PTH1R Mutants Found in Patients with Primary Failure of Tooth Eruption Disrupt G-Protein Signaling

• Published in: PloS one 2016-11, Vol.11 (11), p.e0167033-e0167033
• Main Authors: Subramanian, Hariharan, Döring, Frank, Kollert, Sina, Rukoyatkina, Natalia, Sturm, Julia, Gambaryan, Stepan, Stellzig-Eisenhauer, Angelika, Meyer-Marcotty, Philipp, Eigenthaler, Martin, Wischmeyer, Erhard
• Format: Article
• Language: English
• Subjects: Amino acids; Analysis; Animals; Binding sites; Biochemistry; Biology and Life Sciences; Cell Adhesion Molecules - metabolism; Cell surface; Cyclic AMP; Cyclic AMP - metabolism; Disease; Electrophysiology; Enzyme-linked immunosorbent assay; Eruptions; Failure; Gene Expression Regulation; Gene mutation; GTP-Binding Proteins - genetics; GTP-Binding Proteins - metabolism; HEK293 Cells; Humans; Immunoblotting; Intracellular; Intracellular signalling; Ion channels; Kinases; Medicine and Health Sciences; Microfilament Proteins - metabolism; Mitogen-Activated Protein Kinase 1 - metabolism; Mitogen-Activated Protein Kinase 3 - metabolism; Motility; Mutants; Mutation; Mutation - genetics; Odontology; Oocytes; Oocytes - cytology; Oocytes - metabolism; Orthodontics; Parathyroid; Parathyroid hormone; Parathyroid Hormone - metabolism; Parathyroid hormones; Patients; Peptides; Phosphoproteins - metabolism; Phosphorylation; Physiology; Potassium; Potassium currents; Protein kinase; Proteins; Receptor, Parathyroid Hormone, Type 1 - genetics; Research and Analysis Methods; Signal Transduction; Teeth; Teething; Tooth Diseases - genetics; Tooth Diseases - pathology; Xenopus laevis
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0167033

Primary failure of tooth eruption (PFE) is causally linked to heterozygous mutations of the parathyroid hormone receptor (PTH1R) gene. The mutants described so far lead to exchange of amino acids or truncation of the protein that may result in structural changes of the expressed PTH1R. However, functional effects of these mutations have not been investigated yet. In HEK293 cells, PTH1R wild type was co-transfected with selected PTH1R mutants identified in patients with PFE. The effects on activation of PTH-regulated intracellular signaling pathways were analyzed by ELISA and Western immunoblotting. Differential effects of wild type and mutated PTH1R on TRESK ion channel regulation were analyzed by electrophysiological recordings in Xenopus laevis oocytes. In HEK293 cells, activation of PTH1R wild type increases cAMP and in response activates cAMP-stimulated protein kinase as detected by phosphorylation of the vasodilator stimulated phosphoprotein (VASP). In contrast, the PTH1R mutants are functionally inactive and mutant PTH1R/Gly452Glu has a dominant negative effect on the signaling of PTH1R wild type. Confocal imaging revealed that wild type PTH1R is expressed on the cell surface, whereas PTH1R/Gly452Glu mutant is mostly retained inside the cell. Furthermore, in contrast to wild type PTH1R which substantially augmented K+ currents of TRESK channels, coupling of mutated PTH1R to TRESK channels was completely abolished. PTH1R mutations affect intracellular PTH-regulated signaling in vitro. In patients with primary failure of tooth eruption defective signaling of PTH1R mutations is suggested to occur in dento-alveolar cells and thus may lead to impaired tooth movement.