title: 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.
abstract: 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.
authors.affiliation: Division of Bone and Mineral Metabolism, Charles A. Dana and Thorndike Laboratories, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215, USA.
authors.forename: Z
authors.initials: Z
authors.lastname: Greenberg
authors.forename: A
authors.initials: A
authors.lastname: Bisello
authors.forename: D F
authors.initials: DF
authors.lastname: Mierke
authors.forename: M
authors.initials: M
authors.lastname: Rosenblatt
authors.forename: M
authors.initials: M
authors.lastname: Chorev
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017920:Antiporters; D019556:COS Cells; D002478:Cells, Cultured; D003488:Cyanogen Bromide / Q000737:chemistry; D004262:DNA Restriction Enzymes / Q000378:metabolism; D006801:Humans; D007457:Iodine Radioisotopes; D007930:Leucine / Q000737:chemistry; D008239:Lysine / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008565:Membrane Proteins / Q000737:chemistry; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010281:Parathyroid Hormone / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019920:Photoaffinity Labels; D011487:Protein Conformation; D018016:Receptors, Parathyroid Hormone / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D029701:Saccharomyces cerevisiae Proteins
chemical_list: D017920:Antiporters; D007457:Iodine Radioisotopes; D008565:Membrane Proteins; D010281:Parathyroid Hormone; D019920:Photoaffinity Labels; D018016:Receptors, Parathyroid Hormone; D029701:Saccharomyces cerevisiae Proteins; C099946:VCX1 protein, S cerevisiae; D004262:DNA Restriction Enzymes; D007930:Leucine; D008239:Lysine; D003488:Cyanogen Bromide