Mechanisms of ligand binding to the parathyroid hormone (PTH)/PTH-related protein receptor: selectivity of a modified PTH(1-15) radioligand for GalphaS-coupled receptor conformations

Mol Endocrinol. 2006 Apr;20(4):931-43. doi: 10.1210/me.2005-0349. Epub 2005 Dec 8.


Mechanisms of ligand binding to the PTH/PTHrP receptor (PTHR) were explored using PTH fragment analogs as radioligands in binding assays. In particular, the modified amino-terminal fragment analog, (125)I-[Aib(1,3),Nle8,Gln10,homoarginine11,Ala12,Trp14,Tyr15]rPTH(1-15)NH2, (125)I-[Aib(1,3),M]PTH(1-15), was used as a radioligand that we hypothesized to bind solely to the juxtamembrane (J) portion of the PTHR containing the extracellular loops and transmembrane helices. We also employed (125)I-PTH(1-34) as a radioligand that binds to both the amino-terminal extracellular (N) and J domains of the PTHR. Binding was examined in membranes derived from cells expressing either wild-type or mutant PTHRs. We found that the binding of (125)I-[Aib(1,3),M]PTH(1-15) to the wild-type PTHR was strongly (approximately 90%) inhibited by guanosine 5'-O-(3-thio)triphosphate (GTPgammaS), whereas the binding of (125)I-PTH(1-34) was only mildly (approximately 25%) inhibited by GTPgammaS. Of these two radioligands, only (125)I-[Aib(1,3),M]PTH(1-15) bound to PTHR-delNt, which lacks most of the receptor's N domain, and again this binding was strongly inhibited by GTPgammaS. Binding of (125)I-[Aib(1,3),M]PTH(1-15) to the constitutively active receptor, PTHR-H223R, was only mildly (approximately 20%) inhibited by GTPgammaS, as was the binding of (125)I-PTH(1-34). In membranes prepared from cells lacking Galpha(S) via knockout mutation of Gnas, no binding of (125)I-[Aib(1,3),M]PTH(1-15) was observed, but binding of (125)I-[Aib(1,3),M]PTH(1-15) was recovered by virally transducing the cells to heterologously express Galpha(S). (125)I-PTH(1-34) bound to the membranes with or without Galpha(S). The overall findings confirm the hypothesis that (125)I-[Aib(1,3),M]PTH(1-15) binds solely to the J domain of the PTHR. They further show that this binding is strongly dependent on coupling of the receptor to Galpha(S)-containing heterotrimeric G proteins, whereas the binding of (125)I-PTH(1-34) can occur in the absence of such coupling. Thus, (125)I-[Aib(1,3),M]PTH(1-15) appears to function as a selective probe of Galpha(S)-coupled, active-state PTHR conformations.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • COS Cells
  • Cell Line
  • Cell Membrane / metabolism
  • Chlorocebus aethiops
  • GTP-Binding Protein alpha Subunits, Gs / chemistry*
  • GTP-Binding Protein alpha Subunits, Gs / deficiency
  • GTP-Binding Protein alpha Subunits, Gs / genetics
  • GTP-Binding Protein alpha Subunits, Gs / metabolism*
  • Humans
  • Kinetics
  • LLC-PK1 Cells
  • Ligands
  • Parathyroid Hormone / metabolism
  • Peptide Fragments / metabolism
  • Protein Conformation
  • Radioligand Assay
  • Receptor, Parathyroid Hormone, Type 1 / chemistry*
  • Receptor, Parathyroid Hormone, Type 1 / genetics
  • Receptor, Parathyroid Hormone, Type 1 / metabolism*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Swine


  • Ligands
  • Parathyroid Hormone
  • Peptide Fragments
  • Receptor, Parathyroid Hormone, Type 1
  • Recombinant Proteins
  • GTP-Binding Protein alpha Subunits, Gs