This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.G-protein coupled receptors (GPCRs) are crucial elements in the transduction of information from the extracellular to the intracellular side of a cell. Numerous drugs act via this class of receptors to alter cellular functions. We showed that activation of GPCRs could be monitored by fluorescence resonance energy transfer (FRET) between two variants of the green fluorescent protein (GFP) fused to the receptor. However, the pharmacological profile of this fusion protein is different from that of the native GPCR its kinetics appeared to be slowed down by the large size of the fluorescent reporters. In collaboration with NCMIR at UCSD, we propose to slim down the reporter size by swapping one of the fluorescent proteins with the smaller tetracysteine peptide and test the resulting fusion for its activation kinetics. We will initially test the behavior of this reporter combination in fusion to the A2A receptor, and then extend it to other interesting GPCRs.
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