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

RESEARCH ARTICLE PTH1R Mutants Found in Patients with Primary Failure of Tooth Eruption Disrupt GProtein Signaling Hariharan Subramanian1,2☯, Frank Döring3☯, Sina Kollert3, Natalia Rukoyatkina1,4, Julia Sturm3, Stepan Gambaryan4,5, Angelika Stellzig-Eisenhauer6, Philipp MeyerMarcotty6,7, Martin Eigenthaler1,6‡*, Erhard Wischmeyer3‡ a11111 1 Institute of Clinical Biochemistry and Pathobiochemistry, University of Wuerzburg, Wuerzburg, Germany, 2 Institute of Experimental Cardiovascular Research, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany, 3 Institute of Physiology, AG Molecular Electrophysiology, University of Wuerzburg, Wuerzburg, Germany, 4 Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, St. Petersburg, Russia, 5 Department of Cytology and Histology, St. Petersburg State University, St. Petersburg Russia, 6 Department of Orthodontics, University Clinic of Wuerzburg, Wuerzburg, Germany, 7 Department of Orthodontics, University Clinic of Goettingen, Goettingen, Germany ☯ These authors contributed equally to this work. ‡ These authors are joint senior authors on this work. * OPEN ACCESS Citation: Subramanian H, Döring F, Kollert S, Rukoyatkina N, Sturm J, Gambaryan S, et al. (2016) PTH1R Mutants Found in Patients with Primary Failure of Tooth Eruption Disrupt G-Protein Signaling. PLoS ONE 11(11): e0167033. doi:10.1371/journal.pone.0167033 Editor: Peter A. Friedman, University of Pittsburgh School of Medicine, UNITED STATES Received: April 2, 2016 Accepted: November 8, 2016 Abstract Aim 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. Published: November 29, 2016 Copyright: © 2016 Subramanian et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its supporting information files. Funding: This work was supported by the Interdisziplinäres Zentrum für Klinische Forschung des Universitätsklinikum Würzburg, grant Z-4/113. Publication was supported by the Open Access Publication Fund of the University of Wuerzburg. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Materials and Methods 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. Results 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 PLOS ONE | DOI:10.1371/journal.pone.0167033 November 29, 2016 1 / 16 Disrupted Signaling by PTH1R Mutant Competing Interests: The authors have declared that no competing interests exist. contrast to wild type PTH1R which substantially augmented K+ currents of TRESK channels, coupling of mutated PTH1R to TRESK channels was completely abolished. Conclusions 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. Introduction Parathyroid hormone (PTH), PTH-related peptide (PTHrP) and parathyroid hormone receptor type 1 (PTH1R) play a key role in regulation of bone remodeling and plasma calcium levels as reviewed by Taylor et al. [1]. Various autosomal dominant or recessive mutations in the PTH1R gene have been identified to be associated with different diseases. The most severe clinical disease is a complete loss of PTH1R function resulting in lethal Blomstrand chondrodysplasia characterized by advanced endochondral bone maturation and premature ossification of all skeletal elements [2]. In contrast to this homozygous disease, we and others have described the incidence of heterozygous PTH1R mutations which may inactivate PTH1R function in Primary Failure of Tooth Eruption (PFE) (MIM #125350). PFE is a rare autosomal, non-syndromic disorder with incomplete eruption of mainly posterior teeth and growth deficiency of the alveolar process in the affected region [3, 4]. The developing teeth remain below the occlusion level resulting in a severe lateral open bite situation. Furthermore, movement of the teeth by orthodontic treatment results in fusion of the dental cement with the surrounding bone making further tooth movement impossible [5]. The genetic defects underlying PFE were initially identified by genetic screening in affected families [3]. While the first reported 3 mutations (c.463G>T, c.543+1G>A, c.1050-3C>G) in patients with PFE were predicted to generate loss-of-function proteins [3], the spectrum of PTH1R mutations by now has been expanded by several investigators showing the occurrence of more than 40 potentially pathogenic mutations [4, 6–8]. Furthermore, occurrence of sporadic cases of PTH1R mutations causing PFE have been identified by exome resequencing [9]. The PTH1R mutations identified so far are heterogenous and may result in proteolytic degradation of the PTH1R precursor protein, truncation of the PTH1R protein or single amino acid exchanges within the complex architecture of the receptor. However, until now no functional data on the molecular and cellular effects of the PFE related mutations in eukaryotic cells exist. The PTH1R gene encodes a secretin-like class II G protein-coupled receptor precursor that is processed into a mature receptor protein of a 593 amino acids. The PTH1R receptor belongs to the group of seven-helical-transmembrane receptors and mainly consists of a large extracellular loop with a PTH binding site, seven transmembrane, helically shaped segments and an intracellular signaling domain. PTH1R is highly expressed in osteoblasts and renal tubular cells and regulates calcium homeostasis and bone formation. In addition to PTH agonist, (...truncated)