Browsing by Subject "signal transduction"
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Item Intradimer and interdimer methylation response by bacterial chemoreceptors to attractant stimulus(Texas A&M University, 2007-04-25) Bormans, Arjan FrankThis study focuses on the mechanism of transmembrane signaling by Tar, the aspartate chemoreceptor of Escherichia coli. Like other bacterial chemoreceptors, Tar localizes to the cell membrane and relays information about the external chemical environment through the membrane to a cytoplasmic signaling domain. The output of the signaling domain controls the directional bias of the rotary flagellar motors of the cell. Net movement of a cell in a chemical gradient involves temporal comparison of the current concentration with the concentration in the recent (a few seconds) past. The current concentration is measured as the percent occupancy of the extracellular ligand-binding domain of the receptor, and the past is represented by the extent of covalent methylation of four conserved glutamyl residues in the cytoplasmic domain. Under steady-state conditions, the methylation level corresponds to ligand occupancy. Tar is a dimer, and much evidence suggests that dimers associate into trimers of dimers. Higher-order arrays of receptors form in the presence of the cytoplasmic proteins CheA and CheW. The conformational change generated by ligand binding is transmitted through the membrane by one subunit of a dimer. To examine whether this initially asymmetric signal becomes symmetric within the cytoplasmic domain, I examined aspartate-induced adaptive methylation of the two subunits of mutant Tar receptor heterodimers. In the presence of CheA and CheW, adaptive methylation after addition of aspartate was symmetric, but in their absence, although the level of methylation increased, the rates were different for the two subunits. I also found that cross-talk, at the level of adaptive methylation, occurs between different receptor types even in the absence of CheA and CheW. These results provide support for the idea that a tight association of receptor dimers within trimers of dimers allows for an actively signaling receptor to affect the methylation state, and thus presumably the signaling state, of receptors within a trimer that are not bound to an attractant ligand.Item The Role of Docosahexaenoic Acid in Regulation of Epidermal Growth Factor Receptor Activation and Function(2012-08-30) Turk, Harmony 1985-The epidermal growth factor receptor (EGFR) is a transmembrane receptor tyrosine kinase integral in regulating cell growth, survival, and migration. EGFR signaling, which is dependent on localization of the receptor within lipid rafts, is often hijacked during colon tumorigenesis. Previous work has found that docosahexaenoic acid (DHA) is protective against colon cancer. This fatty acid is proposed to function in part by perturbing lipid rafts and thereby altering cell signaling. The overall objective of this work was to determine whether DHA alters EGFR function and signaling. We assessed EGFR localization and ligand-induced phosphorylation in YAMC cells treated with fatty acids. We found that DHA reduced the localization of EGFR to lipid rafts. Concomitant with altering receptor localization, DHA was found to increase EGFR phosphorylation. However, DHA paradoxically suppressed EGFR signal transduction. We found that DHA uniquely altered EGFR activity, and other long chain polyunsaturated fatty acid did not exert the same effect. We additionally observed similar effects on EGFR activation and signaling by feeding mice a diet enriched in fish oil (high in DHA), and this was attendant with reduced colon tumorigenesis. We next probed the mechanism by which DHA enhances EGFR phosphorylation. We found that DHA facilitates receptor dimerization to increase phosphorylation. We additionally identified Ras activation as the site of perturbation of signal transduction. DHA suppressed signal transduction by both changing the localization of EGFR within the plasma membrane and increasing receptor endocytosis and degradation. Lastly, we extended our observations into a wounding model. Although DHA uniquely altered ligand-stimulated EGFR activity, both DHA and EPA altered EGFR transactivation and signaling upon injury. This culminated in reduced wound healing in DHA and EPA treated cells. In an animal model, we found that diets enriched in either DHA or EPA altered EGFR signaling in the colonocytes of wounded animals. Overall, we found that DHA modifies EGFR signaling, which can be beneficial or detrimental for health depending on the disease state of an individual. These data help elucidate a mechanism by which DHA protects against colon cancer, as well as indicating a potential downside of n-3 PUFA therapy.Item The role of protein-membrane interactions in modulation of signaling by bacterial chemoreceptors(2009-05-15) Draheim, Roger RussellEnvironmental signals are sensed by membrane-spanning receptors that communicate with the cell interior. Bacterial chemoreceptors modulate the activity of the CheA kinase in response to binding of small ligands or upon interaction with substrate-bound periplasmic-binding proteins. The mechanism of signal transduction across the membrane is a displacement of the second transmembrane domain (TM2) a few angstroms toward the cytoplasm. This movement repositions a dynamic transmembrane helix relative to the plane of the cell membrane. The research presented in this dissertation investigated the contribution of TM2-membrane interactions to signaling by the aspartate chemoreceptor (Tar) of Escherichia coli. Aromatic residues that reside at the cytoplasmic polar-hydrophobic membrane interface (Trp-209 and Tyr-210) were found to play a significant role in regulating signaling by Tar. These interactions were subsequently manipulated to modulate the signaling properties of Tar. The baseline signaling state was shown to be incrementally altered by repositioning the Trp-209/Tyr-210 pair. To our knowledge, this is the first example of harnessing membrane-protein interactions to modulate the signal output of a transmembrane receptor in a controlled and predictable manner. Potential long-term applications include the use of analogous mutations to elucidate two-component signaling pathways, to engineer the signaling parameters of biosensors that incorporate chemoreceptors, and to predict the movement of dynamic transmembrane helices in silico.