DEVELOPMENT OF A FLUORESCENCE BASED TESTSYSTEM FOR GPCR ACTIVATION

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 G蛋白偶联受体(GPCRs)是将信息从细胞外传递到细胞内侧的关键元件。许多药物通过这类受体来改变细胞功能。我们发现，GPCRs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，这种融合蛋白的药理特性不同于天然的GPCR，其动力学似乎因荧光报告分子的大尺寸而变慢。与加州大学圣迭戈分校的NCMIR合作，我们建议通过将其中一个荧光蛋白与较小的四半胱氨酸肽交换来瘦身，并测试由此产生的融合的激活动力学。我们将首先测试这种与A2A受体融合的报告组合的行为，然后将其扩展到其他有趣的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.