Novel tools for investigating GPCR-mediated 14-3-3 signaling pathway

Project Summary In addition to well documented G protein dependent andarrestin dependent GPCR signaling pathwaysother cellular effectors are recruited to GPCRsSignal adaptor proteinis one of such cellular effectors engaged with GPCRsproteins are ubiquitously expressed in cellsbut their highest expression is found in the brainAlthough biochemical evidence showsforms complexes with some GPCRsinvestigation of GPCR mediatedsignaling has been largely ignoredLack of ability to assess specificsignaling is a major reason for studies to lag behind studies of G protein andarrestin signaling pathwaysWe have developed a new assay for assessing GPCR mediatedsignaling by measuring GPCR andinteractions in the phasestudyWe demonstrate that GPCR mediatedsignaling is ligandregulatedMultiple GPCRs interact withproteins in response to agonist stimulationGPCR mediatedsignaling is phosphorylation dependentand GPCRinteraction likely takes place after receptor desensitization and internalizationGPCR mediatedsignaling can bearrestin independent and agonists can have different potencies inandarrestin signaling pathwaysGPCRs can also mediateand Rafkinase interactionOur work opens up a new broad realm of previously unappreciated GPCR signal transductionGPCRLinkLight assay cells offer novel tools for GPCR drug discoveryIt is likely that GPCR mediatedsignaling is a more general phenomenon than we have previously realizedIn the Phase II research planwe will continue characterizing and developing a large number of commercial GPCRassay cell lines for brain derived GPCRs including serotonindopamineopioidorexinsomatostatinmuscariniccannabinoidadrenergicand neuropeptide receptorsThese receptorsmediatedsignaling pathway has yet to be characterized individuallyThese GPCRcell lines would be novel assay tools to aid us to study GPCR signaling and to develop new drugsWe will also investigate the potential that metabolic glutamate receptorsGPCR family C membersinteract withproteinsMetabolic glutamate receptorsGRMscan signal through G proteinsbut they do not recruitarrestins and have no GPCR mediatedarrestin signalingWe have showed that GPCR mediatedsignaling pathway can bearrestin independent by using ADRBas an examplecompleted Phaseextra taskThusif we can demonstrate that metabolic glutamate receptors can mediatesignalingwe will have a new approach to target these important receptorsFinding a biasedsignaling ligand would be another important discoveryWe will collaborate with Professor Thomas Sakmar in Rockefeller University for a pilot screenProfSakmar has been studying the human dopamine DreceptorhDRDfor which there are three exon variants in the human populationsDDand DWe will screen for biased ligands that have differential signaling betweenandarrestin signaling pathways by using theDvariant as the modelIf we find biased hits in the screenwe will cross check the hits with different variantsBiased ligands would be valuable probes for characterizing the physiological significance of GPCR mediatedsignaling