Search PubMed⌕ Search

Biomedical subjects

H Metzger

Publications and source records attributed to H Metzger.

At least 19 recordsLinked to original sources
PubMed2001

Kinetic proofreading models for cell signaling predict ways to escape kinetic proofreading.

In the context of cell signaling, kinetic proofreading was introduced to explain how cells can discriminate among ligands based on a kinetic parameter, the ligand-receptor dissociation rate constant. In the kinetic proofreading model of cell signaling, responses occur only when a bound receptor undergoes a complete series of modifications. If the ligand dissociates prematurely, the receptor returns to its basal state and signaling is frustrated. We extend the model to deal with systems where aggregation of receptors is essential to signal transduction, and present a version of the model for systems where signaling depends on an extrinsic kinase. We also investigate the kinetics of signaling molecules, "messengers," that are generated by aggregated receptors but do not remain associated with the receptor complex. We show that the extended model predicts modes of signaling that exhibit kinetic discrimination for some range of parameters but for other parameter values show little or no discrimination and thus escape kinetic proofreading. We compare model predictions with experimental data.

Animals↗
PubMed2001

Unexpected signals in a system subject to kinetic proofreading.

When multivalent ligands attach to IgEs bound to the receptors with high affinity for IgE on mast cells, the receptors aggregate, tyrosines on the receptors become phosphorylated, and a variety of cellular responses are stimulated. Prior studies, confirmed here, demonstrated that the efficiency with which later events are generated from earlier ones is inversely related to the dissociation rate of the aggregating ligand. This finding suggests that the cellular responses are constrained by a "kinetic proofreading" regimen. We have now observed an apparent exception to this rule. Doses of the rapidly or slowly dissociating ligands that generated equivalent levels of tyrosine-phosphorylated receptors comparably stimulated a putatively distal event: transcription of the gene for monocyte chemoattractant protein 1. Possible explanations of this apparent anomaly were explored.

Animals↗
PubMed2001

Spontaneous phosphorylation of the receptor with high affinity for IgE in transfected fibroblasts.

Receptors with high affinity for IgE, FcepsilonRI, which had been transfected into Chinese hamster ovary fibroblasts exhibit an over 20-fold greater spontaneous phosphorylation at physiological temperatures than the same receptors on the widely studied rat mucosal mast cell line, RBL-2H3. This enhanced phosphorylation was not accounted for either by changes in the src-family kinase responsible for the phosphorylation, by reduced activity of phosphatases, or by spontaneous association of the receptors with microdomains. A variety of approaches failed to detect evidence for stable spontaneous aggregates of the receptor. Whereas the altered posttranslational glycosylation of the receptor's principal ectodomain we detected could promote transient spontaneous aggregation and explain the observed effect, other changes in the membrane milieu cannot be excluded. The functional consequences of such spontaneous phosphorylation are considered.

Amino Acid Sequence↗
PubMed2001

Interaction between the unphosphorylated receptor with high affinity for IgE and Lyn kinase.

Chinese hamster ovary fibroblasts previously transfected with the high affinity receptor for IgE (FcepsilonRI) were further transfected with the alpha subunit of the receptor for interleukin 2 (Tac) or with chimeric constructs in which the cytoplasmic domain of Tac was replaced with the C-terminal cytoplasmic domain of either the beta subunit or the gamma subunit of FcepsilonRI. Whereas native Tac failed to affect the aggregation-induced phosphorylation of FcepsilonRI, both chimeric constructs substantially inhibited this reaction. Alternatively, the FcepsilonRI-bearing fibroblasts were transfected with two chimeric constructs in which the cytoplasmic domain of Tac was replaced with a modified short form of Lyn kinase. The Lyn in both of the chimeric constructs had been mutated to remove the sites that are normally myristoylated and palmitoylated, respectively; one of the constructs had in addition been altered to be catalytically inactive. The catalytically active construct enhanced, and the inactive construct inhibited, aggregation-induced phosphorylation of the receptors. All of the chimeric constructs were largely distributed outside the detergent resistant microdomains, and whereas aggregation caused them to move to the domains in part, their aggregation was neither necessary nor enhanced their effects. These results and others indicate that the receptor and Lyn interact through protein-protein interactions that neither are dependent upon either the post-translational modification of the kinase with lipid moieties nor result exclusively from their co-localization in specialized membrane domains.

Adenosine Triphosphate↗
PubMed2001

[Selective pre- intra- and postoperative bile duct diagnosis--an efficient and low complication regimen within the scope of laparoscopic cholecystectomy. Early and late results of a prospective study].

INTRODUCTION: The discussion of diagnostic pre- and intraoperative procedures of the common bile duct (CBD) for laparoscopic cholecystectomy is still open. METHODS: Results of a prospective study of 4000 laparoscopic cholecystectomies were evaluated, adhering to the concept of selective common bile duct examination. In 1999 an inquiry was performed on 1197 patients an average of 38.7 months postoperatively to judge this concept. RESULTS: Three hundred and nineteen patients (8%) examined preoperatively by ERC revealing a CBD stone in 126 cases. The morbidity of this procedure was 3.1%. In 0.18% a lesion of the common bile duct was documented following laparoscopic cholecysTectomy. A intraoperative cholangiography was performed in 67 patients (1.7%) because of unclear anatomy or to exclude a CBD stone. Perioperatively a CBD stone was found in 0.5% of cases. Additionally, in 0.3% the same problem occurred in the long-term follow-up, so that the risk for unknown CBD stones following the selective diagnostic concept is 0.8%. CONCLUSION: The concept of selective diagnostic procedures of the common bile duct shows a low morbidity with sufficient efficiency in avoiding CBD stones as well as duct lesions. Therefore it is recommended for routine use in laparoscopic cholecystectomy.

Cholangiography↗
PubMed2000

Detergent-resistant microdomains offer no refuge for proteins phosphorylated by the IgE receptor.

When the high affinity receptor for IgE and related receptors become aggregated, they emigrate to specialized microdomains of the plasma membrane that are enriched in certain lipids and lipid-anchored proteins. Among the latter are the kinases that initiate signaling cascade(s) by phosphorylating the receptors. In studying the IgE receptor, we explored whether, in addition to their potential role in enhancing the initiation of signaling by the kinase(s), the microdomains might augment the stimulation by excluding phosphatases. In vitro assessment of phosphatase activity, using either a relevant or irrelevant substrate, suggested that the microdomains were deficient in phosphatase activity, but, in vivo, proteins confined to the microdomains were found to be no less vulnerable to dephosphorylation than those outside such domains. In the course of our experiments, we observed that the procedures routinely used to isolate the detergent-resistant domains dissociated the receptor for IgE, thereby artificially accentuating the observed preferential distribution of phosphorylated subunits in the microdomains.

Adaptor Proteins, Signal Transducing↗
PubMed1999

One lyn molecule is sufficient to initiate phosphorylation of aggregated high-affinity IgE receptors.

In response to antigenic stimuli, the multisubunit immune recognition receptors become aggregated and then phosphorylated on their cytoplasmic tyrosines. For the clonotypic receptors of B and T cells and for Fc receptors such as the high-affinity receptor for IgE (FcepsilonRI), a Src family kinase initiates this phosphorylation. We ask whether aggregation of the initiating kinase itself is required for signal transduction or whether, alternatively, a single associated kinase molecule can phosphorylate the receptors in an aggregate. We formulate the alternative molecular mechanisms mathematically and compare predictions with experimental findings on FcepsilonRI-bearing cells expressing varying amounts of the transfected Src family kinase Lyn. The data are consistent with the requirement of only a single Lyn molecule per FcepsilonRI aggregate to initiate signaling and are inconsistent with a mechanism requiring more than one Lyn molecule.

Animals↗
PubMed1999

A quantitative approach to signal transduction.

The high affinity receptor for IgE (FcepsilonRI), is one of a family of immunoreceptors whose antigen-induced clustering leads to a variety of cellular responses. The signaling pathways are enormously complex but by focusing on only the most initial steps, it is now possible to sketch plausible molecular models that relate the interaction of multivalent antigens with the receptor-bound IgE to the earliest cellular events. In this paper, we describe how we have combined quantitative experimentation and mathematical modeling to probe this system further. We also discuss some of the formidable challenges that remain before we can claim reasonably complete understanding of even these early events.

Animals↗
PubMed1998

Characterization of gene expression in resting and activated mast cells.

To characterize gene expression in activated mast cells more comprehensively than heretofore, we surveyed the changes in genetic transcripts by the method of serial analysis of gene expression in the RBL-2H3 line of rat mast cells before and after they were stimulated through their receptors with high affinity for immunoglobulin E (FcepsilonRI). A total of 40,759 transcripts derived from 11,300 genes were analyzed. Among the diverse genes that had not been previously associated with mast cells and that were constitutively expressed were those for the cytokine macrophage migration inhibitory factor neurohormone receptors such as growth hormone- releasing factor and melatonin and components of the exocytotic machinery. In addition, several dozen transcripts were differentially expressed in response to antigen-induced clustering of the FcepsilonRI. Included among these were the genes for preprorelaxin, mitogen-activated protein kinase kinase 3, and the dual specificity protein phosphatase, rVH6. Significantly, the majority of genes differentially expressed in this well-studied model of mast cell activation have not been identified before this analysis.

Animals↗
PubMed1998

An unusual mechanism for ligand antagonism.

The ratio of late to early events stimulated by the mast cell receptor for immunoglobulin E (IgE) correlated with the affinity of a ligand for the receptor-bound IgE. Because excess receptors clustered by a weakly binding ligand could hoard a critical initiating kinase, they prevented the outnumbered clusters engendered by the high-affinity ligands from launching the more complete cascade. A similar mechanism could explain the antagonistic action of some peptides on the activation of T cells.

2,4-Dinitrophenol↗
PubMed1998

Eureka! And other pleasures.

At first one is very pleased at being invited to write a Prefatory Chapter, but as the delivery deadline draws closer one begins to think, "Oh my God! What on earth can I say that all but family members and few close friends will not find a great bore?" One solution is to write a scientific essay, but I concluded that that was a cop-out. I decided that perhaps the best tack to follow was to try to convey to the reader the personal characteristics I bring to my science and to other aspects of my professional career. The writing of this chapter has certainly convinced me that my particular background influenced what problems I chose to work on and how I approached their solution, but I hope that my results have a more ecumenical significance. There's been much written recently about how one's cultural background affects one's science, but I think that thesis can also be exaggerated. Science is a method of inquiry that by using certain guidelines permits rational individuals to observe Nature in a way that their findings will agree and have permanence. We shouldn't be diffident about defending that claim of objectivity.

Allergy and Immunology↗
PubMed1997

Exploiting the difference between intrinsic and extrinsic kinases: implications for regulation of signaling by immunoreceptors.

When receptors must interact with an extrinsic kinase to initiate signaling, the kinase can play a regulatory role that is not available to intrinsic receptor kinases. Whether control is exercised at this level depends critically on the amount of kinase available to the receptors and on the potential for redistribution of the kinase during signaling. This study demonstrates that the high affinity receptor for IgE (Fc epsilonRI) on rat basophilic leukemia cells is regulated by its initiating kinase. We present a mathematical model that allows for the reversible recruitment of extrinsic kinases to phosphorylated immunoreceptor tyrosine-based activation motifs. By comparing model predictions to experimental time courses of phosphorylation, we infer that Lyn is limiting, that redistribution occurs after receptors are aggregated, and that the redistribution makes the relationship between tyrosine phosphorylation and receptor aggregation nonlinear.

Animals↗
PubMed1997

The unique domain as the site on Lyn kinase for its constitutive association with the high affinity receptor for IgE.

Aggregation of the high affinity receptor for IgE (FcepsilonRI) leads to the phosphorylation of tyrosines on the beta and gamma chains of the receptor by the Src family kinase Lyn. We have studied the interaction between Lyn and the FcepsilonRI in vivo using a transfection-based approach. FcepsilonRI were stably transfected into Chinese hamster ovary cells. The small amount of endogenous Src family kinase was sufficient to phosphorylate receptor tyrosines upon extensive aggregation of FcepsilonRI but not after addition of dimers of IgE. Upon stable co-transfection of Lyn kinase into the cells, dimers were now able to stimulate receptor phosphorylation and the response to more extensive aggregation was enhanced. In contrast, co-transfection with catalytically inactive Lyn inhibited the aggregation-induced phosphorylation by the endogenous kinase, and a quantitatively similar inhibition was observed in cells transfected with the SH4-containing unique domain of Lyn. Consistent with the results of others using alternative approaches, our additional studies using a yeast two-hybrid system detected a direct interaction between intact Lyn or its unique domain and the C-terminal cytoplasmic domain of the beta chain but not with the receptor's other cytoplasmic domains.

Animals↗
PubMed1997

Characterization of protein-tyrosine phosphatases that dephosphorylate the high affinity IgE receptor.

An early event that follows aggregation of the high affinity receptor for IgE (FcepsilonRI) is the phosphorylation of protein tyrosines, especially those on the beta- and gamma-subunits of the receptor. Disaggregation of the receptors leads to their rapid dephosphorylation, but even stably aggregated receptors undergo continual rounds of phosphorylation and dephosphorylation. We developed assays to study dephosphorylation of the receptors and other cellular proteins. Whole cell extracts dephosphorylated both subunits of the receptors rapidly and were as active against aggregated as against disaggregated FcepsilonRI. Upon disaggregation, the in vivo dephosphorylation of the FcepsilonRI and several other proteins followed first-order kinetics with closely similar rate constants despite substantial differences in the extent of phosphorylation. These results suggest that the level of phosphorylation of FcepsilonRI is largely controlled by the aggregation-induced action of kinase(s) and not from changes in susceptibility to or activity of the phosphatases. Much of the total phosphatase is lost when the cells are permeabilized, but the rate of dephosphorylation of disaggregated FcepsilonRI was comparable in intact and permeabilized cells. Thus, much of the activity utilized by the cell to dephosphorylate the FcepsilonRI is likely to be associated with the plasma membrane.

Animals↗
PubMed1997

Biochemical evidence that the phosphorylated tyrosines, serines, and threonines on the aggregated high affinity receptor for IgE are in the immunoreceptor tyrosine-based activation motifs.

Activation of cells mediated by the high affinity receptor for IgE leads to rapid phosphorylation of tyrosines (and later other residues) on the receptor's beta and gamma subunits, and there is circumstantial evidence that the tyrosines modified are in the so-called immunoreceptor tyrosine-based activation motifs (ITAMs). We identified and quantitated the residues phosphorylated on the subunits of the native receptor by comparing the properties of peptides derived from the receptors radiolabeled in vivo or in vitro with those of synthetic peptides. Our results with receptors labeled in vivo confirm that only the tyrosines in the ITAMs of beta and gamma became phosphorylated, and preferentially, those in the canonical YXX(L/I) sequences. The extent of phosphorylation of the canonical tyrosines was of the same order of magnitude, but the amino-terminal canonical tyrosine in the ITAM of the beta subunit was consistently phosphorylated to a lesser degree. The non-canonical ITAM tyrosine in the beta subunit was considerably less phosphorylated. Phosphorylation of serine (on beta) and threonine (on gamma) also occurred mainly in the ITAMs, but selectively at some positions whose characteristics seem to be conserved among other receptors containing ITAMs. The studies with receptor complexes isolated and radiolabeled in vitro gave similar results for phosphorylation of tyrosines, suggesting that the latter, much simpler system is a useful model for more detailed studies.

Amino Acid Sequence↗