External GFP fusion enables FRET based binding for addiction related receptors

DESCRIPTION provided by applicant The rise in popularity of fluorescent based assays have in addition to the increased environmental restrictions on radioactive materials have led to a parallel decline in the availability of radioactive ligands and the associated equipment for receptor binding assays Although there is an abundance of fluorescent based functional assays for G coupled protein receptors GPCRs there are few fluorescent based receptor binding assays Early attempts to fuse fluorescent proteins to GPCRs yielded poor results such that today fusion to the C terminal tail is the preferred method of labeling receptors GPCRs with large extracellular domains usually have a cleavable signal peptide that enables cell surface expression We have developed a method for efficient surface expression for receptors with short N terminal tails We also have developed fluorescent ligands for the mu opioid receptor MOR that will enable the development of a FRET based binding assay for MOR As many of the GPCRs associated with drug addiction opioid dopamine cannabinoid orexins etc have short N terminal tails the development of FRET LRET based kits will allow ready access to pharmacological determinants such as measuring affinity constants and receptor occupancy determining competitive vs noncompetitive interactions Such measurements form the backbone to the study of any GPCR and are in danger of becoming lost due to diminished availability of radioligands We plan to develop stable cell lines expressing N terminal labeled GPCRS that can be sold as stand alone cell lines for GPCR studies such as receptor hetero homodimerization or internalization studies Additionally fluorescent labeled ligands specific to GPCRs and FRET based binding kits composed of both the cells lines expressing fluorescent receptor and the fluorescent ligand can be prepared This project will provide several novel products firstly the fluorescent labeled GPCRs secondly the fluorescent labeled ligands and thirdly the binding assays that result from the combination of the two entities Development of FRET LRET based binding assays for GPCRs with short N terminal tails will enable laboratories to continue pharmacological studies without the environmental concerns and costs associated with radioactive materials PUBLIC HEALTH RELEVANCE Receptors are signaling molecules that cross the cell membrane For most of the receptors associated with addiction addition of a florescent tag has been limited to the portion of the receptor on the inside of the cell We have optimized signal sequences used in other receptors involved in addiction that allows us to attach the fluorescent tag to the external portion of the receptor Thus we may now develop fluorescent based tests that in the past were limited to the use of radioactive materials The environmental cost of radioactive materials has resulted in declining availability of such compounds and it is essential that replacement techniques be developed to continue to determine vital pharmacological measurements