Weaver, RE, Mobarec, JC, Wigglesworth, MJ et al. (2 more authors) (2017) 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. Biochemical Pharmacology, 127. pp. 71-81. ISSN 0006-2952
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.
Metadata
Item Type: | Article |
---|---|
Authors/Creators: |
|
Copyright, Publisher and Additional Information: | © 2016 The Author(s). Published by Elsevier Inc. This is an open access article under the terms of the Creative Commons Attribution License (CC-BY). |
Keywords: | Parathyroid Hormone; Pth; Gpcr; Agonist; Receptor; Tip39 |
Dates: |
|
Institution: | The University of Leeds |
Academic Units: | The University of Leeds > Faculty of Biological Sciences (Leeds) > School of Biomedical Sciences (Leeds) |
Depositing User: | Symplectic Publications |
Date Deposited: | 16 Dec 2016 16:32 |
Last Modified: | 23 Jun 2023 22:19 |
Published Version: | https://doi.org/10.1016/j.bcp.2016.12.013 |
Status: | Published |
Publisher: | Elsevier |
Identification Number: | 10.1016/j.bcp.2016.12.013 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:109551 |