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

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• 作者：Richard E. Weaver^a ; Juan C. Mobarec^c ; Mark J. Wigglesworth^b ; ¹ ; Christopher A. Reynolds^c ; Dan Donnelly^a ; ^{d.donnelly@leeds.ac.uk}
• 关键词：Parathyroid hormone ; PTH ; GPCR ; Agonist ; Receptor ; TIP39
• 刊名：Biochemical Pharmacology
• 出版年：2017
• 出版时间：1 March 2017
• 年：2017
• 卷：127
• 期：Complete
• 页码：71-81
• 全文大小：2708 K
• 卷排序：127

文摘

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.