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Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay
Published on: July 28, 2014
Quantitative and realtime correlation between receptor aggregation and intracellular calcium signal transduction
1Faculty of Pharmaceutical Sciences, Nagoya City University, Japan.
Insights
Cellular calcium signals correlate with antigen properties. Optimal hapten density maximizes signals, but antigen size affects responses differently across cell types, suggesting specific receptor aggregate structures are key.
Area of Science:
- Immunology
- Cell Biology
- Biophysics
Background:
- Intracellular calcium signals are crucial for immune cell activation.
- Antigen structure, including hapten density and molecular size, influences receptor-mediated signaling.
- Understanding these relationships is vital for deciphering immune responses.
Purpose of the Study:
- To quantitatively correlate intracellular calcium signals with antigen hapten density and molecular size.
- To investigate differences in antigen recognition between hapten-specific murine B cells (TP67.21) and rat basophilic leukemia cells (RBL-2H3).
- To elucidate the role of receptor aggregation in calcium signal induction and abrogation.
Main Methods:
- Utilized hapten-specific murine B cells (TP67.21) and rat basophilic leukemia cells (RBL-2H3) expressing hapten-specific IgE.
- Measured intracellular calcium signals in response to varying hapten densities and molecular sizes of antigens.
- Employed two fluorescent calcium probes and confocal microscopy to observe receptor mobilization during signal abrogation.
Main Results:
- Both cell lines showed similar dependence of calcium signal magnitude on hapten density, with an optimal density for maximum signal.
- Cellular responses to antigen molecular size differed: TP67.21 cells responded better to larger antigens, while RBL-2H3 cells favored smaller antigens.
- Calcium signaling was abrogated by excess hapten, affecting both influx and intracellular release.
- Observation revealed that large receptor clusters were unaffected by hapten during abrogation, while smaller clusters are necessary for signal induction.
Conclusions:
- Optimal hapten density is a conserved factor for calcium signal induction across different cell types.
- Distinct cellular responses to antigen size suggest cell-specific optimal receptor aggregate structures.
- Smaller receptor clusters, not large ones, are essential for initiating calcium signals.
- Receptor mobilization and aggregation dynamics play a critical role in regulating immune cell signaling.
Abstract:
Quantitative correlation between intracellular calcium signals and hapten density or molecular size of antigens was studied for two cell lines; hapten-specific murine B cells (TP67.21) and rat basophilic leukemia cells (RBL-2H3) with hapten-specific IgE. Magnitude of the induced calcium signal in both cells exhibited the same dependence on hapten density of antigen molecules and there existed an optimal hapten density which induced the maximum amount of calcium signal for both cells. However, they responded differently to antigens of various molecular size. In contrast to TP67.21 cells which showed larger response to larger antigen molecules, RBL-2H3 cells showed the largest response to the smallest antigen. This may possibly suggest that there exists an optimal structure of receptor aggregates for each cell. Calcium signal induced in each cell by multivalent antigen was rapidly abrogated by addition of excess hapten and this abrogation occurred both in transmembrane influx and the release from intracellular stores. We directly observed the mobilization of receptor molecules during this calcium signal abrogation at single cell level by using two fluorescent calcium probes, whose fluorescence wavelength ranges have least overlap, and confocal microscopy. During this abrogation, large clusters of receptor molecules were not affected by hapten molecules. We, therefore, conclude that these large clusters are inactive in the induction of calcium signal and smaller clusters of receptor molecules are necessary for calcium signal induction.
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