. Together these results may give insight into the activation of other prokaryotic response regulators.

Bacterial Proteins↗
PubMed1993

Activation of the phosphosignaling protein CheY. II. Analysis of activated mutants by 19F NMR and protein engineering.

The Escherichia coli CheY protein is activated by phosphorylation, and in turn alters flagellar rotation. To investigate the molecular mechanism of activation, an extensive collection of mutant CheY proteins was analyzed by behavioral assays, in vitro phosphorylation, and 19F NMR chemical shift measurements. Substitution of a positively charged residue (Arg or Lys) in place of Asp13 in the CheY activation site results in activation, even for mutants which cannot be phosphorylated. Thus phosphorylation plays an indirect role in the activation mechanism. Lys109, a residue proposed to act as a conformational "switch" in the activation site, is required for activation of CheY by either phosphorylation or mutation. The 19F NMR chemical shift assay described in the preceding article (Drake, S. K., Bourret, R. B., Luck, L. A., Simon, M. I., and Falke, J. J. (1993) J. Biol Chem. 268, 13081-13088) was again used to monitor six phenylalanine positions in CheY, including one position which probed the vicinity of Lys109. Mutations which activate CheY were observed to perturb the Lys109 probe, providing further evidence that Lys109 is directly involved in the activating conformational change. Two striking contrasts were observed between activation by mutation and phosphorylation. (i) Each activating mutation generates a relatively localized perturbation in the activation site region, whereas phosphorylation triggers a global structural change. (ii) The perturbation of the Lys109 region observed for activating mutations is not detected in the phosphorylated protein. These results are consistent with a two-step model of activated CheY docking to the flagellar switch.

Bacterial Proteins↗
PubMed1993

Activation of phospholipase C beta 2 by the alpha and beta gamma subunits of trimeric GTP-binding protein.

Cotransfection assays were used to show that the members of the GTP-binding protein Gq class of alpha subunits could activate phospholipase C (PLC) beta 2. Similar experiments also demonstrated that G beta 1 gamma 1, G beta 1 gamma 5, and G beta 2 gamma 5 could activate the beta 2 isoform of PLC but not the beta 1 isoform, while G beta 2 gamma 1 did not activate PLC beta 2. To determine which portions of PLC beta 2 are required for activation by G beta gamma or G alpha, a number of PLC beta 2 deletion mutants and chimeras composed of various portions of PLC beta 1 and PLC beta 2 were prepared. We identified the N-terminal segment of PLC beta 2 with amino acid sequence extending to the end of the Y box as the region required for activation by G beta gamma and the C-terminal region as the segment containing amino acid sequences required for activation by G alpha. Furthermore, we found that coexpression of G alpha 16 and G beta 1 gamma 1 but not G beta 1 gamma 5 in COS-7 cells was able to synergistically activate recombinant PLC beta 2. We suggest that G alpha 16 may act together with free G beta 1 gamma 1 to activate PLC beta 2, while G alpha 16 may form heterotrimeric complexes with G beta 1 gamma 5 and be stabilized in an inactive form. We conclude that the regions of PLC beta 2 required for activation by G beta gamma and G alpha are physically separate and that the nature of the G beta subunit may play a role in determining the relative specificity of the G beta gamma complex for effector activation while the nature of the G gamma subunit isoform may be important for determining the affinity of the G beta gamma complex for specific G alpha proteins.

Amino Acid Sequence↗
PubMed1993

Specific interactions of chemoattractant factor receptors with G-proteins.

Stimulation of leukocytes with chemoattractant ligands activates phospholipid turnover and calcium release, ultimately leading to chemotaxis, degranulation, and the inflammatory response. The leukocyte response to these ligands is transduced by the interaction of transmembrane receptors with GTP-binding regulatory proteins (G-proteins). To examine the mechanisms of signal transduction by these receptors, we transfected cDNA clones encoding the receptors for the active cleavage product of the fifth component of complement (C5a) and platelet-activating factor (PAF) into COS-7 cells, then measured the production of inositol phosphates (IP) in response to stimulation with these chemoattractant ligands. Cells transfected with the C5a receptor showed no increase in IP production when stimulated with ligand (5-120 nM). However, in cells co-transfected with these receptors and with the cDNA for G alpha 16, a G-protein alpha subunit that is specific to cells of hematopoietic lineage, addition of ligand caused up to a 5-fold increase in IP production. This interaction was specific, as co-transfection of receptors with the G-proteins G alpha q or G alpha 11 did not allow ligand-dependent increase in IP production. In contrast, ligand-dependent activation of IP production was seen in COS cells transfected solely with the PAF receptor. These results indicate that the C5a receptor utilizes signaling pathways distinct from the PAF receptor and suggest that a pertussis toxin-resistant G-protein, G alpha 16, may play a role in the leukocyte response to inflammatory ligands.

Animals↗
PubMed1993

Identification of critical regions on phospholipase C-beta 1 required for activation by G-proteins.

In order to determine which portion of phosphoinositide-specific phospholipase C (PLC)-beta 1 is required for activation by G alpha q, a series of specific deletions and truncations of PLC-beta 1 cDNA were prepared. After transfection of COS-7 cells with these cDNA clones, the activity and localization of the expressed proteins were determined. Specific deletions in the C-terminal end of the protein did not lead to loss of intrinsic enzymatic activity but did result in loss of the ability to be activated by G alpha q. The region required for activation was localized to the amino acid sequence corresponding to residues 903-1142 of PLC-beta 1. This region was further subdivided into two sequences; one extending from residues Thr-903 to Gln-1030 that was required for particulate fraction association as well as for activation by G alpha q and the other extending from residues Gln-1030 to Leu-1142 that was required for interaction with G alpha subunits. These results were confirmed by the observation that the C-terminal portion of PLC-beta 1, when co-expressed with the muscarinic acetylcholine receptor type 1 or the alpha 1C-adrenergic receptor in COS-7 cells, markedly inhibited ligand-induced release of inositol phosphates. In an in vitro system, two peptides derived from the G-protein interaction region at the C terminus were found to inhibit the guanosine 5'-3-O-(thio)triphosphate-dependent activation of PLC-beta 1 by G alpha q. This further localized the sites on PLC-beta 1 which are involved in interaction with G-protein alpha subunits.

Amino Acid Sequence↗
PubMed1993

Mutational analysis of G protein alpha subunit G(o) alpha expressed in Escherichia coli.

G protein-mediated signal transduction is dependent on alpha subunit interactions with beta gamma subunits, receptors, effectors, magnesium ions, and guanine nucleotides. The interdependence of these interactions can be probed by mutational analysis. We developed large scale screening procedures in recombinant Escherichia coli to identify and characterize novel mutations in G(o) alpha. Random mutations were generated by polymerase chain reaction in the amino-terminal 56 amino acids of G(o) alpha. Guanine nucleotide binding properties of the mutants were assayed in situ and in crude extracts of recombinant E. coli. beta gamma interactions were assayed by pertussis toxin mediated ADP-ribosylation. Efficacy of the screening procedures was evaluated by studying properties of wild-type G(o) alpha and site-directed mutations that were characterized previously in other G proteins. Several novel mutants with altered GTP binding characteristics and reduced ability to interact with beta gamma had been isolated from the randomly generated mutant library. ADP-ribosylation of mutants R10G, K21N, and K35E was significantly reduced, whereas two of the mutants bearing multiple amino acid substitutions were refractory to modification. Mutant K35E also exhibited reduced affinity to guanosine 5'-(3-O-thio)triphosphate at submicromolar concentrations of magnesium. These experiments demonstrate the feasibility of using large scale random mutagenesis in the studies of G protein function.

Adenosine Diphosphate Ribose↗
PubMed1993

Structure of the bovine transducin gamma subunit gene and analysis of promoter function in transgenic mice.

Transducin, the major photoreceptor guanine nucleotide-binding protein (G protein), is composed of three polypeptides: alpha, beta and gamma subunits. The transducin gamma subunit (T gamma) is expressed preferentially in photoreceptors. To study the control mechanisms for photoreceptor-specific expression of the T gamma gene, clones of the bovine T gamma gene were isolated from a bacteriophage genomic library, and the structure of the gene, including a portion of its 5'-flanking region, was characterized. The gene consists of three exons and two introns. The first intron is 91 base pairs (bp) long and is located in the region corresponding to the 5'-untranslated sequence of the T gamma mRNA. The second intron is 5.3 kilobases (kb) long and splits the protein-coding region centrally. A bovine Alu-type repetitive sequence and putative Ret-1 and AP-1 binding site sequences are located in the 5'-flanking region. To investigate promoter function, 1.4 kb of DNA from the 5'-flanking region was joined to the prokaryotic chloramphenicol acetyltransferase (CAT) gene, and the chimeric bovine T gamma gene was used to generate a line of transgenic mice. CAT activity was readily detected in the retinas of the transgenic mice, but was absent in brain, heart, kidney, liver, lung, spleen and other tissues. These results suggest that the 1.4 kb 5'-flanking region of the bovine T gamma gene contains conserved sequence elements that direct tissue-specific expression. Human T gamma cDNA clones were characterized, and a short homologous region of the human T gamma gene promotor was obtained by polymerase chain reaction (PCR) amplification for comparison with the bovine promoter.

Animals↗
PubMed1993

Identification, chromosomal location, and genome organization of mammalian G-protein-coupled receptors.

Vertebrate G-protein-coupled receptors are encoded by a diverse multigene family. Thirteen distinct G-protein-coupled receptors (Gpcr) were cloned from mouse germline cDNA following amplification in the polymerase chain reaction (PCR) with degenerate oligonucleotide primers complementary to the third and sixth transmembrane domains. Eleven Gpcr clones were mapped to single sites in the mouse genome following interspecific backcross analysis. One clone was mapped to two sites and another was not polymorphic in the cross and could not be mapped. Gpcr loci were well dispersed throughout the mouse genome and mapped to chromosomes 1, 2, 3, 4, 5, 8, 9, 10, 13, 17, and 18. Six Gpcr clones likely represent mouse homologs of already identified receptors, one Gpcr clone may identify a third type of IL-8 receptor, and three Gpcr clones appear to encode novel G-protein-coupled receptors. Further, three factor crosses and Southern blot analyses demonstrated that Gpcr16 maps proximally within the Spr deletion on mouse chromosome 1, near the Vil and Bcg loci. Human chromosomal locations for most Gpcr loci could be predicted based on linkage homologies that have been identified between human and mouse. Mapping additional G-protein-coupled receptors against the panel of murine interspecific backcrosses should expand our understanding of mammalian Gpcr gene evolution and genome organization.

Amino Acid Sequence↗
PubMed1993

Computer simulation of the phosphorylation cascade controlling bacterial chemotaxis.

We have developed a computer program that simulates the intracellular reactions mediating the rapid (nonadaptive) chemotactic response of Escherichia coli bacteria to the attractant aspartate and the repellent Ni2+ ions. The model is built from modular units representing the molecular components involved, which are each assigned a known value of intracellular concentration and enzymatic rate constant wherever possible. The components are linked into a network of coupled biochemical reactions based on a compilation of widely accepted mechanisms but incorporating several novel features. The computer motor shows the same pattern of runs, tumbles and pauses seen in actual bacteria and responds in the same way as living bacteria to sudden changes in concentration of aspartate or Ni2+. The simulated network accurately reproduces the phenotype of more than 30 mutants in which components of the chemotactic pathway are deleted and/or expressed in excess amounts and shows a rapidity of response to a step change in aspartate concentration similar to living bacteria. Discrepancies between the simulation and real bacteria in the phenotype of certain mutants and in the gain of the chemotactic response to aspartate suggest the existence of additional as yet unidentified interactions in the in vivo signal processing pathway.

Aspartic Acid↗
PubMed1993

Intermolecular complementation of the kinase activity of CheA.

CheA is a dimeric autophosphorylating protein kinase that plays a critical role in the signal transduction network controlling chemotaxis in Escherichia coli. The autophosphorylation reaction was analysed using mutant proteins defective in kinase and regulatory functions. Proteins in which the site of autophosphorylation was mutated (CheA48HQ) or missing (CheAs) were found to phosphorylate the kinase-defective mutant, CheA470GK. The kinetics of this reaction support the hypothesis that autophosphorylation is the result of trans-phosphorylation within a dimer. The carboxy-terminal portion of CheA was previously shown to be dispensable for autophosphorylation, but required for regulation in response to environmental signals transmitted through a transducer and CheW. Mixing of CheA48HQ or CheA470GK with a truncated protein lacking this regulatory domain demonstrated that regulated autophosphorylation requires the presence of both carboxy-terminal portions in a CheA dimer. These results indicate that the dimeric form of CheA plays an integral role in signal transduction in bacterial chemotaxis.

Bacterial Proteins↗
PubMed1993

The carboxy-terminal portion of the CheA kinase mediates regulation of autophosphorylation by transducer and CheW.

The CheA kinase is a central protein in the signal transduction network that controls chemotaxis in Escherichia coli. CheA receives information from a transmembrane receptor (e.g., Tar) and CheW proteins and relays it to the CheB and CheY proteins. The biochemical activities of CheA proteins truncated at various distances from the carboxy terminus were examined. The carboxy-terminal portion of CheA regulates autophosphorylation in response to environmental signals transmitted through Tar and CheW. The central portion of CheA is required for autophosphorylation and is also presumably involved in dimer formation. The amino-terminal portion of CheA was previously shown to contain the site of autophosphorylation and to be able to transfer the phosphoryl group to CheB and CheY. These studies further delineate three functional domains of the CheA protein.

Bacterial Proteins↗