- molecular immunology
- biophysical chemistry
- cell Biology
The Baird/Holowka laboratory employs a broad range of biochemical, biophysical, and immunological methods to investigate the structure and molecular mechanisms of cell surface receptors in the immune response. Biophysical and biochemical studies are carried out in conjunction with measurements of cellular activities so as to determine what features are critical for the initiation and regulation of signal transduction. As described below, current studies focus mainly on three receptor systems that operate in immunological and other inflammatory responses. The goal of these integrated studies is to understand complex biological systems on a molecular level.
The mast cell surface receptor FceRI binds immunoglobulin E (IgE) with high affinity to mediate the release of histamine from intracellular granules during the allergic immune response. The critical event in the process is the aggregation of a few receptor molecules, which is initiated in vivo by the molecular bridging of receptor-bound IgE with oligovalent ligands (antigens). Ongoing studies are examining the importance of structural orientations of crosslinked IgE-receptor complexes in determining triggering vs. non-triggering configurations. The kinetics and thermodynamics of binding and crosslinking between cell-bound IgE and structurally defined oligovalent ligands are measured with fluorescence methods and analyzed in detail with realistic theoretical models. Quantitative fluorescence confocal microscopy and bright fluorescent probes allow monitoring of the lateral diffusion of membrane receptors and changes in the distribution and dynamics of receptors on the cell surface that accompany receptor cross-linking and cellular activation. These approaches have revealed interactions between cross-linked receptors and other cellular components that may be involved in the signal transduction mechanism. A variety of biochemical methods are being used to investigate the molecular composition of the complexes formed with the receptors. The signalling activities induced by activated receptors (e.g., protein kinases and phosphatases, phospholipases, Ca2+ mobilization) are measured in parallel and related to the physical changes observed. A major current thrust of these integrated studies is elucidating the apparent importance of specialized membrane domains in the receptor-mediated signal transduction process.
The receptor for antigen on T-cells (TCR) provides the means by which T-cells recognize and kill the target cells bearing the foreign antigens, or stimulate other immunological responses to fend off the invasion. Similar to FceRI receptors, crosslinking of TCR receptors appears to play an important role in cellular activation, and many of the same biochemical activities are involved in the signal transduction process. These similarities as well as the structural and mechanistic features unique to the TCR system are being investigated with the experimental methods established and under development in this laboratory. For example, fluoresence resonance energy transfer (FRET) is being used to map the dispositions of TCR subunits and monitor TCR aggregation together with accessory molecules. TCR association with membrane domains and cytoskeleton is also being investigated.
The soluble cytokine interleukin-1 (IL-1) stimulates a large variety of cells in the manifestation of immunological and inflammatory responses. FRET measurements in the Baird/Holowka lab recently revealed that IL-1 causes aggregation of its receptor. Further characterization of this process is ongoing and the roles of cytoskeletal interactions and endocytosis in receptor function are being investigated.
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