title: Backbone-methylated analogues of the principle receptor binding region of human parathyroid hormone. Evidence for binding to both the N-terminal extracellular domain and extracellular loop region.
abstract: We have used backbone N-methylations of parathyroid hormone (PTH) to study the role of these NH groups in the C-terminal amphiphilic alpha-helix of PTH (1-31) in binding to and activating the PTH receptor (P1R). The circular dichroism (CD) spectra indicated the structure of the C-terminal alpha-helix was locally disrupted around the methylation site. The CD spectra differences were explained by assuming a helix disruption for four residues on each side of the site of methylation and taking into account the known dependence of CD on the length of an alpha-helix. Binding and adenylyl cyclase-stimulating data showed that outside of the alpha-helix, methylation of residues Asp30 and Val31 had little effect on structure or activities. Within the alpha-helix, disruption of the structure was associated with increased loss of activity, but for specific residues Val21, Leu24, Arg25, and Leu28 there was a dramatic loss of activities, thus suggesting a more direct role of these NH groups in correct P1R binding and activation. Activity analyses with P1R-delNT, a mutant with its long N-terminal region deleted, gave a different pattern of effects and implicated Ser17, Trp23, and Lys26 as important for its PTH activation. These two groups of residues are located on opposite sides of the helix. These results are compatible with the C-terminal helix binding to both the N-terminal segment and also to the looped-out extracellular region. These data thus provide direct evidence for important roles of the C-terminal domain of PTH in determining high affinity binding and activation of the P1R receptor.
authors.affiliation: Institute for Biological Sciences, National Research Council, Ottawa, Ontario K1A 0R6 Canada.
authors.forename: Jean-René
authors.initials: JR
authors.lastname: Barbier
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Gardella
authors.forename: Thomas
authors.initials: T
authors.lastname: Dean
authors.forename: Susanne
authors.initials: S
authors.lastname: MacLean
authors.forename: Zhanna
authors.initials: Z
authors.lastname: Potetinova
authors.forename: James F
authors.initials: JF
authors.lastname: Whitfield
authors.forename: Gordon E
authors.initials: GE
authors.lastname: Willick
mesh_terms: D000262:Adenylyl Cyclases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002942:Circular Dichroism; D006801:Humans; D008745:Methylation; D008969:Molecular Sequence Data; D010281:Parathyroid Hormone / Q000737:chemistry / Q000378:metabolism*; D013552:Swine
chemical_list: D010281:Parathyroid Hormone; D000262:Adenylyl Cyclases