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 -cells by the endolysosomal protein Clec16a. GLP1R agonists are valuable therapeutic agents for diabetes based upon their direct effects on -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 -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 -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 -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 -cell mass related to changes in -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 -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.