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

Richard E. Weaver, Juan C. Mobarec, Mark J. Wigglesworth, Christopher A. Reynolds, Dan Donnelly

Research output: Contribution to journalArticle

5 Citations (Scopus)
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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-PTH2 interaction. To address this, we utilised the different pharmacological profiles of TIP39 and PTH(1–34) at PTH2 and 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-PTH2 complex 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.

Original languageEnglish
Pages (from-to)71-81
Number of pages11
JournalBiochemical Pharmacology
Volume127
Early online date22 Dec 2016
DOIs
Publication statusPublished - 1 Mar 2017
Externally publishedYes

Bibliographical note

This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Keywords

  • Agonist
  • GPCR
  • Parathyroid hormone
  • PTH
  • Receptor
  • TIP39

ASJC Scopus subject areas

  • Biochemistry
  • Pharmacology

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