DEVELOPMENT OF A FLUORESCENCE BASED TESTSYSTEM FOR GPCR ACTIVATION

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. G-protein coupled receptors (GPCRs) play a crucial role in the transduction of information from the extracellular environment. We monitored activation of GPCRs by fluorescence resonance energy transfer (FRET) between two variants of the green fluorescent protein (CFP and YFP) inserted into the A2A receptor. However, the tagging with two bulky fluorescent proteins eliminates coupling to adenylyl cyclase and slows down the pharmacokinetics of the receptor. In collaboration with NCMIR, we developed a less disruptive FRET-based approach by replacing one of the fluorescent protein with the much smaller tetracysteine tag, expressed the resulting fusion protein in HeLa and HEK293 cells and studied its behavior during receptor activation. Radioligand binding parameters and adenylylcyclase activation were identical to those of the wild-type A2A-receptor. Real-time monitoring of the activation switch of the A2A-receptor uncovered activation kinetics with at 66-88 msec time constant. This novel approach is a significant improvement to the dual GFP-based approach as it enables us to determine accurate pharmacological parameters of GPCRs in living cells within seconds. While the insertion of two bulky fluorescent proteins (CFP and YFP) disturbed the normal pharmacological kinetics of the receptor and affected the downstream signalling, the replacement of YFP with the smaller tetracysteine peptide allowed us to recover almost completely the activity features of the wild type, untagged version. This approach has a considerable potential for generalization to other members of the GPCRA family and further showcases the advantages of small, genetically encoded molecular tags. A synopsis of these data is presented in the manuscript ¿ A FlAsH-Based Approach to Determine G Protein-Coupled Receptor Activation in Living Cells¿, (Nature Methods, vol.2, no.3, 171-176, 2005). During the previous funding period we have also completed the characterization of the adenosine antagonist theophylline comparing radioligand binding and FRET data. In collaboration with Jean-Pierre Vilardaga, we have conducted a side study using the alpha 2A-receptor chimera used in the Nature Methods paper. Here we compared different ligands systematically broken down by individual hydroxyl-groups from Nor-epinephrine via Dopamin down to tyramin and catechol, investigated their signal at the receptor site and compared it with the signal they give at the G-protein level. We observed a correlation amongst the receptor activation signal, the substitution pattern of hydroxyl-groups and their activation potential of the G-protein.