Project Summary/Abstract Cardiovascular disease (CVD) is the major cause of death in the United Stand and is particularly prevalent in patients with type-2 diabetes mellitus (T2DM). Recently, agonists of the glucagon-like peptide-1 receptor (GLP-1R), a G protein-coupled receptor (GPCR) and common target for the treatment of T2DM, have shown promising cardiovascular benefits including a significant reduction in CVD-associated morbidity and mortality. However, broader clinical trials of approved GLP-1R agonists, many of which are synthetic peptides, have yielded mixed results. Thus, there is a critical need to understand the underlying mechanism driving the different modes of action for the same class of reagents to design more targeted and effective therapeutics. Notably, many synthetic peptide agonists of GLP-1R have demonstrated biased agonism, i.e., a ligand drives preference for certain signaling pathways relative to the endogenous agonist, GLP-1. This phenomenon is seen in many other GPCRs. For example, the Gellman lab replaced α residues with β residues on the N- terminus of the PTH peptide and observed a G-protein bias when treating cells expressing the PTH receptor. I propose to build on this strategy to develop novel synthetic peptide agonists of GLP-1R that are biased for the Gs and Gq pathways as tools to better understand GLP-1R signaling. I will design and synthesize α/β- peptides analogies of GLP-1 by replacing the first eight amino acid residues at the N-terminus, individually, with β residues. Cell-based assays will be performed to measure cAMP production (Gs), calcium mobilization (Gq), and β-arrestin-1, 2 recruitment, which represent the major signaling pathways of GLP-1R, to characterize the signaling profile and determine their biased relative to GLP-1. Next, I hypothesize that the biased activation of GLP-1R is the result of unique post-translational modifications (PTMs) and protein-protein interactions that arise after peptide binding and that downstream expression and phosphorylation of intracellular proteins is altered as a result. To test this hypothesis, I will use mass spectrometry (MS)-based proteomics to characterize the PTMs of purified GLP-1R that arise after treatment with the Gs and Gq biased peptides as well as β-arrestin-1 and -2 biased peptides previously developed in the Gellman group. A second MS assay will be performed after co-immunoprecipitation of the receptor to investigate unique receptor-protein interactions that arise after peptide binding. Finally, a third MS assay will access downstream changes in protein expression and phosphorylation for an in-depth understanding of the signal transduction produced by biased agonist binding. The results of this proposal will help elucidate the mechanisms driving the signal transduction of GPCRs and aid in the development of safer, more effective therapeutics.