View Abstract

A4650 - Molecular Analysis of ADGRF5, an Adhesion G Protein-Coupled Receptor Required for Alveolar Homeostasis
Author Block: J. P. Bridges1, K. Brown1, A. Filuta1, W. Miller2; 1Pediatrics, Cincinnati Childrens Hosp Med Ctr, Cincinnati, OH, United States, 2Molecular Genetics, University of Cincinnati, Cincinnati, OH, United States.
Background: We have previously demonstrated that the G protein-coupled receptor Adgrf5 (Gpr116) regulates alveolar homeostasis in mice. The ability to pharmacologically manipulate the Adgrf5 pathway, both positively and negatively, would be a major therapeutic advance for patients with lung diseases associated with pulmonary surfactant disorders. The goal of this study was to determine the molecular determinants of ADGRF5 essential for activation in vitro and in transgenic mouse models. Methods: G protein-coupled assays (calcium transients and inositol phosphate (IP) conversion assays) were performed in primary AT2 cells and in HEK293 cells transiently expressing wild-type ADGRF5 or chimeric cDNAs that harbored alanine substitutions at sites predicted to be essential for receptor function. A synthetic peptide corresponding to the first 10 amino acids in the ectodomain of the C-terminal Fragment of Adgrf5 (termed GAP10) and a scrambled control peptide were used in activity assays with the chimeric ADGRF5 mutants in vitro and nebulized to WT and Adgrf5H991A mice to determine the impact of ADGRF5 activation on surfactant pool sizes in vivo. Results: Alanine mutation analysis identified four key amino acids within the ectodomain and two in the second extracelullar loop of ADGRF5 that are required for activation. We also identified a conserved amino acid, H991, that is essential for cleavage of the wild-type receptor into N- and C-terminal fragments. To test the hypothesis that cleavage of ADGRF5 is required for activation in vivo, we generated mice that express the H991A mutant (Adgrf5H991A) via CRISPR/Cas9 editing. Analysis of 4-week old Adgrf5H991A/H991A mice revealed increased pulmonary surfactant and airspace enlargement, identical to the phenotype observed in Adgrf5-/- mice, indicating that cleavage of Adgrf5 is essential for receptor function in vivo. Activation of ADGRF5 via GAP10 nebulization to WT mice suppressed alveolar surfactant pools and elicited calcium transients in primary AT2 cells from Adgrf5H991A mice. Conclusion: Structure/function studies in vitro and in vivo identified critical residues in ADGRF5 essential for activation. While the endogenous ligand of ADGRF5 is unknown, our data support a model whereby binding of a ligand to the NTF results in separation of the NTF from the CTF, revealing a cryptic tethered peptide that binds the second extracellular loop of ADGRF5, culminating in activation and suppression of surfactant secretion from AT2 cells. Ongoing studies are focused on identification of the endogenous ligand and intracellular signaling events mediating ADGRF5-regulated exocytosis in AT2 cells.