Elsevier

Peptides

Volume 53, March 2014, Pages 243-249
Peptides

Molecular cloning and functional characterization of the diapause hormone receptor in the corn earworm Helicoverpa zea

https://doi.org/10.1016/j.peptides.2013.11.005Get rights and content

Highlights

  • The gene encoding diapause hormone receptor of Helicoverpa zea was identified.

  • H. zea diapause hormone receptor was expressed in a heterologous reporter system.

  • The receptor was pharmacologically characterized by 68 ligands including peptidomimetics.

  • The ligand activities on the receptor were correlated with previous in vivo studies.

Abstract

The diapause hormone (DH) in the heliothine moth has shown its activity in termination of pupal diapause, while the orthology in the silkworm is known to induce embryonic diapause. In the current study, we cloned the diapause hormone receptor from the corn earworm Helicoverpa zea (HzDHr) and tested its ligand specificities in a heterologous reporter system. HzDHr was expressed in Chinese Hamster Ovary (CHO) cells, which were co-transfected with the aequorin reporter, and was used to measure the ligand activities. A total of 68 chemicals, including natural DH analogs and structurally similar peptide mimetics, were tested for agonistic and antagonistic activities. Several peptide mimetics with a 2-amino-7-bromofluorene-succinoyl (2Abf-Suc) N-terminal modification showed strong agonistic activities; these mimetics included 2Abf-Suc-F[dA]PRLamide, 2Abf-Suc-F[dR]PRLamide, 2Abf-Suc-FKPRLamide and 2Abf-Suc-FGPRLamide. Antagonistic activity was found in the ecdysis triggering hormone in Drosophila melanogaster (FFLKITKNVPRLamide). Interestingly, HzDHr does not discriminate between DH (WFGPRLamide C-terminal motif) and another closely related endogenous peptide, pyrokinin 1 (FXPRXamide; a C-terminal motif that is separate from WFGPRLamide). We provide large-scale in vitro data that serve as a reference for the development of agonists and antagonists to disrupt the DH signaling pathway.

Introduction

Neuropeptides are major controllers of various physiological and developmental events in insects, such as metabolism, reproduction, diapause, molting, growth, and metamorphosis. Among more than 40 different types of neuropeptides in insects [5], [10], [24], a large group carrying the C-terminal amino acid motif PRXamide is generally further categorized into three groups: cardioacceleratory peptide (CAPA) [23], ecdysis triggering hormone (ETH) [25], and pyrokinin/pheromone biosynthesis activating-neuropeptide (PK/PBAN) [7]. Likewise, the receptors for these peptides, G protein coupled receptors (GPCR), are clustered in the phylogeny [19], [22], indicating that the C-terminal motifs are an ancestral signature that is conserved for proper ligand–receptor interactions. Furthermore, the PRXamide C-terminal motif is also found in the mammalian neuropeptide neuromedin U, and its receptor is also clustered with insect PRXamide receptors [21].

The PRXamide peptides are further categorized by variations of the C-terminal motifs: CAPA contains FPRXamide; ETH has PRXamide generally with a K at the −6 position [25]; and PK/PBAN is known for FXPRXamide [7]. The PK/PBAN groups of peptides are further categorized into two subgroups; PK1 includes the diapause hormone (PK1/DH) with the WFGPRLamide motif, and PK2 includes PBAN (PK2/PBAN) with the general consensus FXPRLamide excluding the PK1 motif [7]. While the ligands are categorized by their conserved C-terminal motifs, the receptors for the ligands are similarly distinguished in the phylogeny as coevolutionary events between the ligands and the receptors.

DH was originally described for the induction of embryonic diapause by acting on developing oocytes during the pupal–adult development of the mother in Bombyx mori [6], [29]. Subsequently, DH-like peptides were found in heliothine moths, including Helicoverpa zea [12], H. assulta [3] and Heliothis virescens [28]. However, the bioactivity of DH in the heliothine moths was found to break pupal diapause, opposite to the action of DH in B. mori. The DH activity that breaks pupal diapause was found in H. armigera [32], H. assulta [33], H. virescens [28] and H. zea [30]. The DH signaling pathway, for either inducing or breaking diapause, which enables insects continue life in regularly recurring harsh environments, may serve as an excellent target system for controlling insect pests. This concept has been demonstrated by disruption of the diapause of H. zea by using DH agonistic or antagonistic peptidomimetics [30].

Understanding the properties of ligand interaction with the target receptor lays a foundation for rational design of peptidomimetics and potentially simple chemical compounds. Several PRXamide mimetics have previously been tested on in vivo systems of lepidopteran species for their various bioactivities [13], [14], [15], [17], which may have occurred through several different PRXamide receptors including the DH receptor (DHr). Furthermore, the in vivo activities of peptidomimetics are the consequences of complex phenomena, such as biostability, and bioavailability, and receptor specificity of the compounds. In this study, we provide in vitro activities of a total 68 peptidomimetics and PRXamide analogs on the DHr of a notorious pest species H. zea.

Section snippets

Chemicals and Insect

All pyrokinin analogs and peptide mimetics were synthesized as described previously by Nachman et al. [13], [14], [15], [17]. The peptides from Tribolium castaneum were synthesized by Genescript (Piscataway, NJ). For culturing Chinese Hamster Ovary cells, DMEM/F12 medium, fetal bovine serum (FBS), Fungizone® and Penicillin/Streptomycin, and coelenterazine for an aequorin functional assay were purchased from Gibco® Cell Culture at Life Technologies™ (Grand Island, NY). TransIT®-LT1 Transfection

Molecular characterization of HzDHr

The HzDHr cDNA contained a 1581 bp open reading frame (ORF), encoding a 516 amino acid protein (Fig. S1), with predicted molecular weights of 58.91 kDa and a pI of 9.34. The HzDHr sequence was deposited into GenBank (accession number KC182787). The ORF contained seven transmembrane domains, which is a typical signature of GPCRs. Multiple sequence alignment (Fig. 2) with the DHrs of D. plexippus, B. mori, O. thyellina and Ostrinia nubilalis [18] showed that DHr is highly conserved in the

Discussion

Previous studies have shown that the PK1/DH receptor and the PK2/PBAN receptor generally distinguish their respective ligands DH and PBAN in Lepidoptera. The DHr of B. mori expressed in a Xenopus oocyte was activated by the DH of B. mori and distinguished other Bombyx FXP(R/K)Lamide peptides by revealing a 20–50× higher EC50 [4]. Similarly, the PBAN receptor from H. virescens showed ∼20× higher sensitivity to PBAN than that to the DHs of B. mori and Manduca sexta [8]. In dipteran insects, D.

Acknowledgements

We thank Dr. Ladislav Šimo, Dong Hun Kim, and Joshua Urban for technical assistance. The research was supported in part by a US-Israel Binational Agricultural Research and Development Fund (BARD) grant (IS-4205-09C) (RJN) as well as a USDA-NIFA grant no. 2011-67013-30199 (RJN). This paper is contribution no. 14-123-J from the Kansas Agricultural Experiment Station.

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