PROKR2互作蛋白SNAPIN对其功能的调控机制研究

PROKR2 belongs to the family of GPCRs，which involved in various physiological processes which included gastrointestinal mobility, angiogenesis, circadian rhythm regulation and migration of neural stem cell. Recently,studies had shown that mutations in PROKR2 were associated with the Kallmann syndrome (KS) and/or idiopathic hypogonadotropic hypogonadism (IHH). Current studies mainly focus on its structure and functions, few was known about its regulating proteins and proteins participating in its signaling pathways.More and more GPCR interactive proteins (GIPs) which play an important regulating function were found ,the C terminus of GPCRs was GIPs’ important anchor regions.In the previous research, we used the cytoplasmic C-terminal tail of human PROKR2（333F-384K）as a bait and screened the interactive proteins from the human cDNA library through the yeast two-hybrid system.This reaserch main to test the interaction between PROKR2 and SNAPIN; To construct the truncated mutants according to their function areas and to find the interaction areas or sites between them through related experiments; To investigate the role that SNAPIN played in the receptor function of PROKR2,which not only can help us understand the regulatory mechanisms of PROKR2's function, provide the theoretical basis for the genetic diseases caused by the mutations on the interactive region of PROKR2, but also to promote the discovery of some new genes related to the KS / IHH.

PROKR2属于GPCR家族成员之一，可参与平滑肌收缩、血管生成、近日节律调节及促神经干细胞迁移。近期研究表明其突变与卡尔曼综合征（KS）和/或特发性促性腺激素型性腺功能减退（IHH）发病有关。目前研究多集中在其结构与功能上，而其调节蛋白及信号通路蛋白研究几乎空白。越来越多对GPCRs有重要调节功能的互作蛋白（GIPs）被发现，GPCRs的C末端正是GIPs重要锚定区。前期我们利用酵母双杂交技术，以人PROKR2的C末端（333F-384K）为饵，从人cDNA文库中筛选出其互作蛋白SNAPIN（国内外未见报道）。本课题欲进一步验证两者之间的相互作用；根据功能区构建截短突变、通过相关实验寻找互作区域/位点；研究SNAPIN在PROKR2受体功能中的作用，不仅可增强对PROKR2功能调节的了解，为因PROKR2互作区碱基突变所致遗传性疾病的病因学提供解释，且可促一些新KS/IHH相关基因的发现

PROKR2属于GPCR家族成员之一，其与配体Prokinticins结合后，参与平滑肌收缩、血管生成、近日节律调节、及促进神经干细胞迁移等。近期研究表明其突变与卡尔曼综合征（KS）和/或特发性促性腺激素型性腺功能减退（IHH）发病有关。目前针对PROKR2的研究多集中在其基因突变对受体功能的影响方面，而对其调节蛋白及信号通路蛋白的研究几乎是片空白。越来越多的对GPCRs有重要调节功能的互作蛋白（GIPs）被发现，而GPCRs的C末端正是GIPs的重要锚定区。我们利用酵母双杂交技术，以人PROKR2的C末端（333F-384K）为“饵”，从人cDNA文库中筛选出其互作蛋白SNAPIN，验证了两者之间的互作关系并发现两者在细胞膜上呈现共定位，丰富了hPROKR2的GIPs库信息。进一步寻找到两者的互作位点位于SNAPIN分子中含H1与H2区的片段（37-136aa）及hPROKR2的343YFK345及351HWR353，提供了新的蛋白互作位点。在功能上，研究发现两者的相互作用参与了hPROKR2的再循环过程，SNAPIN有助于hPROKR2内吞之后的重新上膜，破坏两者之间的相互作用有可能使内吞的hPROKR2进入降解途径。