Browsing by Subject "Receptors, Muscarinic"
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Item FRET-Based Conformational Sensor for the m1 Muscarinic Cholinergic Receptor(2012-07-20) Chang, Seungwoo; Ross, Elliott M.Signaling behaviors of G-protein-mediated signaling are the outcome of a regulated cycle of GTP binding and hydrolysis. Binding of GTP, which activates G proteins, is promoted by an agonist-activated receptor; and hydrolysis of bound GTP, which deactivates G proteins, is accelerated by a GTPase-activating protein (GAP). These two processes need to be coordinately regulated to achieve fast turning on and off of signaling with robust signal output. I developed an optical conformational sensor for the m1 muscarinic cholinergic receptor (M1), a prototypical G protein-coupled receptor (GPCR), to study how receptor activity is regulated by the coordinated action of agonist, G protein and GAP. To create the sensor, I adopted an underlying design originally developed by the Lohse group. The sensor exploits intramolecular fluorescence resonance energy transfer, FRET, to monitor activation-associated conformational changes in intracellular loop 3 of the receptor. In the sensor, a CFP FRET donor and a labeling site for FlAsH (fluorescein-based biarsenical dye) FRET acceptor are engineered into the M1 receptor at the C terminus and loop 3, respectively. The development proceeded through several distinct optimization steps that probably reflect general considerations for developing such sensors for class A GPCRs. After optimizing the labeling conditions to approach stoichiometric derivatization by FlAsH, I found that the fluorescence response of the sensor depended on: (1) the location of the FlAsH labeling site in loop 3; (2) the length of the C-terminal region, which apparently acts as a lever arm, prior to placement of the CFP; and (3) the choice among circularly permuted CFP moieties. Finally, based on a homology-modeled structure of the M1 receptor, placement of the FlAsH site and the length of its flexible linkers were re-optimized to prevent interference with binding of the sensor to G?q. The sensor retained essentially wild-type agonist binding and signaling activity of the M1 receptor in living cells and cell membranes. Fluorescence responses of the sensor to muscarinic agonists paralleled their cellular efficacies. The sensor in living cells faithfully reported agonist-driven conformational change of the M1 receptor. Therefore, the FRET-based sensor proves to be a useful tool to investigate the mechanisms by which conformational dynamics of the M1 receptor is regulated by agonist in living cells and membranes. Effects of G?q on the conformation of the M1 receptor could not be determined because stable interaction between the M1 receptor and G?q could not be detected in cells or cell membranes either by fluorescence change or by agonist binding affinity; this is also true for wild-type receptor. Although the sensor reconstituted in phospholipid vesicles retained wild-type agonist binding and signaling function, fluorescence response to agonist was not detected in the vesicles. I demonstrated that solubilization of the sensor denatured a substantial fraction of the sensor, resulting in low fractional ligand binding activity of in vitro labeled sensor and thus artifactually low fluorescence response to agonist.Item Starvation Response in Caenorhabditis elegans(2009-01-14) Kang, Chanhee; Avery, LeonWhen the supply of environmental nutrients is limited, multicellular animas can make physiological and behavioral changes so as to cope with nutrient starvation. Although starvation response is essential for the survival of animals during nutrient deprivation, uncontrolled or uncoordinated starvation responses could be harmful. Autophagy, a lysosomal degradation pathway for long-lived proteins and cytoplasmic organelles, is known to be an important starvation response, which promotes both cell and organism survival by providing fundamental building blocks to maintain energy homeostasis during starvation. Under different conditions, however, autophagy may instead act to promote cell death through an autophagic cell death pathway. Why autophagy acts in some instances to promote survival but in others to promote death is poorly understood. Here I show that physiological levels of autophagy act to promote survival in Caenorhabditis elegans during starvation, whereas insufficient or excessive levels of autophagy contribute to death. I find that inhibition of autophagy decreases survival of wildtype worms during starvation. Furthermore, I find that in gpb-2 starvationhypersensitive mutants, starvation induces excessive autophagy in pharyngeal muscles, which in turn, causes damage that may contribute to death. These results demonstrate that, depending on level of its activation, autophagy can have either prosurvival or prodeath functions, providing in vivo evidence that an uncontrolled starvation response could be harmful to animals. Thus, it is important that animals ensure that their starvation response is coordinated between individual cells. However, the mechanisms by which animals sense starvation systemically remain elusive. Here I use gpb-2 mutants to identify molecules and mechanisms that modulate starvation signaling. I found that specific amino acids could suppress the starvation-induced death of gpb-2 mutants, and that MGL-1 and MGL-2, C. elegans homologs of metabotropic glutamate receptors, were involved. MGL-1 and MGL-2 acted in AIY and AIB neurons respectively. Treatment with leucine suppressed starvation-induced stress resistance and life span extension in wild-type worms, and mutation of mgl-1 and mgl-2 abolished these effects of leucine. Theses results suggest that metabotropic glutamate receptor homologs in AIY and AIB neuron may modulate a systemic starvation response in C. elegans.