Unexpected signals in a system subject to kinetic proofreading
- Section on Chemical Immunology, Arthritis and Rheumatism Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892
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Contributed by Henry Metzger
Abstract
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.
Footnotes
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↵ * Present address: The Wistar Institute, Room 489, 3601 Spruce Street, Philadelphia, PA 19104.
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↵ † Present address: LI-COR, Inc., 4308 Progressive Avenue, P.O. Box 4000, Lincoln, NE 68504.
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↵ ‡ To whom reprint requests should be addressed. E-mail: metzgerh{at}exchange.nih.gov.
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See commentary on page 6989.
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↵ § In most anti-hapten systems the forward rate constants, k +1, are more or less similar and the intrinsic affinity is largely governed by the reverse rate constant, k −1 (13). We confirmed this for the DNP and NP antigens used here by measuring the binding of the iodinated derivatives. We obtained a k +1 of ≈5 × 105 M−1⋅sec−1 for the binding of the DNP antigen to adherent cells such as used here (Results). That calculation was based on solving the equation for the rate at which equilibrium is achieved for a reversible reaction between reactants having an affinity (K a) of ≥1010 M−1. The latter value was estimated from separate experiments in which the binding of the DNP-modified antigen to cells in suspension was examined (data not shown). We were unable to determine k +1 for the NP antigen because of the high concentration of this antigen required to observe appreciable binding and the rapid rate of dissociation during washing of adherent cells. However, it is notable that the stimulation of the initial phosphorylation of FcɛRI induced by the NP-conjugated antigen occurs with similar kinetics as with the high affinity derivative (Fig. 1 Lower). Using cell suspensions, we estimated an avidity of ≈5 × 106 M−1 for the NP antigen.
- Abbreviations:
- FcɛRI,
- high-affinity receptor for IgE;
- MCP-1,
- monocyte chemoattractant protein 1;
- DNP,
- 2,4-dinitrophenyl;
- NP,
- 2-nitrophenyl;
- PY,
- phosphotyrosine
- Copyright © 2001, The National Academy of Sciences
