Elsevier

Peptides

Volume 28, Issue 4, April 2007, Pages 887-892
Peptides

Development of a rat parathyroid hormone 2 receptor antagonist

https://doi.org/10.1016/j.peptides.2006.12.002Get rights and content

Abstract

The parathyroid hormone 2 (PTH2) receptor is a Family B G-protein coupled receptor most highly expressed within the brain. Current evidence suggests that tuberoinfundibular peptide of 39 residues (TIP39) is the PTH2 receptor's endogenous ligand. To facilitate investigation of the physiological function of the PTH2 receptor/TIP39 system, we have developed a novel PTH2 receptor antagonist, by changing several residues within the amino terminal domain of TIP39. Histidine4, tyrosine5, tryptophan6, histidine7-TIP39 binds the PTH2 receptor with high affinity, has over 30-fold selectivity for the rat PTH2 receptor over the rat PTH1 receptor and displays no detectable agonist activity. This ligand should be useful for in vivo investigation of PTH2 receptor function.

Introduction

The parathyroid hormone 2 (PTH2) receptor is a member of the Family B group of G-protein coupled receptors (GPCRs) [6]. Its distribution has been mapped in detail, both in the central nervous system and in a number of peripheral organs [16], [18]. Based on its distribution in the brain it may play a role in regulation of neuroendocrine, emotion, auditory, and pain related processes. Its peripheral distribution suggests involvement in endocrine, cardiovascular and reproductive function [20]. However, there is relatively little experimental data relevant to its physiological role. The few published studies have used administration of pharmacological doses of its putative agonist, tuberoinfundibular peptide of 39 residues (TIP39) [1], [9], [10], [13], [14], [21], or sequestration of TIP39 with an antibody [1]. A potent and specific antagonist would be a particularly useful reagent for investigation of PTH2 receptor function.

The PTH2 receptor has about 50% sequence identity with the PTH1 receptor [17]. The PTH1 receptor is also referred to as the PTH/PTHrP receptor. It is the receptor responsible for parathyroid hormone (PTH) regulation of calcium homeostasis, and for many effects of parathyroid hormone-related protein (PTHrP) on skeletal development and tissue remodeling [11]. PTH1 receptors are potently activated by both PTH and PTHrP. The human PTH2 receptor is potently activated by both PTH and TIP39 [19], whereas PTH is a weak partial agonist at the rat PTH2 receptor and TIP39 a potent agonist [15]. PTHrP does not cause detectable activation of PTH2 receptors. Peptides containing the first 34 or 36 residues of PTH or PTHrP have full potency and affinity at the PTH1 receptor, and have been used in most studies of receptor function. The carboxyl terminal half of these peptides is largely responsible for high affinity binding to the receptor and the amino terminal half for activation of the receptor [2]. Ligand residues that provide PTH2 receptor discrimination between PTH and PTHrP have been identified. Either a hybrid peptide composed of the first 21 residues of PTHrP followed by 13 residues of PTH or an analog of PTHrP in which the phenylalanine normally present at residue 23 of PTHrP is replaced by tryptophan, which is present at that position in PTH, bind to but do not activate the human PTH2 receptor [4]. These human PTH2 receptor antagonists are full and potent agonists at the human PTH1 receptor. Their properties at the rat PTH2 receptor have not been described. They may not be ideal reagents for investigating PTH2 receptor function in rodents because of the relatively low potency of PTH for the rat PTH2 receptor, and because of the confound created by their activation of the PTH1 receptor.

Deletion of residues from the amino terminus of PTH or PTHrP reduces their ability to activate the PTH1 receptor while having relatively small effects on their binding affinity. PTH(7-34), PTHrP(7-36), and several analogs of these peptides are PTH1 receptor antagonists (Ref. [3] and references therein). We reasoned that similar modifications of TIP39 might generate a PTH2 receptor antagonist. Removing amino terminal residues from TIP39 did reduce its ability to activate the PTH2 receptor, but it also reduced the peptide's affinity for the PTH2 receptor such that a peptide with no detectable agonist activity, TIP(7-39), had too low an affinity to be useful as a PTH2 receptor antagonist [7]. Surprisingly, removing amino terminus residues from TIP39 increased its affinity for the PTH1 receptor, and TIP(7-39) is a potent PTH1 receptor antagonist [8].

We have now taken a third approach to development of a PTH2 receptor antagonist. Instead of removing residues from the amino terminus, receptor activating, domain of TIP39, we examined the effect of changing some of the residues to ones with different charge or bulk. Using this approach, we have developed a potent and selective PTH2 receptor antagonist.

Section snippets

Preparation of TIP39 mutations

A bovine TIP39 cDNA was cloned into the vector pet32 (Novagen, EMD Biosciences, San Diego, CA) to produce a hexahistidine containing thioredoxin fusion protein. Site-directed mutagenesis was performed using the Stratagene Quick-Change mutagenesis strategy. A 50 μl PCR reaction mixture contained 50 ng wild-type pet-TIP39 plasmid DNA, 125 ng of designed mutagenic upper and lower primers, 10 mM dNTPs, five units of PfuTurbo DNA Polymerase (Stratagene, LaJolla CA), and reaction buffer supplied with

Results

We performed site directed mutagenesis on a bovine TIP39 cDNA cloned as a histidine tagged fusion protein in a bacterial expression vector. After identification of mutants by sequencing the inserts in selected clones, protein expression was induced in bacterial cultures, the expressed proteins were purified on a nickel column, and then digested with Factor Xa. The desired peptides were then purified by reverse-phase HPLC. Their purity was assessed and sequence confirmed by high resolution

Discussion

We have identified a peptide analog of TIP39 that is a potent and selective antagonist of the rat PTH2 receptor. It has over 30-fold selectivity for the rat PTH2 receptor over the rat PTH1 receptor and causes no detectable activation of either receptor in our very sensitive assays. The antagonist has significantly greater affinity for the PTH2 receptor than previously described antagonists. Previously described PTH analogs are also either not selective for the PTH2 receptor or act as agonists

Acknowledgements

This research was supported by the intramural program of the NIH, National Institute of Mental Health. DNA sequencing was provided by the National Institute of Neurological Diseases Intramural Sequencing Core Facility. Mass Spectrometry was performed in the National Institute of Neurological Diseases Intramural Protein/Peptide Sequencing Facility. We thank Samuel Hoare for helpful discussion.

References (21)

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