Elsevier

Peptides

Volume 25, Issue 10, October 2004, Pages 1819-1830
Peptides

Structure–activity relationships and structural conformation of a novel urotensin II-related peptide

https://doi.org/10.1016/j.peptides.2004.04.019Get rights and content

Abstract

Urotensin II (UII) has been described as the most potent vasoconstrictor peptide and recognized as the endogenous ligand of the orphan G protein-coupled receptor GPR14. Recently, a UII-related peptide (URP) has been isolated from the rat brain and its sequence has been established as H-Ala-Cys-Phe-Trp-Lys-Tyr-Cys-Val-OH. In order to study the structure–function relationships of URP, we have synthesized a series of URP analogs and measured their binding affinity on hGPR14-transfected cells and their contractile activity in a rat aortic ring bioassay. Alanine substitution of each residue of URP significantly reduced the binding affinity and the contractile activity of the peptides, except for the Ala8-substituted analog that retained biological activity. Most importantly, d-scan of URP revealed that [d-Trp4]URP abrogated and [d-Tyr6]URP partially suppressed the UII-evoked contractile response. [Orn5]URP, which had very low agonistic efficacy, was the most potent antagonist in this series. The solution structure of URP has been determined by 1H NMR spectroscopy and molecular dynamics. URP exhibited a single conformation characterized by an inverse γ-turn comprising residues Trp-Lys-Tyr which plays a crucial role in the biological activity of URP. These pharmacological and structural data should prove useful for the rational design of non-peptide ligands as potential GPR14 agonists and antagonists.

Introduction

Urotensin II (UII) is a cyclic peptide that has been originally isolated from the caudal neurosecretory organ (termed urophysis) of teleost fish [53]. Characterization of UII from the brain of a frog has subsequently shown that a UII gene is also present in tetrapods and that this gene is expressed in the CNS [14]. In fact, the cDNA, encoding the UII precursor, has now been cloned in various species of vertebrates including carp [51], frog [16], mouse [17], rat [17], pig [47], monkey [21] and human [16]. The structure of the cyclic hexapeptide located in the C-terminal portion of UII has been fully conserved from fish to mammals while the sequence of the N-terminal region is highly variable (Fig. 1).

Pharmacological studies have shown that UII exercices a large array of spasmogenic actions. Notably, in fish and amphibians, UII provokes smooth muscle contraction in various vascular, intestinal and urogenital tissues [15]. In mammals, UII exerts a complex myotropic action on smooth muscles. Early studies have shown that UII produces relaxation of the mouse anococcygeus muscle [25] and causes hypotension in anesthetized rat [26]. More recently, UII has been found to exert potent contractile activity in rat, porcine, dog and primate vascular tissues [2], [8], [19] and primate airway smooth muscles [29]. In addition, intracerebroventricular or intracerebral administration of UII elicits various cardiovascular [36], [41], endocrine [24] and behavioral effects [24].

Using a reverse pharmacology approach, four groups have concomitantly shown that UII is the cognate ligand for the orphan G protein-coupled receptor GPR14 [2], [40], [47], [50]. The UII receptor is widely expressed in various peripheral organs including the cardiovascular system, the kidney, the bladder, as well as the central nervous system [2], [11], [24], [40], [44], [61], [63]. The variety of effects of UII and the broad expression pattern of its receptors indicate that UII may be involved in physiopathological processes. Indeed, recent studies suggest that UII may play a role in smooth muscle cell proliferation [56], [66], cardiac fibrosis and hypertrophy [64], [70], heart failure [66], cardiac remodeling [6], [20], atherosclerosis [62] and renal dysfunction [63].

A recent report has demonstrated the existence, in mouse, rat and human, of a paralog of UII named UII-related peptide (URP) [60] (Fig. 1). In the rat brain, the URP isoform has been purified and sequenced whereas the UII isoform has not yet been isolated [60]. Further, synthetic URP exhibits a higher binding affinity and a slightly higher potency to increase Ca2+ mobilization in human GPR14-transfected cells than the UII variant [60], suggesting that the biological effects previously attributed to UII could actually be exerted, at least in part, by its paralog URP.

In the present report, we have studied the structure–activity relationships of a series of URP analogs using a radioligand binding assay with hGPR14-transfected cells and an aortic ring contraction assay, and we have determined the solution conformation of URP by 1H NMR and molecular dynamics.

Section snippets

Chemicals and reagents

All l-amino acid residues, preloaded polyethylene glycol–polystyrene resins (Fmoc-Val-PEG-PS, Fmoc-Ala-PEG-PS), dichloromethane (DCM), 1-hydroxybenzotriazole (HOBt) and trifluoroacetic acid (TFA) were purchased from Applera-France (Courtaboeuf, France). Fmoc-3-iodo-Tyr-OH, Fmoc-Cys(Acm)-OH and all d-amino acid residues were obtained from Bachem Biochimie (Weil am Rhein, Germany). Fmoc-Pen(Acm)-OH was from VWR (Fontenay-sous-Bois, France). N,N-dimethylformamide (DMF), isopropanol (iPrOH) and

Peptide analysis

RP-HPLC analysis of hUII, URP and URP analogs revealed that the purity of all peptides was higher than 98% (Table 1). For all peptides, the molecular weight observed by MS analysis agreed with the theoretical values (Table 1). These physicochemical data ensured that pharmacological and functional information as well as structural parameters were undoubtedly assigned to the considered analog.

Structure–activity relationships

The binding affinity of URP (compound 1) on human GPR14-transfected cells was slightly higher than that

Discussion

Soon after the UII precursor had been characterized in human [16], several studies have shown that UII is a natural ligand of the orphan receptor GPR14 [2], [40], [47], [50]. Recently, a UII-related peptide called URP has been isolated from the rat brain [60] and the URP precursor cDNA has been cloned in human, rat and mouse [60]. Binding and functional studies have revealed that URP, like UII, is a potent agonist of GPR14 [60]. In the present report, we describe the structure–activity

Acknowledgments

This study was supported by INSERM and the Ministère de la Jeunesse, de l’Education Nationale et de la Recherche. D.C. and C.D. were recipients of fellowships from INSERM-SERVIER laboratories.

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    Symbols and abbreviations are in accord with the recommendations of the IUPAC-IUB Joint Commission on Biochemical Nomenclature: Nomenclature and Symbolism for Amino Acids and Peptides, Biochem. J. 1984, 219, 345–373, and of A Short Guide to Abbreviations and Their Use in Peptide Science, J. Peptide Sci. 2003, 9, 1–8. Additional abbreviations are as follows: RP-HPLC, reversed-phase high-performance liquid chromatography; NMR, nuclear magnetic resonance; MALDI-TOF-MS, matrix-assisted laser desorption ionization-time of flight mass spectrometry. All optically active amino acids are of the l-configuration unless otherwise noted.

    1

    Authors who equally contributed to this work.

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