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.
Highlights
The recent increase in structural information for class B G proteincoupled receptor (GPCR), encompassing both the extracellular domain and the transmembrane helical bundle, can be used to interpret pharmacological studies of class B peptide hormones
Our focus is on the parathyroid hormone 2 receptor (PTH2), a Family B G proteincoupled receptor (GPCR) which is potently activated by its endogenous neuropeptide TIP39 (‘‘tuberoinfundibular peptide of 39 residues”)
The aim of this study was to use site-directed mutagenesis to substitute selected PTH2 residues in the J domain, with those found in PTH1, in order to identify residues in PTH2 that play a role in ligand selection through the recognition of the N-terminal region of TIP39
Summary
The recent increase in structural information for class B GPCRs, encompassing both the extracellular domain and the transmembrane helical bundle, can be used to interpret pharmacological studies of class B peptide hormones. Our focus is on the parathyroid hormone 2 receptor (PTH2), a Family B G proteincoupled receptor (GPCR) which is potently activated by its endogenous neuropeptide TIP39 (‘‘tuberoinfundibular peptide of 39 residues”). Human PTH2 is activated by parathyroid hormone (PTH) and shares 50% sequence identity with PTH1, the. We seek to identify the key interactions that govern the selective activation of PTH2 by TIP39. Like other Family B GPCRs, PTH2 is activated by peptide agonists via a two site interaction model [2,3] in which the ligand’s
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