Hh通路在动物胚胎发育和物质能量代谢中发挥重要作用，该通路异常会导致发育缺陷和严重疾病，对其信号传递机制的研究具有重要的生物学和临床指导意义。但是，目前该通路分子机制仍不明确。一个主要问题是信号如何从SMO向下游传递。根据我们对GRK2和Gpr161的大量前期工作和文献报道，我们认为GRK2-Gpr161可能是Hh通路信号传递的重要分子开关，二者协同作用精细调控初级纤毛中PKA活性，实现对信号传递的调节。本项目拟应用斑马鱼和成纤维细胞系为材料，通过CRISPR-Cas9、FRET、CoIP-MS以及常规生化、分子和细胞手段，从Gpr161功能、GRK2-Gpr161互作机制、初级纤毛PKA活性和通路活性、GRK2招募激活机制等角度细致揭示Hh通路下游信号传递的分子机制，为全面认识该通路提供重要理论知识，为该通路相关疾病机制研究、药物分子靶点筛选以及临床实践提供重要理论依据和线索。

Hedgehog (Hh) signaling pathway plays crucial roles in the embryonic development of most animals as well as in tissue homeostasis, metabolism and physiological processes in juveniles and adults. Aberrant Hh pathway activity has been implicated in many human disorders including birth defects and cancers. However, the mechanism of the Hh pathway is still not clear. One key question is that how Hh signal is transduced from activated Smoothened (SMO) to GLI transcription factor. Our GRK2 results and preliminary data of Gpr161 indicate that GRK2-GPR161 may function as a key molecular switch to control the signal transduction downstream of SMO by delicate regulating the PKA activity in primary cilia (PC), the crucial cell organelle for Hh pathway in vertebrate. In our study, to elucidate the mechanism of GRK2-GPR161 in controlling vertebrate Hh pathway, the gpr161 mutants in both zebrafish and NIH3T3 fibroblast cell will be generated by CRIPSR-Cas9 and well characterized by genetic and cell biological methods. The GRK2-GPR161 interaction will be assayed genetically and biochemically. The potential phosphorylation site of GPR161 by GRK2 will be identified by CoIP-MS, and be examined for their biological significance. The ciliary PKA biosensor will be employed to analyze the corresponding relation between the Hh activity and the ciliary PKA activity by Fluorescence Resonance Energy Transfer (FRET). In addition, the mechanism of recruitment and activation of GRK2 by SMO will be investigated. These works will: 1) unveil the mechanism of Hh signal transduction downstream of SMO; 2) provide potential targets for medicine discovery and clues for clinical practice of Hh pathway related human disorders.