Endosomal regulation of GLP-1 receptor function in beta cells by Clec16a

 描述(申请人提供)：在这项R03申请中提出的研究将集中在内溶酶体蛋白Clec16a在胰腺细胞中的内体周转，GLP1R是一种G蛋白偶联受体。GLP1R激动剂是治疗糖尿病的有价值的药物，基于它们对？细胞的直接作用；然而，这些作用往往受到抑制GLP1R依赖的信号转导机制的限制。因此，减缓配体结合的GLP1R内溶酶体翻转的策略可能有利于延长GLP1R激动剂的活性。Clec16a先前已被证明是？细胞自噬的关键调节因子，但内溶酶体蛋白在自噬和内体转运过程中常常扮演互补的角色。事实上，我们的初步研究表明，Clec16a调节胰腺细胞内胞液相示踪物的运输，并被蛋白激酶A(PKA)磷酸化，以响应GLP1R激动剂的治疗。此外，我们发现GLP1R激动剂的急性治疗显著增加了胰腺特异性Clec16a基因敲除胰岛的胰岛素释放。因此，我们假设Clec16a通过直接参与配体结合的GLP1受体的内溶酶体降解来负向调节GLP1R信号转导。具体目的I.在体内确定Clec16a缺失对GLP1R信号的生理影响。这一目标将利用使用条件性Clec16a loxP等位基因的活体和隔离胰岛中使用GLP1R激动剂治疗后的？细胞特异性功能丧失实验。实验将集中于葡萄糖耐量和胰岛素分泌的测定，以及与ç细胞增殖和/或存活率变化相关的？细胞团的形态分析。特定目的II.研究Clec16a依赖 GLP1R内切酶转运和降解的调控。实验将通过活细胞成像分析来确定Clec16a在配体结合的GLP1Rs通过内溶酶体途径周转中的作用。此外，还将确定依赖于PKA的Clec16a磷酸化对调节溶酶体GLP1R运输/降解的调节作用。通过完成这些目标中描述的工作，我们将建立Clec16a和内溶酶途径降解GLP1R的作用和作用机制。我们将确定抑制内体途径以减轻Ex-4介导的信号转导的中断以改善ç细胞功能的效用，这可能为加强糖尿病患者的GLP1R激动剂治疗开辟一条新的途径。此外，这些研究可能会建议基于GLP1的新方法来治疗患有CLEC16A基因多态的糖尿病患者。

 DESCRIPTION (provided by applicant): The research proposed in this R03 application will focus on the endosomal turnover of the glucagon- like peptide 1 receptor (GLP1R), a G-protein coupled receptor, in pancreatic ß-cells by the endolysosomal protein Clec16a. GLP1R agonists are valuable therapeutic agents for diabetes based upon their direct effects on ß-cells; however, these effects are often self-limited by mechanisms to inactivate GLP1R-dependent signaling. Strategies to slow endolysosomal turnover of ligand bound GLP1Rs could therefore be beneficial to extend the activity of GLP1R agonists. Clec16a has been previously shown to be a key regulator of autophagy in ß-cells, but endolysosomal proteins can often play complementary roles in both autophagy and endosomal trafficking. Indeed, our preliminary studies reveal that Clec16a regulates trafficking of endocytic fluid-phase tracers in pancreatic ß-cells and is also phosphorylated by protein kinase A (PKA) in response to GLP1R agonist treatment. Furthermore, we find that acute treatment with GLP1R agonists markedly enhances insulin release in pancreas-specific Clec16a knockout islets. Therefore, we hypothesize that Clec16a negatively regulates GLP1R signaling through a direct role in the endolysosomal degradation of ligand bound GLP1 receptors. Specific Aim I. Determine the physiologic impact of loss of Clec16a on GLP1R signaling in vivo. This aim will utilize ß-cell specific loss-of-function experiments in vivo using a conditional Clec16a loxP allele, as well as in isolated islets, following treatment with GLP1R-agonists. Experiments will focus on determinations of glucose tolerance and insulin secretion, as well as morphologic analysis of ß-cell mass related to changes in ß-cell proliferation and/or survival. Specific Aim II. To investigate Clec16a-dependent regulation of GLP1R endolysosomal trafficking and degradation. Experiments will determine the role of Clec16a in turnover of ligand-bound GLP1Rs through the endolysosomal pathway by live cell imaging assays. In addition, the regulatory role of PKA-dependent phosphorylation of Clec16a to regulate trafficking/degradation of GLP1R at the lysosome will be determined. By completing the work described in these aims, we will establish the role and functional mechanism for GLP1R-degradation by Clec16a and the endolysosomal pathway. We will determine the utility of inhibition of the endosomal pathway to relieve the breaks on Ex-4 mediated signaling to improve ß-cell function, which could open a new pathway to enhance GLP1R-agonist therapy for patients with diabetes. Furthermore, these studies may suggest new GLP1-based approaches to treat diabetic patients with CLEC16A polymorphisms.