Mapping the bimolecular interface of the parathyroid hormone (PTH)-PTH1 receptor complex: spatial proximity between Lys(27) (of the hormone principal binding domain) and leu(261) (of the first extracellular loop) of the human PTH1 receptor

Biochemistry. 2000 Jul 18;39(28):8142-52. doi: 10.1021/bi000195n.


In an effort to characterize the bimolecular interface between parathyroid hormone (PTH) and its human receptor PTH1-Rc (hPTH1-Rc), we previously identified two contact sites in the receptor: one for position 1 and another for position 13 (located at the ends of the principal activation domain) in PTH(1-34). The present study reports a third, novel "contact site" between hPTH1-Rc and Lys(27) of PTH(1-34). Lys(27) is located in the principal binding domain of the hormone (residues 25-34). The photoreactive PTH(1-34) analogue K27 contains a benzophenone (BP) moiety on Lys(27). The analogue binds to stably transfected HEK 293/C-21 cells (which express a high level of recombinant hPTH1-Rc) and stimulates adenylyl cyclase activity with a potency similar to PTH(1-34). In addition, (125)I-K27 cross-links effectively and specifically to the hPTH1-Rc. Enzymatic (Glu-C and Lys-C) and chemical (CNBr and BNPS-skatole) digestions of the photoconjugate between (125)I-K27 and hPTH1-Rc were performed. In addition, photoconjugates involving the bioactive mutants [L261M]- and [R262K]-hPTH1-Rc, transiently expressed in COS-7 cells, were also digested. The data obtained clearly identify L(261) or R(262) of the first extracellular loop of hPTH1-Rc as the contact site for Lys(27) in the hormone. On the basis of (i) the similarity in molecular mass between the CNBr digest of the (125)I-K27-[L261M]hPTH1-Rc conjugate and free (125)I-K27 and (ii) the failure to cross-link (125)I-K27 to a bioactive mutant receptor [L261A]hPTH1-Rc, we conclude that L(261) is the cross-linking site. These results provide the first demonstration of an interaction between the principal binding domain of PTH and the first extracellular loop of hPTH1-Rc. Revealing proximity of Lys(27) (in PTH) to L(261) (in hPTH1-Rc) provides additional insight into the nature of the ligand-receptor bimolecular interface and clearly illustrates that the extracellular loops of the receptor contribute to the specificity of the PTH-PTH1-Rc interaction. Taken together with previous studies, the new findings add important constraints on the possible positioning of the C-terminal helix of PTH (which contains the principal binding domain) relative to the first extracellular loop and the distal C-terminal helix of the large extracellular amino terminal domain of the PTH1-Rc.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antiporters
  • COS Cells
  • Cells, Cultured
  • Cyanogen Bromide / chemistry
  • DNA Restriction Enzymes / metabolism
  • Humans
  • Iodine Radioisotopes
  • Leucine / chemistry
  • Lysine / chemistry
  • Magnetic Resonance Spectroscopy
  • Membrane Proteins / chemistry
  • Molecular Sequence Data
  • Mutagenesis
  • Parathyroid Hormone / chemistry*
  • Parathyroid Hormone / genetics
  • Parathyroid Hormone / metabolism
  • Photoaffinity Labels
  • Protein Conformation
  • Receptors, Parathyroid Hormone / chemistry*
  • Receptors, Parathyroid Hormone / genetics
  • Receptors, Parathyroid Hormone / metabolism
  • Reproducibility of Results
  • Saccharomyces cerevisiae Proteins*


  • Antiporters
  • Iodine Radioisotopes
  • Membrane Proteins
  • Parathyroid Hormone
  • Photoaffinity Labels
  • Receptors, Parathyroid Hormone
  • Saccharomyces cerevisiae Proteins
  • VCX1 protein, S cerevisiae
  • DNA Restriction Enzymes
  • Leucine
  • Lysine
  • Cyanogen Bromide