G-protein coupled receptors (GPCRs) are, as a class, the most common target of clinically used drugs. The standard model of GPCR function holds that receptors, G-proteins and effector molecules interact with each other sequentially and transiently (by collision coupling), allowing each receptor to activate several G-protein molecules, and each G-protein subunit to activate several effector molecules. Recent work has, however, cast doubt on the generality of collision coupling. Instead, it has been suggested that GPCR signaling molecules can be precoupled in """"""""signalosome"""""""" complexes that remain intact during signaling. Preassembly of complexes could facilitate rapid signaling and provide the receptor-effector specificity necessary for normal cell function. The long term objective of this research is to understand the spatial arrangement and temporal dynamics of signaling molecules in living cells. Accordingly, the goal of this project is to determine if GPCR signaling is mediated by collision coupling, by stable complexes of signaling molecules, or by a combination of these mechanisms. We have developed a simple technique to detect interactions between membrane proteins and to quantify the stability of such interactions in live cells. This technique measures changes in the lateral mobility of a membrane protein when a potential interacting partner is experimentally immobilized. Transmembrane proteins (e.g. GPCRs and ion channels) are immobilized in intact cells, and the lateral mobility of potentially interacting proteins is measured by monitoring fluorescence recovery after photobleaching. We will use this method together with standard electrophysiological techniques to test specific hypotheses about GPCR signaling complexes.
The specific aims are (i) to test the hypothesis that inactive GPCRs and G-protein heterotrimers form specific complexes that facilitate signaling; (2) to test the hypothesis that G-protein heterotrimers form complexes with inwardly-rectifying potassium (GIRK) channels; (3) to test the hypothesis that RGS proteins accelerate signal onset by forming stable complexes with GPCRs and/or G-protein heterotrimers; and (4) to determine if G-proteins dissociate into component Get and Gbg subunits during signaling. Currently available drugs act at the first step of GPCR signaling, namely binding of the drug (or a blocker) to the receptor. It is anticipated that future therapeutic drugs will target the subsequent steps of signaling. Development of such drugs will require a detailed understanding of these steps, e.g. when and where receptors, G-proteins and effector molecules interact with each other to transmit signals. The goal of this project is to provide this information. ? ? ?
|Wan, Qingwen; Okashah, Najeah; Inoue, Asuka et al. (2018) Mini G protein probes for active G protein-coupled receptors (GPCRs) in live cells. J Biol Chem 293:7466-7473|
|Martin, Brent R; Lambert, Nevin A (2016) Activated G Protein G?s Samples Multiple Endomembrane Compartments. J Biol Chem 291:20295-20302|
|Brown, Nicole E; Lambert, Nevin A; Hepler, John R (2016) RGS14 regulates the lifetime of G?-GTP signaling but does not prolong G?? signaling following receptor activation in live cells. Pharmacol Res Perspect 4:e00249|
|Zhang, Maoxiang; Davis, Jason E; Li, Chunman et al. (2016) GGA3 Interacts with a G Protein-Coupled Receptor and Modulates Its Cell Surface Export. Mol Cell Biol 36:1152-63|
|Donthamsetti, Prashant; Quejada, Jose Rafael; Javitch, Jonathan A et al. (2015) Using Bioluminescence Resonance Energy Transfer (BRET) to Characterize Agonist-Induced Arrestin Recruitment to Modified and Unmodified G Protein-Coupled Receptors. Curr Protoc Pharmacol 70:2.14.1-14|
|Lan, Tien-Hung; Wu, Guangyu; Lambert, Nevin A (2015) Lateral diffusion contributes to FRET from lanthanide-tagged membrane proteins. Biochem Biophys Res Commun 464:244-8|
|Lan, Tien-Hung; Liu, Qiuju; Li, Chunman et al. (2015) BRET evidence that ?2 adrenergic receptors do not oligomerize in cells. Sci Rep 5:10166|
|Chen, Yuanyuan; Tang, Hong; Seibel, William et al. (2015) A High-Throughput Drug Screening Strategy for Detecting Rhodopsin P23H Mutant Rescue and Degradation. Invest Ophthalmol Vis Sci 56:2553-67|
|Masuho, Ikuo; Martemyanov, Kirill A; Lambert, Nevin A (2015) Monitoring G Protein Activation in Cells with BRET. Methods Mol Biol 1335:107-13|
|Yeatman, Holly R; Lane, J Robert; Choy, Kwok Ho Christopher et al. (2014) Allosteric modulation of M1 muscarinic acetylcholine receptor internalization and subcellular trafficking. J Biol Chem 289:15856-66|
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