title: Residue 19 of the parathyroid hormone (PTH) modulates ligand interaction with the juxtamembrane region of the PTH-1 receptor.
abstract: Recent data suggest that the binding of parathyroid hormone (PTH)-(1-34) to the PTH-1 receptor (P1R) involves a high-affinity interaction between the C-terminal (15-34) domain of the ligand and the amino-terminal extracellular (N) domain of the receptor and a low-affinity interaction between the N-terminal (1-14) portion of PTH and the juxtamembrane (J) region of the receptor, with the latter interaction giving rise to signal transduction. We investigated whether residues C-terminal of position 14 in PTH(1-34) contribute to the J component of the interaction mechanism by comparing the capacity of PTH analogues N-terminally modified to improve J domain affinity and C-terminally truncated at position 14, 20, or 34 to stimulate cAMP formation in COS-7 cells transiently transfected with P1R-delNt, a P1R construct that lacks most of the N domain. In these cells, the potency of [M]PTH(1-34) (M = Ala(1,3,12),Gln(10),Har(11),Trp(14),Arg(19)) was 120-fold greater than that of [M]PTH(1-14) (EC(50)s = 3.0 +/- 0.8 and 360 +/- 90 nM, respectively) but was equal to that of [M]PTH(1-20) (EC(50) = 2.3 +/- 0.3 nM). Reverting the Arg(19) substitution of [M]PTH(1-20) to the native Glu reduced cAMP signaling potency on P1R-delNt by 12-fold (EC(50) of [M]PTH(1-20)-Glu(19) = 27 +/- 4 nM), and it decreased the analog's capacity to inhibit the binding of the J domain-selective radioligand, (125)I-[Aib(1,3),Nle(8),M,Tyr(21)]ratPTH(1-21), to the full-length P1R stably expressed in LLC-PK1 cells by 40-fold. The Glu(19) --> Arg modification, however, did not affect the capacity of PTH(15-31) to inhibit the binding of the N domain-selective radioligand (125)I-bPTH(3-34) to the full-length receptor. The overall data suggest that residues (15-20) of PTH, and particularly residue 19, contribute to the capacity of the N-terminal portion of the ligand to interact with the juxtamembrane region of the receptor. The NMR data presented in the accompanying manuscript suggests that this role could involve intramolecular effects on secondary structure in the N-terminal portion of the ligand.
authors.affiliation: Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114, USA.
authors.forename: Masaru
authors.initials: M
authors.lastname: Shimizu
authors.forename: Naoto
authors.initials: N
authors.lastname: Shimizu
authors.forename: Janet C
authors.initials: JC
authors.lastname: Tsang
authors.forename: Brian D
authors.initials: BD
authors.lastname: Petroni
authors.forename: Ashok
authors.initials: A
authors.lastname: Khatri
authors.forename: John T
authors.initials: JT
authors.lastname: Potts
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Gardella
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001120:Arginine / Q000737:chemistry* / Q000235:genetics; D001667:Binding, Competitive / Q000235:genetics; D019556:COS Cells; D002417:Cattle; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism; D002522:Chlorocebus aethiops; D018698:Glutamic Acid / Q000235:genetics; D006801:Humans; D018374:LLC-PK1 Cells; D008024:Ligands; D010281:Parathyroid Hormone / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000235:genetics; D011869:Radioligand Assay; D051381:Rats; D018016:Receptors, Parathyroid Hormone / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D013552:Swine
chemical_list: D008024:Ligands; D010281:Parathyroid Hormone; D010446:Peptide Fragments; D018016:Receptors, Parathyroid Hormone; C025076:parathyroid hormone (1-34), bovine; D018698:Glutamic Acid; D001120:Arginine