Biomedical subjects

M I Simon

Publications and source records attributed to M I Simon.

At least 19 recordsLinked to original sources

PubMed2004

Comparative analysis of transcriptional profiles between two apoptotic pathways of light-induced retinal degeneration.

Light exposure can exacerbate the condition of a variety of human retinal diseases by increasing the rate of photoreceptor cell death. How light negatively affects photoreceptor cell survival is not yet fully understood. Previous studies involving light damage models have revealed two independent apoptotic pathways: low levels of light induce retinal degeneration in the arrestin -/- mouse via constitutive activation of the phototransduction cascade, whereas strong light exposure to the retina, such as in an albino eye, elicits photoreceptor cell death via activator protein (AP-1) induction. In order to better understand the initial gene expression changes underlying light damage, dark-reared arrestin -/- and albino BALB/c mice were exposed to constant white light (2000 lux), and their retinal morphology was assessed as a function of time. The expression profiles of retinal transcripts were then compared between dark-adapted and light-exposed arrestin -/-, pigmented wild-type and BALB/c mice at a time point when morphological changes were minimal. As expected, the dark-adapted samples showed little difference in expression pattern between the three genotypes. Among the genes differentially regulated by light in BALB/c, but not arrestin -/- retinas, were c-fos and other stress-induced early response genes. In both mouse models, a marked increase in expression of the bZIP family of transcription factors was observed. Our results show a select group of unique and overlapping sets of genes induced by light in the two mouse models. These expression changes may constitute the underlying initiating events leading to the two distinct mechanisms of light damage.

Animals↗

PubMed2004

Recoverin regulates light-dependent phosphodiesterase activity in retinal rods.

The Ca2+-binding protein recoverin may regulate visual transduction in retinal rods and cones, but its functional role and mechanism of action remain controversial. We compared the photoresponses of rods from control mice and from mice in which the recoverin gene was knocked out. Our analysis indicates that Ca2+-recoverin prolongs the dark-adapted flash response and increases the rod's sensitivity to dim steady light. Knockout rods had faster Ca2+ dynamics, indicating that recoverin is a significant Ca2+ buffer in the outer segment, but incorporation of exogenous buffer did not restore wild-type behavior. We infer that Ca2+-recoverin potentiates light-triggered phosphodiesterase activity, probably by effectively prolonging the catalytic activity of photoexcited rhodopsin.

Adaptation, Physiological↗

PubMed2003

Methacholine-induced airway hyperresponsiveness is dependent on Galphaq signaling.

Airway function in health and disease as well as in response to bronchospastic stimuli (i.e., irritants, allergens, and inflammatory mediators) is controlled, in part, by cholinergic muscarinic receptor regulation of smooth muscle. In particular, the dependence of airway smooth muscle contraction/relaxation on heterotrimeric G protein-coupled receptor signaling suggests that these events underlie the responses regulating airway function. Galphaq-containing G proteins are proposed to be a prominent signaling pathway, and the availability of knockout mice deficient of this subunit has allowed for an investigation of its potential role in airway function. Airway responses in Galphaq-deficient mice (activities assessed by both tracheal tension and in vivo lung function measurements) were attenuated relative to wild-type controls. Moreover, ovalbumin sensitization/aerosol challenge of Galphaq-deficient mice also failed to elicit an allergen-induced increase in airway reactivity to methacholine. These findings indicate that cholinergic receptor-mediated responses are dependent on Galphaq-mediated signaling events and identify Galphaq as a potential target of preventative/intervening therapies for lung dysfunction.

Airway Resistance↗

PubMed2001

Gene expression profiles of light-induced apoptosis in arrestin/rhodopsin kinase-deficient mouse retinas.

To examine the molecular processes that lead to light-induced retinal degeneration, mutant mice deficient in arrestin and rhodopsin kinase were raised in the dark and then subjected to relatively low doses of white light. The kinetics of the subsequent induction of apoptosis, change in mRNA transcript level, and photoreceptor cell death were monitored. Analysis of transcript profiles identified clusters of genes that responded differently to illumination, including a cluster of photoreceptor-specific genes that showed marked decreases in levels long before morphological damage could be readily ascertained. The behaviors of other gene clusters demonstrate the coordinate induction of stress gene responses early in the course of irradiation. There was little, if any, change in transcript levels corresponding to genes associated with the initiation of apoptosis or antiapoptotic effects. Transcript analysis provides insight into the patterns of gene expression that are associated with the different stages of retinal degeneration in this model system.

Animals↗

PubMed2001

A diverse family of GPCRs expressed in specific subsets of nociceptive sensory neurons.

In vertebrates, peripheral chemosensory neurons express large families of G protein-coupled receptors (GPCRs), reflecting the diversity and specificity of stimuli they detect. However, somatosensory neurons, which respond to chemical, thermal, or mechanical stimuli, are more broadly tuned. Here we describe a family of approximately 50 GPCRs related to Mas1, called mrgs, a subset of which is expressed in specific subpopulations of sensory neurons that detect painful stimuli. The expression patterns of mrgs thus reveal an unexpected degree of molecular diversity among nociceptive neurons. Some of these receptors can be specifically activated in heterologous cells by RFamide neuropeptides such as NPFF and NPAF, which are analgesic in vivo. Thus, mrgs may regulate nociceptor function and/or development, including the sensation or modulation of pain.

Amino Acid Sequence↗

PubMed2001

RGS9-1 is required for normal inactivation of mouse cone phototransduction.

PURPOSE: To test the hypothesis that Regulator of G-protein Signaling 9 (RGS9-1) is necessary for the normal inactivation of retinal cones. METHODS: Mice having the gene RGS9-1 inactivated in both alleles (RGS9-1 -/-) were tested between the ages 8-10 weeks with electroretinographic (ERG) protocols that isolate cone-driven responses. Immunohistochemistry was performed with a primary antibody against RGS9-1 (anti-RGS9-1c), with the secondary conjugated to fluorescein isothiocyanate, and with rhodamine-conjugated peanut agglutinin. RESULTS: (1) Immunohistochemistry showed RGS9-1 to be strongly expressed in the cones of wildtype (WT is C57BL/6) mice, but absent from the cones of RGS9-1 mice. (2) Cone-driven b-wave responses of dark-adapted RGS9-1 -/- mice had saturating amplitudes and sensitivities in the midwave and UV regions of the spectrum equal to or slightly greater than those of WT (C57BL/6) mice. (3) Cone-driven b-wave and a-wave responses of RGS9-1 -/- mice recovered much more slowly than those of WT after a strong conditioning flash: for a flash estimated to isomerize 1.2% of the M-cone pigment and 0.9% of the UV-cone pigment, recovery of 50% saturating amplitude was approximately 60-fold slower than in WT. CONCLUSIONS: (1) The amplitudes and sensitivities of the cone-driven responses indicate that cones and cone-driven neurons in RGS9-1 -/- mice have normal generator currents. (2) The greatly retarded recovery of cone-driven responses of RGS9-1 -/- mice relative to those of WT mice establishes that RGS9-1 is required for normal inactivation of the cone phototransduction cascades of both UV- and M-cones.

Animals↗

PubMed2001

Conditionally expressed G alpha 15 couples to endogenous receptors in GH3 cells.

The mammalian G proteins G15 and G16 couple a wide variety of receptors to phospholipase C (PLC) in co-transfected systems, and it has been suggested that they can be used as tools in agonist-screening systems. Using the reversed tetracycline-controlled transactivation system we generated rat pituitary GH3 cell clones that expressed Galphal5 and Galpha16 conditionally to study the coupling of endogenous receptors to both G proteins. In cells expressing moderate levels of Galpha15, activation of various endogenous receptors increased inositol phosphate production, whereas conditional expression of Galpha16 had no significant effect on agonist-dependent PLC activity. Activation of PLC through Galpha15 in response to carbachol did not increase cytosolic [Ca2+] ([Ca2+]i) but stimulated protein kinase C. While carbachol decreased the secretory activity in non-induced GH3 cells, it increased secretion in cells expressing Galpha15. Our data demonstrate that Galpha15 has a higher functional promiscuity than Galpha16 when studied in a system that preserves physiological G protein and receptor levels. In addition, Galpha15-mediated coupling of a receptor to PLC can change the cellular response to receptor agonists, indicating that downstream cellular functions can be used to detect receptor activation in screening systems employing a promiscuous G protein.

Animals↗

PubMed2001

Nucleotide binding by the histidine kinase CheA.

To probe the structural basis for protein histidine kinase (PHK) catalytic activity and the prospects for PHK-specific inhibitor design, we report the crystal structures for the nucleotide binding domain of Thermotoga maritima CheA with ADP and three ATP analogs (ADPNP, ADPCP and TNP-ATP) bound with either Mg(2+) or Mn(2+). The conformation of ADPNP bound to CheA and related ATPases differs from that reported in the ADPNP complex of PHK EnvZ. Interactions of the active site with the nucleotide gamma-phosphate and its associated Mg(2+) ion are linked to conformational changes in an ATP-lid that could mediate recognition of the substrate domain. The inhibitor TNP-ATP binds CheA with its phosphates in a nonproductive conformation and its adenine and trinitrophenyl groups in two adjacent binding pockets. The trinitrophenyl interaction may be exploited for designing CheA-targeted drugs that would not interfere with host ATPases.

Adenosine Diphosphate↗

PubMed2001

COS-l, a putative two-component histidine kinase of Candida albicans, is an in vivo virulence factor.

The human fungal pathogen, Candida albicans, has three putative histidine kinases showing homology to those of plants, bacteria and other fungi. We have constructed a homozygous deletion strain and a hemizygous reconstituted strain of one of these histidine-kinase-encoding genes, COS-1, in C. albicans. Neither strain showed any growth defect in a number of liquid media nor increased resistance or sensitivity to a number of antifungal drugs. Importantly, we show that the COS-1 homozygous disruption strain had significantly reduced virulence in a systemic murine model of candidosis. Thus, COS-1 appears to be an in vivo virulence factor and may represent a novel target for the development of antifungal drugs.

Animals↗

PubMed2000

Modules in the photoreceptor RGS9-1.Gbeta 5L GTPase-accelerating protein complex control effector coupling, GTPase acceleration, protein folding, and stability.

RGS (regulators of G protein signaling) proteins regulate G protein signaling by accelerating GTP hydrolysis, but little is known about regulation of GTPase-accelerating protein (GAP) activities or roles of domains and subunits outside the catalytic cores. RGS9-1 is the GAP required for rapid recovery of light responses in vertebrate photoreceptors and the only mammalian RGS protein with a defined physiological function. It belongs to an RGS subfamily whose members have multiple domains, including G(gamma)-like domains that bind G(beta)(5) proteins. Members of this subfamily play important roles in neuronal signaling. Within the GAP complex organized around the RGS domain of RGS9-1, we have identified a functional role for the G(gamma)-like-G(beta)(5L) complex in regulation of GAP activity by an effector subunit, cGMP phosphodiesterase gamma and in protein folding and stability of RGS9-1. The C-terminal domain of RGS9-1 also plays a major role in conferring effector stimulation. The sequence of the RGS domain determines whether the sign of the effector effect will be positive or negative. These roles were observed in vitro using full-length proteins or fragments for RGS9-1, RGS7, G(beta)(5S), and G(beta)(5L). The dependence of RGS9-1 on G(beta)(5) co-expression for folding, stability, and function has been confirmed in vivo using transgenic Xenopus laevis. These results reveal how multiple domains and regulatory polypeptides work together to fine tune G(talpha) inactivation.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed2000

Direct genetic demonstration of G alpha 13 coupling to the orphan G protein-coupled receptor G2A leading to RhoA-dependent actin rearrangement.

G2A is an orphan G protein-coupled receptor (GPCR), expressed predominantly in T and B cells and homologous to a small group of GPCRs of unknown function expressed in lymphoid tissues. G2A is transcriptionally induced in response to diverse stimuli, and its ectopic expression suppresses transformation of B lymphoid precursors by BCR-ABL. G2A induces morphological transformation of NIH 3T3 fibroblasts. Microinjection of constructs encoding G2A into Swiss 3T3 fibroblasts induces actin reorganization into stress fibers that depends on RhoA, but not CDC42 or RAC. G2A elicits RhoA-dependent transcriptional activation of serum response factor. Direct evaluation of RhoA activity demonstrates elevated levels of RhoA-GTP in G2A-expressing cells. Microinjection of embryonic fibroblasts derived from various G alpha knockout mice establishes a requirement for G alpha 13 but not G alpha 12 or G alpha q/11 in G2A-induced actin rearrangement. In conclusion, G2A represents a family of GPCRs expressed in lymphocytes that may link diverse stimuli to cytoskeletal reorganization and transcriptional activation through a pathway involving G alpha 13 and RhoA.

Actins↗

PubMed2000

Molecular mechanism of the inhibition of phospholipase C beta 3 by protein kinase C.

Activation of protein kinase C (PKC) can result from stimulation of the receptor-G protein-phospholipase C (PLCbeta) pathway. In turn, phosphorylation of PLCbeta by PKC may play a role in the regulation of receptor-mediated phosphatidylinositide (PI) turnover and intracellular Ca(2+) release. Activation of endogenous PKC by phorbol 12-myristate 13-acetate inhibited both Galpha(q)-coupled (oxytocin and M1 muscarinic) and Galpha(i)-coupled (formyl-Met-Leu-Phe) receptor-stimulated PI turnover by 50-100% in PHM1, HeLa, COSM6, and RBL-2H3 cells expressing PLCbeta(3). Activation of conventional PKCs with thymeleatoxin similarly inhibited oxytocin or formyl-Met-Leu-Phe receptor-stimulated PI turnover. The PKC inhibitory effect was also observed when PLCbeta(3) was stimulated directly by Galpha(q) or Gbetagamma in overexpression assays. PKC phosphorylated PLCbeta(3) at the same predominant site in vivo and in vitro. Peptide sequencing of in vitro phosphorylated recombinant PLCbeta(3) and site-directed mutagenesis identified Ser(1105) as the predominant phosphorylation site. Ser(1105) is also phosphorylated by protein kinase A (PKA; Yue, C., Dodge, K. L., Weber, G., and Sanborn, B. M. (1998) J. Biol. Chem. 273, 18023-18027). Similar to PKA, the inhibition by PKC of Galpha(q)-stimulated PLCbeta(3) activity was completely abolished by mutation of Ser(1105) to Ala. In contrast, mutation of Ser(1105) or Ser(26), another putative phosphorylation target, to Ala had no effect on inhibition of Gbetagamma-stimulated PLCbeta(3) activity by PKC or PKA. These data indicate that PKC and PKA act similarly in that they inhibit Galpha(q)-stimulated PLCbeta(3) as a result of phosphorylation of Ser(1105). Moreover, PKC and PKA both inhibit Gbetagamma-stimulated activity by mechanisms that do not involve Ser(1105).

Cyclic AMP-Dependent Protein Kinases↗

PubMed2000

Polarity exchange at the interface of regulators of G protein signaling with G protein alpha-subunits.

RGS proteins are GTPase-activating proteins (GAPs) for G protein alpha-subunits. This GAP activity is mediated by the interaction of conserved residues on regulator of G protein signaling (RGS) proteins and Galpha-subunits. We mutated the important contact sites Glu-89, Asn-90, and Asn-130 in RGS16 to lysine, aspartate, and alanine, respectively. The interaction of RGS16 and its mutants with Galpha(t) and Galpha(i1) was studied. The GAP activities of RGS16N90D and RGS16N130A were strongly attenuated. RGS16E89K increased GTP hydrolysis of Galpha(i1) by a similar extent, but with an about 100-fold reduced affinity compared with non-mutated RGS16. As Glu-89 in RGS16 is interacting with Lys-210 in Galpha(i1), this lysine was changed to glutamate for compensation. Galpha(i1)K210E was insensitive to RGS16 but interacted with RGS16E89K. In rat uterine smooth muscle cells, wild type RGS16 abolished G(i)-mediated alpha(2)-adrenoreceptor signaling, whereas RGS16E89K was without effect. Both Galpha(i1) and Galpha(i1)K210E mimicked the effect of alpha(2)-adrenoreceptor stimulation. Galpha(i1)K210E was sensitive to RGS16E89K and 10-fold more potent than Galpha(i1). Analogous mutants of Galpha(q) (Galpha(q)K215E) and RGS4 (RGS4E87K) were created and studied in COS-7 cells. The activity of wild type Galpha(q) was counteracted by wild type RGS4 but not by RGS4E87K. The activity of Galpha(q)K215E was inhibited by RGS4E87K, whereas non-mutated RGS4 was ineffective. We conclude that mutation of a conserved lysine residue to glutamate in Galpha(i) and Galpha(q) family members renders these proteins insensitive to wild type RGS proteins. Nevertheless, they are sensitive to glutamate to lysine mutants of RGS proteins. Such mutant pairs will be helpful tools in analyzing Galpha-RGS specificities in living cells.

Adenylyl Cyclases↗

PubMed2000

Muscarinic inhibition of calcium current and M current in Galpha q-deficient mice.

Activation of M(1) muscarinic acetylcholine receptors (M(1) mAChR) inhibits M-type potassium currents (I(K(M))) and N-type calcium currents (I(Ca)) in mammalian sympathetic ganglia. Previous antisense experiments suggested that, in rat superior cervical ganglion (SCG) neurons, both effects were partly mediated by the G-protein Galpha(q) (Delmas et al., 1998a; Haley et al., 1998a), but did not eliminate a contribution by other pertussis toxin (PTX)-insensitive G-proteins. We have tested this further using mice deficient in the Galpha(q) gene. PTX-insensitive M(1) mAChR inhibition of I(Ca) was strongly reduced in Galpha(q) -/- mouse SCG neurons and was fully restored by acute overexpression of Galpha(q). In contrast, M(1) mAChR inhibition of I(K(M)) persisted in Galpha(q)-/- mouse SCG cells. However, unlike rat SCG neurons, muscarinic inhibition of I(K(M)) was partly PTX-sensitive. Residual (PTX-insensitive) I(K(M)) inhibition was slightly reduced in Galpha(q) -/- neurons, and the remaining response was then suppressed by anti-Galpha(q/11) antibodies. Bradykinin (BK) also inhibits I(K(M)) in rat SCG neurons via a PTX-insensitive G-protein (G(q) and/or G(11); Jones et al., 1995). In mouse SCG neurons, I(K(M)) inhibition by BK was fully PTX-resistant. It was unchanged in Galpha(q) -/- mice but was abolished by anti-Galpha(q/11) antibody. We conclude that, in mouse SCG neurons (1) M(1) mAChR inhibition of I(Ca) is mediated principally by G(q), (2) M(1) mAChR inhibition of I(K(M)) is mediated partly by G(q), more substantially by G(11), and partly by a PTX-sensitive G-protein(s), and (3) BK-induced inhibition of I(K(M)) is mediated wholly by G(11).

Animals↗

PubMed2000

Genomic characterization of the human heterotrimeric G protein alpha, beta, and gamma subunit genes.

Heterotrimeric guanine nucleotide binding proteins (G proteins) transduce extracellular signals received by transmembrane receptors to effector proteins. Each subunit of the G protein complex is encoded by a member of one of three corresponding gene families. Currently, 16 different members of the alpha subunit family, 5 different members of the beta subunit family, and 11 different members of the gamma subunit family have been described in mammals. Here we have identified and characterized Bacterial Artificial Chromosomes (BACs) containing the human homologs of each of the alpha, beta, and gamma subunit genes as well as a G alpha11 pseudogene and a previously undiscovered G gamma5-like gene. The gene structure and chromosome location of each gene was determined, as were the orientations of paired genes. These results provide greater insight into the evolution and functional diversity of the mammalian G protein subunit genes.

Amino Acid Sequence↗

PubMed2000

Robust perfect adaptation in bacterial chemotaxis through integral feedback control.

Integral feedback control is a basic engineering strategy for ensuring that the output of a system robustly tracks its desired value independent of noise or variations in system parameters. In biological systems, it is common for the response to an extracellular stimulus to return to its prestimulus value even in the continued presence of the signal-a process termed adaptation or desensitization. Barkai, Alon, Surette, and Leibler have provided both theoretical and experimental evidence that the precision of adaptation in bacterial chemotaxis is robust to dramatic changes in the levels and kinetic rate constants of the constituent proteins in this signaling network [Alon, U., Surette, M. G., Barkai, N. & Leibler, S. (1998) Nature (London) 397, 168-171]. Here we propose that the robustness of perfect adaptation is the result of this system possessing the property of integral feedback control. Using techniques from control and dynamical systems theory, we demonstrate that integral control is structurally inherent in the Barkai-Leibler model and identify and characterize the key assumptions of the model. Most importantly, we argue that integral control in some form is necessary for a robust implementation of perfect adaptation. More generally, integral control may underlie the robustness of many homeostatic mechanisms.

Acclimatization↗

PubMed2000

Mice lacking G-protein receptor kinase 1 have profoundly slowed recovery of cone-driven retinal responses.

G-Protein receptor kinase 1 (GRK1) ("rhodopsin kinase") is necessary for the inactivation of photoactivated rhodopsin, the light receptor of the G-protein transduction cascade of rod photoreceptors. GRK1 has also been reported to be present in retinal cones in which its function is unknown. To examine the role of GRK1 in retinal cone signaling pathways, we measured in mice having null mutations of GRK1 (GRK1 -/-) cone-driven electroretinographic (ERG) responses, including an a-wave component identified as the field potential generated by suppression of the circulating current of the cone photoreceptors. Dark-adapted GRK1 -/- animals generated cone-driven ERGs having saturating amplitudes and sensitivities in both visible and UV spectral regions similar to those of wild-type (WT) mice. However, after exposure to a bright conditioning flash, the cone-driven ERGs of GRK1 -/- animals recovered 30-50 times more slowly than those of WT mice and similarly slower than the cone-driven ERGs of mice homozygously null for arrestin (Arrestin -/-), whose cone (but not rod) response recoveries were found to be as rapid as those of WT. Our observations argue that GRK1 is essential for normal deactivation of murine cone phototransduction and provide the first functional evidence for a major role of a specific GRK in the inactivation of vertebrate cone phototransduction.

Animals↗

PubMed2000

Co-expression of Gbeta5 enhances the function of two Ggamma subunit-like domain-containing regulators of G protein signaling proteins.

Regulators of G protein signaling (RGS) stimulate the GTPase activity of G protein Galpha subunits and probably play additional roles. Some RGS proteins contain a Ggamma subunit-like (GGL) domain, which mediates a specific interaction with Gbeta5. The role of such interactions in RGS function is unclear. RGS proteins can accelerate the kinetics of coupling of G protein-coupled receptors to G-protein-gated inwardly rectifying K(+) (GIRK) channels. Therefore, we coupled m2-muscarinic acetylcholine receptors to GIRK channels in Xenopus oocytes to evaluate the effect of Gbeta5 on RGS function. Co-expression of either RGS7 or RGS9 modestly accelerated GIRK channel kinetics. When Gbeta5 was co-expressed with either RGS7 or RGS9, the acceleration of GIRK channel kinetics was strongly increased over that produced by RGS7 or RGS9 alone. RGS function was not enhanced by co-expression of Gbeta1, and co-expression of Gbeta5 alone had no effect on GIRK channel kinetics. Gbeta5 did not modulate the function either of RGS4, an RGS protein that lacks a GGL domain, or of a functional RGS7 construct in which the GGL domain was omitted. Enhancement of RGS7 function by Gbeta5 was not a consequence of an increase in the amount of plasma membrane or cytosolic RGS7 protein.

Animals↗