Project Summary G protein-coupled receptors (GPCRs) transmit the extracellular stimuli into intracellular signals, by which they can orchestrate a myriad of cellular and physiological processes. GPCRs form the largest superfamily of surface receptors and their aberrant function causes diseases such as cancer, asthma, hypertension, endocrine and musculoskeletal dysfunction. Adhesion GPCRs (aGPCRs) are the second largest but the least studied family of GPCRs and have recently gained much interest due to their direct or indirect involvement in various diseases. In order to target aGPCRs therapeutically, we need to understand the mechanisms by which they are activated and the intracellular signaling cascades that they initiate. aGPCRs have an unusually long N-terminal fragment (NTF) that is cleaved during their biosynthesis but stays bound to the rest of the receptor non-covalently. The processes taking place after binding of extracellular ligands to this NTF can vary for different aGPCRs. While such interactions can stabilize a certain conformation and trigger or inhibit signaling in some aGPCRs, it can also dissociate the NTF and unmask a small tethered peptide (stalk) on the very N-terminus of the remaining receptor. We showed that the NTF of ADGRG2/GPR64, an orphan aGPCR, functions as an inhibitor and its deletion results in a receptor that is constitutively activated by its stalk. We provided compelling evidence that GPR64 regulates secretion of parathyroid hormone, a master regulator of bone metabolism by human-derived parathyroid adenoma cells. Via molecular and cellular assays, we have demonstrated that NTF-deficient GPR64 elevates cAMP levels by activating adenylyl cyclase, interacts with -arrestins, becomes ubiquitinated and internalizes via unknown mechanisms. A mutant that lacks this tethered peptide and NTF (stalk-less) is devoid of constitutive activity but responds to the exogenously added synthetic tethered peptide and interacts with - arrestins. The underlying mechanism for recruitment of -arrestins by stalk-less GPR64 in the absence of G- protein signaling is not clear. These published and preliminary data combined with our current knowledge of another closely related aGPCR, ADGRG1/GPR56 lead us to hypothesize that specific structural elements control signaling, endocytic pathways and physiological functions of these aGPCRs. To shed light on the pharmacological and physiological characteristics of these receptors, we will: (1) Determine the impact of NTF and its cleavage on the binding of tethered peptides and trafficking of GPR64 and GPR56; (2) Identify the structural motifs and intracellular regulators that control G-protein and -arrestin signaling downstream of GPR64 and GPR56 and (3) Determine the effects of GPR64 signaling and its NTF and stalk on bone development in Zebrafish, a low vertebrate model organism. These novel translational studies will greatly bolster our understanding of aGPCR pharmacology and lay the foundati...