DEVELOPMENT OF A FLUORESCENCE BASED TESTSYSTEM FOR GPCR ACTIVATION

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 研究中心，而研究中心不一定是研究者所在的机构。 G蛋白偶联受体（GPCR）是细胞外到细胞内的信息转导的关键元件。 许多药物通过这类受体来改变细胞功能。我们发现，GPCR的激活可以通过与受体融合的绿色荧光蛋白（GFP）的两个变体之间的荧光共振能量转移（FRET）来监测。然而，该融合蛋白的药理学特征与天然GPCR的药理学特征不同  其动力学似乎因荧光报道分子的大尺寸而减慢。在UCSD与NCMIR的合作中，我们建议通过将其中一种荧光蛋白与较小的四半胱氨酸肽交换来减小报告分子的大小，并测试所得融合的活化动力学。我们将首先测试这种报告基因组合与A2 A受体融合的行为，然后将其扩展到其他感兴趣的GPCR。

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) are crucial elements in the transduction of information from the extracellular to the intracellular side of a cell. Numerous drugs act via this class of receptors to alter cellular functions. We showed that activation of GPCRs could be monitored by fluorescence resonance energy transfer (FRET) between two variants of the green fluorescent protein (GFP) fused to the receptor. However, the pharmacological profile of this fusion protein is different from that of the native GPCR  its kinetics appeared to be slowed down by the large size of the fluorescent reporters. In collaboration with NCMIR at UCSD, we propose to slim down the reporter size by swapping one of the fluorescent proteins with the smaller tetracysteine peptide and test the resulting fusion for its activation kinetics. We will initially test the behavior of this reporter combination in fusion to the A2A receptor, and then extend it to other interesting GPCRs.