FRET-Based Conformational Sensor for the m1 Muscarinic Cholinergic Receptor

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Title: FRET-Based Conformational Sensor for the m1 Muscarinic Cholinergic Receptor
Author: Chang, Seungwoo
Abstract: 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 . [Keywords : FRET , conformational sensor , m1 muscarinic cholinergic , GPCR , FlAsH]
URI: http : / /hdl .handle .net /2152 .5 /1019
Date: 2012-07-20


FRET-Based Conformational Sensor for the m1 Muscarinic Cholinergic Receptor. Graduate School of Biomedical Sciences. Available electronically from http : / /hdl .handle .net /2152 .5 /1019 .

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