CCKAR的磷酸化编码对胰岛β细胞的功能调控及机制

The cholecystokinin A receptor (CCKAR), a G protein-coupled receptor (GPCR) and the main target for CCK binding in pancreatic beta cells, has been demonstrated to play important roles in the functional regulation of beta cells and has been considered as a potential pharmaceutical target for the treatment of diabetes mellitus. Upon activation, the CCKAR initiates multiple signaling pathways, regulating diverge physiological functions. Most GPCRs signal through both G protein and arrestin pathways. Whereas the G protein-mediated signaling pathways of CCKAR have been extensively studied, the arrestin-regulated pathways remain to be further explored. Our previous results revealed that CCKAR regulates both insulin secretion and anti-apoptotic effects in pancreatic beta cells through arrestin-mediated pathways. However, the detailed mechanism remains unclear. Phosphorylation of GPCR by intracellular kinases plays essential roles in the regulation of receptor functions by promoting interactions of the receptor with arrestins. Therefore, based on the newly-developed concepts of biased signaling and barcode hypothesis in GPCR, we hypothesize that the CCKAR in pancreatic beta cells could be differentially phosphorylated by multiple kinases and the kinase-specific phosphorylation patterns that resemble different barcodes dictate different arrestin-mediated pathways, which are correlated to divergent physiological functions. In the current project, we will thoroughly investigate the phosphorylation barcodes of CCKAR as well as the underlying mechanism by which CCKAR phosphorylation barcodes regulate beta cell functions. Our results will contribute to a better understanding of the pharmacological properties of CCKAR and further provide a theoretical basis for the development of CCKAR ligands-based novel drugs for the treatment of diabetes mellitus.

胆囊收缩素A受体（CCKAR）是G蛋白偶联受体，其激活对胰岛β细胞的功能起显著改善作用，被认为是糖尿病新型疗法的潜在药物靶点。CCKAR激活多条信号通路，目前研究主要集中在G蛋白依赖性信号通路，而对另一重要信号分子arrestin介导的通路鲜有报道。我们前期研究发现β细胞中CCKAR通过arrestin介导的信号通路调控胰岛素分泌和抑制细胞凋亡，但具体的机制尚不清楚。胞内激酶引发的受体磷酸化是招募arrestin的决定性因素。结合GPCR前沿研究中最新的磷酸化编码的概念，我们推测CCKAR可被多种激酶差异性磷酸化，不同的磷酸化模式通过arrestin介导不同信号通路，调控不同的生理功能。本项目将联合分子和细胞生物学技术，应用基因敲除动物模型，研究CCKAR的磷酸化编码对胰岛β细胞功能调控机制，旨在深度解析CCKAR的药理学特性，为更好的发展CCKAR配体作为糖尿病治疗新型药物奠定理论基础。

胰岛β细胞上表达的胆囊收缩素A受体（CCKAR）激活后可以显著促进胰岛β细胞增殖和胰岛素分泌，可以有效调控胰岛β细胞功能，因此其具备作为糖尿病治疗新型靶点的潜力。我们前期研究中发现CCKAR通过β-arrestin介导的信号通路调控胰岛B细胞功能，但具体的作用机制和生理意义尚不明确。在本课题中，我们发现CCKAR可以被多种激酶差异性磷酸化，其中GRK2和GRK6介导的不同的磷酸化编码调控β-arrestin不同的功能构象。我们利用SILAC的实验方法精准鉴定了GRK2和GKR6在CCKAR的磷酸化位点，并联合分子生物学和细胞生物学技术，明确了GRK6在促进高糖刺激的胰岛素分泌以及胰岛B细胞增殖等方面的功能，并进一步阐明了β-arrestin识别CCKAR磷酸化编码以及与下游信号分子的作用模式。我们通过构建CCKAR和不同信号分子的融合蛋白，制备慢病毒感染OLETF糖尿病大鼠模型，证实了CCKAR通过β-arrestin信号通路改善胰岛功能，揭示了CCKAR的磷酸化编码对胰岛功能调控的生理意义。我们同时研究了其他GPCR如生长抑素受体和促肾上腺皮质激素释放激素受体等在胰岛细胞环路中的调控功能，为研究GPCR超家族在胰岛中的生理学功能及其磷酸化编码的生理意义提供了新的思路。