External GFP fusion enables FRET based binding for addiction related receptors

 描述（由申请人提供）：除了对放射性材料的环境限制增加之外，基于荧光的测定的普及已经导致用于放射性物质的放射性配体（和相关设备）的可用性的平行下降。 受体结合测定。虽然存在大量的基于荧光的G偶联蛋白受体（GPCR）功能测定，但很少有基于荧光的受体结合测定。将荧光蛋白融合到GPCR的早期尝试产生了差的结果，使得今天融合到C-末端尾是标记受体的优选方法。具有大的胞外结构域的GPCR通常具有能够实现细胞表面表达的可切割信号肽。我们已经开发了一种方法，有效的表面表达受体短N-末端尾巴。我们还开发了μ阿片受体（莫尔）的荧光配体，这将使得能够开发基于FRET的莫尔结合测定。许多与药物成瘾相关的GPCR（阿片类药物、多巴胺、大麻素、食欲素等）具有短的N-末端尾部，基于FRET/LRET的试剂盒的开发将允许容易地获得药理学决定因素，例如测量亲和常数和受体占有率，确定竞争性与非竞争性相互作用。这种测量形成了任何气相色谱-化学反应研究的支柱，并且由于放射性配体的可用性减少而有丢失的危险。我们计划开发表达N-末端标记的GPCRS的稳定细胞系，其可以作为用于GPCR研究的独立细胞系出售，例如受体异源/同源二聚化或内化研究。另外，可以制备特异于GPCR的荧光标记配体和基于FRET的结合试剂盒，所述结合试剂盒由表达荧光受体和荧光配体的细胞系组成。该项目将提供几种新产品;首先是荧光标记的GPCR，其次是荧光标记的配体，第三是由两种实体组合产生的结合测定。开发基于FRET/LRET的具有短N-末端尾的GPCR结合试验将使实验室能够继续进行药理学研究，而无需考虑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.