Binding Domain of Human Parathyroid Hormone Receptor:  From Conformation to Function^,

Abstract

A 31 amino acid fragment of the extracellular N-terminus of the human G-protein coupled receptor for parathyroid hormone (PTH1R) has been structurally characterized by NMR and molecular dynamics simulations. The fragment PTH1R[168-198] includes residues 173-189, shown by photoaffinity cross-linking to be a contact domain with position 13 of parathyroid hormone (PTH). The structure of PTH1R[168-198], determined in a micellar solution of dodecylphosphocholine to mimic the membrane environment, consists of three alpha-helices, separated by a well-defined turn and a flexible region. The topological orientation of PTH1R[168-198] was determined from nitroxide-radical induced relaxation of NMR signals utilizing 5- and 16-doxylstearic acid. The C-terminal helix (residues 190-196), consisting of seven amino acids of the first transmembrane domain, is very hydrophobic and embedded in the lipid core. This helix is preceded by a well-defined turn, forming an approximate 90 degrees bend, placing the other helices (residues 169-176 and 180-189), both of which are amphipathic, on the surface of the micelle. In this orientation, many hydrophilic residues of the receptor, including Glu177, Arg179, Arg181, Glu182, Asp185, and Arg186, are projecting toward the solvent available to form complementary Coulombic interactions with the polar residues of the principal binding domain of the ligand (e.g., Arg25, Lys26, Lys27, Asp30, and His32). Given that the binding domain of PTH adopts an amphipathic alpha-helix which lies on the membrane, we visualize ligand binding as a two stage process involving a nonspecific hydrophobic interaction of amphipathic helices with the membrane, followed by two-dimensional diffusion leading to highly specific, ligand-receptor interaction.

Methionine acts as a “magnet” in photoaffinity crosslinking experiments

“…The region 169-176 of PTHR1 was shown by NMR to adopt an alpha-helical structure on the surface of the membrane [21]. Our recent results suggest that this helix might re-arrange and fold back towards the helical bundle during ligand-binding [8].…”

“…Crosslinked ligandreceptor conjugates were isolated, partially purified, digested with Endo-F and CNBr, and analyzed by SDS-PAGE. For native PTHR1 crosslinked to either Bpa 11 -PTH or Bpa 21 Fig. 3A and B).…”

ASBMR 27th Annual Meeting M001–M525

“…The applicability of such estimates has not been validated in nonWestern Caucasian populations. The aim of this study was to assess the ability of BMD measurements, to identify subjects with prevalent vertebral fractures, based on measurements at the hip or forearm in a local non-Western Caucasian population, and compare these estimates to those derived from Western counterparts [1][2][3] . 460 subjects (301 women and 159 men), aged 65-85 years, were randomly recruited from a large metropolitan area.…”

“…BMD precision error was calculated as the root-mean-square standard deviation (RMS-SD) and coefficient of variation (RMS-%CV) for the repeated measurements. To directly compare the precision error between the devices, all Hologic values were converted to Lunarequivalent BMD (1). The 4 technologists who worked with both Hologic and Lunar Abstracts systems had more experience using Hologic (>5 yrs, 3 yrs, 3 yrs, 3 yrs) than Lunar (< 4 months for all).…”

Purification and Characterization of a Receptor for Human Parathyroid Hormone and Parathyroid Hormone-related Peptide

“…Understanding hormone-PTH1R and PTH1R-G protein interactions has relied largely on determination of functional consequences resulting from mutations in either the hormone or the receptor (13,(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36), analysis of receptor fragments after cross-linking to radioiodinated, p-benzoyl-L-phenylalaninemodified ligands (13,(37)(38)(39)(40), crystallographic resolution of PTH (41), and NMR of PTH (42)(43)(44)(45)(46)(47)(48)(49)(50)(51)(52)(53)(54)(55)(56)(57)(58), PTHrP (59 -68), and short segments of the PTH1R (see Refs. 69 and 70 and for review see Refs.…”