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Real-time detection of G protein activation using monomolecular Gγ FRET sensors.
MedLine Citation:
PMID:  23336397     Owner:  NLM     Status:  Publisher    
Abstract/OtherAbstract:
Abstract G protein-coupled receptors (GPCRs) are involved in many diseases, and are the target of a large percentage of modern drugs. While only a few fluorescence resonance energy transfer (FRET) sensors have been made for real-time detection of GPCR activation, the human genome encodes roughly 950 GPCRs and a need for a broad real-time detection system is needed. In this study, we developed and characterized a new type of G protein sensor based on the Gγ subunit containing an intra-molecular FRET pair to allow detection of real-time G protein activation in multiple cell lines using time-resolved fluorescence spectroscopy. Our Gγ sensors were able to detect G protein activation to aluminum fluoride, a G protein activator, in human embryonic kidney 293 cells (HEK293). Moreover, our sensors were able to couple to the bradykinin B(2), parathyroid type 1 and adrenergic 2A GPCRs and detect G protein activation in response to the cognate ligands; bradykinin, parathyroid and noradrenaline hormones, respectively. Furthermore, our sensors also detected ligand-independent G protein activation by fluid shear stress. G protein inhibitors, pertussis toxin and guanosine 5' [β-thio] diphosphate, inhibited the FRET response to G protein stimulants indicating that the sensor response is specific to G protein activation. These findings suggest that the described Gγ sensors can be used to monitor real-time G protein activation by a variety of G protein stimulants or inhibitors.
Authors:
Jose Candelario; Mirianas Chachisvilis
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Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2013-1-22
Journal Detail:
Title:  Journal of receptor and signal transduction research     Volume:  -     ISSN:  1532-4281     ISO Abbreviation:  J. Recept. Signal Transduct. Res.     Publication Date:  2013 Jan 
Date Detail:
Created Date:  2013-1-22     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  9509432     Medline TA:  J Recept Signal Transduct Res     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
Affiliation:
La Jolla Bioengineering Institute , San Diego, CA , USA.
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