title: High affinity binding of the peptide agonist TIP-39 to the parathyroid hormone 2 (PTH2) receptor requires the hydroxyl group of Tyr-318 on transmembrane helix 5.
abstract: TIP39 ("tuberoinfundibular peptide of 39 residues") acts via the parathyroid hormone 2 receptor, PTH2, a Family B G protein-coupled receptor (GPCR). Despite the importance of GPCRs in human physiology and pharmacotherapy, little is known about the molecular details of the TIP39-PTH2interaction. To address this, we utilised the different pharmacological profiles of TIP39 and PTH(1-34) at PTH2and its related receptor PTH1: TIP39 being an agonist at the former but an antagonist at the latter, while PTH(1-34) activates both. A total of 23 site-directed mutations of PTH2, in which residues were substituted to the equivalent in PTH1,were made and pharmacologically screened for agonist activity. Follow-up mutations were analysed by radioligand binding and cAMP assays. A model of the TIP39-PTH2complex was built and analysed using molecular dynamics. Only Tyr318-Ile displayed reduced TIP39 potency, despite having increased PTH(1-34) potency, and further mutagenesis and analysis at this site demonstrated that this was due to reduced TIP39 affinity at Tyr318-Ile (pIC50=6.01±0.03) compared with wild type (pIC50=7.81±0.03). The hydroxyl group of the Tyr-318's side chain was shown to be important for TIP39 binding, with the Tyr318-Phe mutant displaying 13-fold lower affinity and 35-fold lower potency compared with wild type. TIP39 truncated by up to 5 residues at the N-terminus was still sensitive to the mutations at Tyr-318, suggesting that it interacts with a region within TIP39(6-39). Molecular modelling and molecular dynamics simulations suggest that the selectivity is based on an interaction between the Tyr-318 hydroxyl group with the carboxylate side chain of Asp-7 of the peptide.
authors.affiliation: School of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK.
authors.forename: Richard E
authors.initials: RE
authors.lastname: Weaver
authors.affiliation: School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, UK.
authors.forename: Juan C
authors.initials: JC
authors.lastname: Mobarec
authors.affiliation: GlaxoSmithKline, New Frontiers Science Park North, Third Avenue, Harlow CM19 5AW, UK.
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Wigglesworth
authors.affiliation: School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, UK.
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Reynolds
authors.affiliation: School of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK. Electronic address: d.donnelly@leeds.ac.uk.
authors.forename: Dan
authors.initials: D
authors.lastname: Donnelly
mesh_terms: D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D009479:Neuropeptides / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D017433:Protein Structure, Secondary; D011869:Radioligand Assay; D044168:Receptor, Parathyroid Hormone, Type 1 / Q000737:chemistry / Q000378:metabolism; D044183:Receptor, Parathyroid Hormone, Type 2 / Q000819:agonists / Q000737:chemistry / Q000378:metabolism*; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics
chemical_list: D009479:Neuropeptides; D044168:Receptor, Parathyroid Hormone, Type 1; D044183:Receptor, Parathyroid Hormone, Type 2; C400136:tuberoinfundibular peptide 39; D014443:Tyrosine
keywords: Agonist; GPCR; PTH; Parathyroid hormone; Receptor; TIP39