title: A salt bridge between Arg-20 on parathyroid hormone (PTH) and Asp-137 on the PTH1 receptor is essential for full affinity.
abstract: Parathyroid hormone (PTH) acts via the receptor PTH1 and plays an important role in calcium homeostasis. PTH's interaction with the N-terminal domain of PTH1 is mediated in part by Arg-20 on the peptide which forms a number of interactions with the receptor: a charge-charge interaction with Asp-137; hydrogen bonds with the backbone of Asp-29 and Met-32; and hydrophobic interactions with Met-32 and Gln-37. The aim of this work was to establish the importance of the charge-charge interaction through the combined use of modified peptide ligands, site-directed mutations of the receptor, and pharmacological assays. The substitution of Arg-20 with norleucine resulted in a 50-fold reduction in potency at PTH1 and Asp-137-Glu while, in contrast, both Asp-137-Asn and Asp-137-Ala receptors were largely insensitive to this ligand modification. The effect of this removal of the positive charge as position 20 could be partially rescued at PTH1 and Asp-137-Glu, but not Asp-137-Asn and Asp-137-Ala, through a substitution of peptide position 20 with ornithine. The latter two receptors, which have no negative charge at position 137, displayed potency for PTH that was reduced by 40- and 117-fold, respectively. These data demonstrate that a negative charge at residue-137 is important for interacting with ligands containing a positive charge at residue-20, and that the Arg-20 interaction with Asp-137, observed in the crystal structure of the isolated N-terminal domain of PTH1, is likely to be present in the full length receptor where it provides an important affinity- and potency-generating interaction through a salt bridge.
authors.affiliation: School of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK.
authors.forename: Richard E
authors.initials: RE
authors.lastname: Weaver
authors.affiliation: GlaxoSmithKline, New Frontiers Science Park North, Third Avenue, Harlow CM19 5AW, UK.
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Wigglesworth
authors.affiliation: School of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK. Electronic address: d.donnelly@leeds.ac.uk.
authors.forename: Dan
authors.initials: D
authors.lastname: Donnelly
mesh_terms: D001120:Arginine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001224:Aspartic Acid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D010281:Parathyroid Hormone / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D044168:Receptor, Parathyroid Hormone, Type 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055672:Static Electricity
chemical_list: C473449:PTH1R protein, human; D010281:Parathyroid Hormone; D044168:Receptor, Parathyroid Hormone, Type 1; D001224:Aspartic Acid; D001120:Arginine
keywords: Agonist; GPCR; Osteoporosis; PTH; Parathyroid hormone; Receptor