title: 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.
abstract: 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.
authors.affiliation: Endocrine Unit, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114, USA.
authors.forename: Thomas
authors.initials: T
authors.lastname: Dean
authors.forename: Agnes
authors.initials: A
authors.lastname: Linglart
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Mahon
authors.forename: Murat
authors.initials: M
authors.lastname: Bastepe
authors.forename: Harald
authors.initials: H
authors.lastname: Jüppner
authors.forename: John T
authors.initials: JT
authors.lastname: Potts
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Gardella
mesh_terms: D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000737:chemistry* / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D018374:LLC-PK1 Cells; D008024:Ligands; D010281:Parathyroid Hormone / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D011487:Protein Conformation; D011869:Radioligand Assay; D044168:Receptor, Parathyroid Hormone, Type 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013552:Swine
chemical_list: D008024:Ligands; D010281:Parathyroid Hormone; D010446:Peptide Fragments; D044168:Receptor, Parathyroid Hormone, Type 1; D011994:Recombinant Proteins; D019205:GTP-Binding Protein alpha Subunits, Gs