SUMMARY The proposed research focuses on a subset of G protein-coupled receptors (GPCRs) that are naturally modulated by long polypeptide hormones (family B GPCRs). These receptors control many important metabolic functions. For example, glucagon-like peptide-1 (GLP-1) activation of the cognate GPCR, the GLP-1R, helps to control glucose levels in the bloodstream. Synthetic analogues of GLP-1, including liraglutide and semaglutide (each > 30 residues), are used to treat type 2 diabetes. Parathyroid hormone (PTH) activates the parathyroid hormone receptor 1 (PTHR1) and thereby helps to control calcium and phosphate levels in the bloodstream. Agonists of the PTHR1, teriparatide and abaloparatide (each 34 residues), are used to treat osteoporosis. A major focus of the proposed studies is to elucidate how the information encoded in polypeptide hormones is "read out" by B family GPCRs and transmitted to intracellular partner proteins. This question is of great interest in terms of basic understanding of signal transduction at the molecular level. Insights that emerge from the proposed research could support the development of new agonists with tailored signaling properties and thereby illuminate a path to new therapeutic agents with minimized side effects. Recent discoveries have led us to formulate a new hypothesis regarding the bioactive forms of the natural GLP-1R agonists, GLP-1(7-36) and exendin-4 (Ex-4), in complex with the GLP-1R, and to extend this hypothesis to other hormone-activated GPCRs (Aims 1 and 2). We propose that retention of a specific mode of agonist mobility is essential for activity. Although it is widely understood that GPCRs are mobile proteins, and that agonists transmit information via binding to GPCRs by inducing specific receptor conformations, which are recognized by cytosolic partner proteins, our hypothesis constitutes a new perspective. The current view is that the bioactive form of a peptide agonist comprises a single, specific conformation, while our hypothesis includes specific dynamic modes to define the agonist bioactive form. The distinction between these two hypotheses is significant because they make different predictions with regard to designing new agonists. Our hypothesis highlights the need to maintain specific modes of agonist flexibility, while the traditional view emphasizes rigidification. Therefore, the experimental program we propose could lead to highly impactful outcomes. Aim 3 builds on capabilities established during prior years of support via R01 GM056414, involving replacement of selected α-amino acid residues with β-amino acid residues. We will try to develop potent and protease-resistant inverse agonists of constitutively active natural variants of the PTHR1 that are associated with Jansen's Metaphyseal Chondrodysplasia, for which there is no current therapy.