N-Terminal Phosphorylation of Parathyroid Hormone (PTH) Abolishes Its Receptor Activity

Abstract

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.

Why Current PTH Assays Mislead Clinical Decision Making in Patients with Secondary Hyperparathyroidism

“…However, n-oxPTH assays will also have limitations. Potential other post-translational PTH modifications such as phosphorylation of certain amino acids of the PTH molecule [53,54] will not be detected by this type of assay system [49][50][51] . What we really need is a true PTH bioassay suitable for routine testinga challenge for scientists working in the field of assay development.…”

Phosphorylation-induced unfolding regulates p19 INK4d during the human cell cycle

“…Alternatively, purified isotope-labeled proteins of interest may be added directly to unlabeled, native cell lysates to study how endogenous cellular proteins such as kinases, phosphatases or proteases act on them [15,16]. Here, we adopted the latter approach to gain mechanistic insights into the phosphorylation behavior of full-length human Figure 2A), other p19 INK4d serines, including Ser76, were unaffected.…”

Small Molecule Inhibited Parathyroid Hormone Mediated cAMP Response by N–Terminal Peptide Binding

“…GPCRs form the largest family of human transmembrane proteins and play a major role in various physiological functions, including cell–cell communication, cell differentiation, metabolism and synaptic transmission. Various signaling molecules including hormones, neurotransmitters, chemokines, ions, tastants and odorants bind to GPCRs 22 and activate the pathways to secondary messengers such as inositol trisphosphate, diacylglycerol, cGMP or cAMP 23 24 25 26 . Agonists such as PTH, PTH related peptide or tuberoinfundibular peptide of 39 residues (TIP39) interact with parathyroid hormone receptors 1 and 2 (PTH1R/2R), both members of class B GPCRs, and activate intracellular signaling, in turn modulating cellular function, including the skeletal, endocrine, cardiovascular and nervous systems 24 27 28 29 30 .…”

“…The elevated levels of calcium may increase in the urine and cause kidney stones. At the molecular level, basic signal transduction starts when PTH binds to its receptors leading to their activation and subsequent generation of secondary messengers 22 24 33 . PTH binding and receptor activation is carried out by the N–terminal part of the peptide 34 .…”

“…It implies that one possible regulation of hormonal activity could be at the ligand level, particularly the N–terminal domain of PTH. Recently, it has been reported that N–terminal phosphorylation at Ser1, Ser3 and Ser17 of PTH abolished receptor activity 24 .…”