N-terminal phosphorylation of parathyroid hormone (PTH) abolishes its receptor activity.

The parathyroid hormone (PTH) is an 84-residue peptide, which regulates the blood Ca(2+) level via GPCR binding and subsequent activation of intracellular signaling cascades. PTH is posttranslationally phosphorylated in the parathyroid glands; however, the functional significance of this processes is not well characterized. In the present study, mass spectrometric analysis revealed three sites of phosphorylation, and NMR spectroscopy assigned Ser1, Ser3, and Ser17 as modified sites. These sites are located at the N-terminus of the hormone, which is important for receptor recognition and activation. NMR shows further that the three phosphate groups remotely disturb the α-helical propensity up to Ala36. An intracellular cAMP accumulation assay elucidated the biological significance of this phosphorylation because it ablated the PTH-mediated signaling. Our studies thus shed light on functional implications of phosphorylation at native PTH as an additional level of regulation.

[1]  F. Hausch.  Structures of class B G protein-coupled receptors: prospects for drug discovery. , 2013, Angewandte Chemie.

[2]  M. Bouvier.  Unraveling the structural basis of GPCR activation and inactivation , 2013, Nature Structural &Molecular Biology.

[3]  C. Reynolds,et al.  The activation of the CGRP receptor. , 2013, Biochemical Society transactions.

[4]  T. Gardella,et al.  Chemistry as an expanding resource in protein science: fully synthetic and fully active human parathyroid hormone-related protein (1-141). , 2012, Angewandte Chemie.

[5]  Michel Bouvier,et al.  Restructuring G-Protein- Coupled Receptor Activation , 2012, Cell.

[6]  A. Beck‐Sickinger,et al.  Genetically encoded photocrosslinkers as molecular probes to study G-protein-coupled receptors (GPCRs). , 2012, Angewandte Chemie.

[7]  R. Rudolph,et al.  Binding specificity of the ectodomain of the parathyroid hormone receptor. , 2011, Biophysical chemistry.

[8]  K. Fuxe,et al.  G-protein-coupled receptor heteromer dynamics , 2010, Journal of Cell Science.

[9]  Yutaka Ito,et al.  Cellular structural biology. , 2010, Current opinion in structural biology.

[10]  R. A. Hall,et al.  Fine-tuning of GPCR activity by receptor-interacting proteins , 2009, Nature Reviews Molecular Cell Biology.

[11]  L. Pietrogrande.  Update on the efficacy, safety, and adherence to treatment of full length parathyroid hormone, PTH (1-84), in the treatment of postmenopausal osteoporosis , 2009, International Journal of Women's Health.

[12]  L. Johnson.  The regulation of protein phosphorylation. , 2009, Biochemical Society transactions.

[13]  M. Wigglesworth,et al.  Ligand-Receptor Interactions at the Parathyroid Hormone Receptors: Subtype Binding Selectivity Is Mediated via an Interaction between Residue 23 on the Ligand and Residue 41 on the Receptor , 2008, Molecular Pharmacology.

[14]  H. Xu,et al.  Molecular recognition of parathyroid hormone by its G protein-coupled receptor , 2008, Proceedings of the National Academy of Sciences.

[15]  R. Rudolph,et al.  High yield production of recombinant native and modified peptides exemplified by ligands for G-protein coupled receptors. , 2008, Protein expression and purification.

[16]  M. Rubin,et al.  An N-terminal molecular form of parathyroid hormone (PTH) distinct from hPTH(1 84) is overproduced in parathyroid carcinoma. , 2007, Clinical chemistry.

[17]  Sylvie Garneau-Tsodikova,et al.  Protein posttranslational modifications: the chemistry of proteome diversifications. , 2005, Angewandte Chemie.

[18]  H. Jüppner,et al.  Parathyroid hormone and parathyroid hormone-related peptide, and their receptors. , 2005, Biochemical and biophysical research communications.

[19]  S. Hoare,et al.  Mechanisms of peptide and nonpeptide ligand binding to Class B G-protein-coupled receptors. , 2005, Drug discovery today.

[20]  L. Rao,et al.  Parathyroid hormone secretion and action: evidence for discrete receptors for the carboxyl-terminal region and related biological actions of carboxyl- terminal ligands. , 2005, Endocrine reviews.

[21]  W. Lee,et al.  The 10th and 11th residues of short length N-terminal PTH(1-12) analogue are important for its optimum potency. , 2004, The journal of peptide research : official journal of the American Peptide Society.

[22]  P. D'amour,et al.  Amino-terminal form of parathyroid hormone (PTH) with immunologic similarities to hPTH(1-84) is overproduced in primary and secondary hyperparathyroidism. , 2003, Clinical chemistry.

[23]  F. R. Bringhurst.  Circulating forms of parathyroid hormone: peeling back the onion. , 2003, Clinical chemistry.

[24]  S. Mammi,et al.  Molecular characterization of a ligand-tethered parathyroid hormone receptor. , 2002, Biophysical chemistry.

[25]  H. Jüppner,et al.  Molecular properties of the PTH/PTHrP receptor , 2001, Trends in Endocrinology & Metabolism.

[26]  T. Bonner,et al.  Comparison of rat and human parathyroid hormone 2 (PTH2) receptor activation: PTH is a low potency partial agonist at the rat PTH2 receptor. , 1999, Endocrinology.

[27]  U. Marx,et al.  Structure-Activity Relation of NH2-terminal Human Parathyroid Hormone Fragments* , 1998, The Journal of Biological Chemistry.

[28]  C M Dobson,et al.  Protein Folding Monitored at Individual Residues During a Two-Dimensional NMR Experiment , 1996, Science.

[29]  T. Bonner,et al.  Identification and Functional Expression of a Receptor Selectively Recognizing Parathyroid Hormone, the PTH2 Receptor (*) , 1995, The Journal of Biological Chemistry.

[30]  T. Dieckmann,et al.  Protein Structure Determination with Three‐ and Four‐Dimensional NMR Spectroscopy , 1994 .

[31]  M. Freeman,et al.  A G protein-linked receptor for parathyroid hormone and parathyroid hormone-related peptide. , 1991, Science.

[32]  H. Kronenberg,et al.  Mutational analysis of the receptor-activating region of human parathyroid hormone. , 1991, The Journal of biological chemistry.

[33]  E. Chen,et al.  A parathyroid hormone-related protein implicated in malignant hypercalcemia: cloning and expression. , 1987, Science.

[34]  S. Rabbani,et al.  Phosphorylation of parathyroid hormone by human and bovine parathyroid glands. , 1984, The Journal of biological chemistry.

[35]  S. Nussbaum,et al.  Parathyroid hormone . renal receptor interactions. Demonstration of two receptor-binding domains. , 1980, The Journal of biological chemistry.

[36]  G. Hendy,et al.  Human parathyroid hormone: immunological properties of the amino terminus and of synthetic fragments. , 1974, Clinical science and molecular medicine.

[37]  Z. Agus,et al.  Effects of parathyroid hormone on renal tubular reabsorption of calcium, sodium, and phosphate. , 1973, The American journal of physiology.

[38]  L. Raisz.  Stimulation of Bone Resorption by Parathyroid Hormone in Tissue Culture , 1963, Nature.

[39]  R. Lefkowitz,et al.  Signalling: Seven-transmembrane receptors , 2002, Nature Reviews Molecular Cell Biology.

[40]  A. Rich,et al.  Biosynthesis of parathyroid hormone. , 1980, Annals of the New York Academy of Sciences.