External GFP fusion enables FRET based binding for addiction related receptors

 描述(申请人提供)：随着基于荧光的分析方法的普及，加上对放射性物质的环境限制增加，放射性配体(及相关设备)的可获得性也相应下降。 受体结合分析。虽然有大量基于荧光的G偶联蛋白受体(GPCRs)功能分析，但很少有基于荧光的受体结合分析。早期试图将荧光蛋白融合到GPCRs上的结果很差，以至于今天融合到C末端尾巴上是标记受体的首选方法。具有大的胞外结构域的GPCRs通常有一个可切割的信号肽，使细胞表面表达。我们开发了一种短尾N末端受体的高效表面表达方法。我们还开发了Mu阿片受体(MOR)的荧光配体，这将使基于FRET的MOR结合试验的发展成为可能。与药物成瘾(阿片类药物、多巴胺、大麻素、食欲素等)有关的许多GPCRs由于FRET/LRET试剂盒具有短的N-末端，因此可以方便地获得药物决定因素，如测量亲和力常数和受体占有率，确定竞争性和非竞争性相互作用。这种测量构成了研究任何GPCR的支柱，并且由于放射性配基的减少而有丢失的危险。我们计划开发稳定表达N-末端标记的GPCRs的细胞系，这些细胞系可以作为独立的细胞系出售，用于GPCR研究，如受体异源/同源二聚化或内化研究。此外，还可以制备GPCRs特异的荧光标记配体和由表达荧光受体的细胞系和荧光配体组成的FRET结合试剂盒。该项目将提供几种新的产品：第一种是荧光标记的GPCRs，第二种是荧光标记的配体，第三种是两种实体相结合的结合分析。开发基于FRET/LRET的短N末端GPCRs结合分析将使实验室能够继续进行药理学研究，而不会产生与放射性材料相关的环境问题和成本。

 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.