Isolation and functional characterization of an allatotropin receptor from Manduca sexta

https://doi.org/10.1016/j.ibmb.2011.06.002Get rights and content

Abstract

Manduca sexta allatotropin (Manse-AT) is a multifunctional neuropeptide whose actions include the stimulation of juvenile hormone biosynthesis, myotropic stimulation, cardioacceleratory functions, and inhibition of active ion transport. Manse-AT is a member of a structurally related peptide family that is widely found in insects and also in other invertebrates. Its precise role depends on the insect species and developmental stage. In some lepidopteran insects including M. sexta, structurally-related AT-like (ATL) peptides can be derived from alternatively spliced mRNAs transcribed from the AT gene. We have isolated a cDNA for an AT receptor (ATR) from M. sexta by a PCR-based approach using the sequence of the ATR from Bombyx mori. The sequence of the M. sexta ATR is similar to several G protein-coupled receptors from other insect species and to the mammalian orexin receptor. We demonstrate that the M. sexta ATR expressed in vertebrate cell lines is activated in a dose-responsive manner by Manse-AT and each Manse-ATL peptide in the rank order ATL-I > ATL-II > ATL-III > AT, and functional analysis in multiple cell lines suggest that the receptor is coupled through elevated levels of Ca2+ and cAMP. In feeding larvae, Manse-ATR mRNA is present at highest levels in the Malpighian tubules, followed by the midgut, hindgut, testes, and corpora allata, consistent with its action on multiple target tissues. In the adult corpora cardiaca–corpora allata complex, Manse-ATR mRNA is present at relatively low levels in both sexes.

Graphical abstract

Highlights

► We isolated the cDNA for the allatotropin receptor (ATR) in Manduca sexta. ► The M. sexta ATR is similar to other insect receptors and to the mammalian orexin receptor. ► The M. sexta ATR expressed in mammalian cells responds to allatotropin and each allatotropin-like peptide. ► The M. sexta ATR is expressed in multiple tissues in feeding larvae, the highest level is in the Malpighian tubules.

Introduction

Juvenile hormone (JH) is a sesquiterpenoid that possesses numerous developmental and physiological functions in insects, including the regulation of molting and metamorphosis in immature insects (Riddiford, 1994) and the control of reproductive events and egg maturation in adult insects (Wyatt and Davey, 1996). The control of JH levels in the insect hemolymph is achieved, in part, by neuropeptides that stimulate or inhibit its biosynthesis by the corpora allata (CA), allatotropin (AT) and allatostatin (AST), respectively (Weaver and Audsley, 2009). A great deal of progress has been made in the characterization of these neuropeptides using biochemical methods and physiological assays performed in vitro. More recently, major advances in studying the full complement of neuropeptides in insects came with the utilization of bioinformatic tools to predict the genes encoding neuropeptides from genome sequences and EST libraries. Neuropeptides can occur as unique components or as members of families based on their sequence similarity whose members often have similar functions.

Insect neuropeptides were first characterized by their biochemical isolation, structural elucidation, and activity in a number of bioassays; and were usually named based on their first known biological activity. AT was first characterized in the tobacco hornworm, Manduca sexta, (and designated Manse-AT) based on its ability to stimulate JH biosynthesis in the CA of adult, female lepidopteran insects in vitro (Kataoka et al., 1989). Subsequently in M. sexta, it was shown that Manse-AT possesses cardioexcitatory activity in pharate adults (Veenstra et al., 1994) and inhibits active ion transport across the larval midgut epithelium in vitro (Lee et al., 1998). Additional functions for Manse-AT in other lepidopterans include the stimulation of JH biosynthesis by the larval CA in some, but not all, lepidopteran insects (Audsley et al., 2000, Li et al., 2002), stimulation of foregut contractions in Helicoverpa armigera and Lacanobia oleracea (Duve et al., 1999, Duve et al., 2000), and the stimulation of ventral diaphragm oscillation in Pseudaletia unipuncta (Koladich et al., 2002). Additional activities were described for Manse-AT, or the endogenous homolog, in other insects including the stimulation of JH biosynthesis by CA of insects from the orders Diptera (Tu et al., 2001, Veenstra and Costes, 1999), Hymenoptera (Rachinsky and Feldlaufer, 2000), Dermaptera (Rankin et al., 2005) and Coleoptera (Abdel-latief and Hoffmann, 2010); the stimulation of myotropic activity in insects from the orders Orthoptera (Paemen et al., 1991), Dictyoptera (Rudwall et al., 2000), Coleoptera (Spittaels et al., 1996), and Hemiptera (Santini and Ronderos, 2007); and the photic entrainment of the circadian clock in an insect from the order Dictyoptera (Petri et al., 2002). Like many neuropeptides, Manse-AT has multiple functions, and its primary role may differ depending on the developmental stage of the insect.

Manse-AT is a member of a peptide family that has been isolated from numerous insect species, deduced from the sequences of homologous genes, or identified in silico from nucleotide sequence databases (Elekonich and Horodyski, 2003, Weaver and Audsley, 2009). Related peptides have been isolated from mollusks (Harada et al., 1993, Li et al., 1993) and annelids (Ukena et al., 1995) based on their myotropic activity, and it has been suggested that the ancestral role for this peptide family is related to its myotropic role (Elekonich and Horodyski, 2003). Despite its widespread appearance in numerous insects, the AT sequence has not been identified in Drosophila melanogaster or other members of that genus (Hewes and Taghert, 2001, Vanden Broeck, 2001), or in the hymenopteran insects Apis mellifera (Hummon et al., 2006) or Nasonia vitripennis (Hauser et al., 2010). In some lepidopteran insects including M. sexta, multiple mRNAs that differ by alternative splicing are transcribed from the AT gene in a stage- and tissue-specific manner and predict the presence of peptides that are structurally related to Manse-AT [ATL peptides; (Abdel-latief et al., 2003, Horodyski et al., 2001, Sheng et al., 2007, Yin et al., 2005)], and whose biological activities overlap with those of Manse-AT (Lee et al., 2002).

Neuropeptides exert effects on their cellular targets by binding to high affinity receptors, the vast majority of which are members of a large family of structurally-related proteins that possess seven hydrophobic membrane-spanning regions, the G protein-coupled receptors (GPCRs). Ligand binding transduces a signal mediated by activation of a GTP-binding protein (G protein) that leads to alterations in the levels of second messengers that, in turn, exert functional changes in the target cell and the organism. The specificity of GPCRs is generally determined by expression of the GPCR in a heterologous cell line or in Xenopus oocytes, and the subsequent demonstration of ligand-dependent changes in second messengers that are monitored by the quantification of their levels, expression of reporter gene constructs, or the opening of channel proteins. The characterization of neuropeptide GPCRs has also been greatly advanced by the use of molecular tools and the determination of genome sequences to identify similar GPCRs in different insects which respond to the homologous neuropeptide.

The AT receptor (ATR) was first identified in the silkmoth, Bombyx mori, (Bommo-ATR) by the systematic screening of GPCRs that were highly expressed in the corpora cardiaca–corpora allata (CC–CA) complex with neuropeptides in a heterologous expression system (Yamanaka et al., 2008). Transcript levels of Bommo-ATR were determined in multiple tissues at two larval stages by quantitative RT-PCR (qPCR), consistent with the multiple functions of the peptide. Bommo-ATR mRNA levels differed between the two larval stages, and levels were highest in the CC–CA, brain, epidermis, and testes. In situ hybridization demonstrated that Bommo-ATR mRNA was present in the CC, but not the CA.

We used the sequence of Bommo-ATR to design oligonucleotide primers to isolate the homologous GPCR from M. sexta (Manse-ATR). In order to determine if Manse-ATR reacted to Manse-AT or the Manse-ATL peptides, we expressed Manse-ATR in a heterologous cell line and tested whether these peptides activate the Manse-ATR and then determined their relative potencies. We showed that Manse-AT and each Manse-ATL mediated peptide-dependent responses in a dose-responsive manner at high affinity. Each of the Manse-ATL peptides exhibited a higher potency to Manse-ATR than Manse-AT. Then, to determine which second messenger the receptor is coupled to, Manse-ATR was expressed in other cell lines to demonstrate that the receptor activation increases both cAMP and Ca2+ levels suggesting that it may act through two distinct G proteins.

Section snippets

Insects

Larvae of the tobacco hornworm, M. sexta, were reared individually on an artificial diet (BioServ) at 25 °C under a long-day (16L:8D) photoperiod (Bell and Joachim, 1976). The day of ecdysis to the fifth larval instar is designated day 0. Wandering larvae, characterized by the appearance of a prominent dorsal vessel, were collected and transferred to plastic vials with vermiculite. These larvae molted into pupae 5 days later. Pharate adults were selected when the pupal cuticle above the wing is

Isolation of an allatotropin receptor (ATR) cDNA

The only insect in which an ATR has been characterized thus far is the silkworm, B. mori (Yamanaka et al., 2008). To identify an ATR from M. sexta, we designed degenerate oligonucleotide primers based on the sequence of the B. mori ATR cDNA (BNGR-A16) to amplify a portion of a putative M. sexta ATR cDNA using day 0, 5th instar larval cDNA. We used this tissue because the larval midgut is a known target of Manse-AT (Lee et al., 1998), and the large size of the larval midgut compared with the

Discussion

In this study, we used the sequence of the B. mori ATR (BNGR-A16) (Yamanaka et al., 2008) to design degenerate oligonucleotide primers to amplify a portion of the homologous cDNA from the larval midgut of M. sexta, and determined the full-length sequence following amplification of the 5′- and 3′-ends using RACE. We predicted that the midgut from feeding M. sexta larvae would be a good source of Manse-ATR mRNA since Manse-AT robustly inhibits active ion transport across the larval midgut

Acknowledgments

This research was supported by a grant from the National Science Foundation IOS-0821930 and research support from the Ohio University College of Osteopathic Medicine Research Committee to F.M.H. We thank M. Parmentier (University of Brussels, Belgium) and M. Detheux (Euroscreen S.A., Belgium) for providing CHO-WTA11 and CHO-PAM28 cells. We gratefully acknowledge the Interuniversity Attraction Poles programs [Belgian Science Policy Grant (P6/14)] and the K.U. Leuven Research Foundation (GOA/11/02

References (67)

  • L. Paemen et al.

    Lom-AG-myotropin: a novel myotropic peptide from the male accessory glands of Locusta migratoria

    Peptides

    (1991)
  • A. Rachinsky et al.

    Responsiveness of honey bee (Apis mellifera L.) corpora allata to allatoregulatory peptides from four insect species

    J. Insect Physiol.

    (2000)
  • S.M. Rankin et al.

    Effects of Manduca allatotropin and localization of Manduca allatotropin-immunoreactive cells in earwigs

    Comp. Biochem. Physiol.

    (2005)
  • L.M. Riddiford

    Cellular and molecular actions of juvenile hormone I. General considerations and premetamorphic actions

    Adv. In Insect Phys.

    (1994)
  • T. Sakurai et al.

    Orexins and orexin receptors: a family of hypothalamic neuropeptides and G protein-coupled receptors that regulate feeding behavior

    Cell

    (1998)
  • L.M. Schwartz et al.

    Hormonal control of rates of metamorphic development in the tobacco hornworm Manduca sexta

    Dev. Biol.

    (1983)
  • Z. Sheng et al.

    Biochemical and molecular characterization of allatotropin and allatostatin from the Eri silkworm, Samia cynthia ricini

    Insect Biochem. Mol. Biol.

    (2007)
  • J. Stables et al.

    A bioluminescent assay for agonist activity at potentially any G-protein-coupled receptor

    Anal. Biochem.

    (1997)
  • M.-P. Tu et al.

    Immunolocalization and possible effect of a moth allatotropin-like substance in a fly, Phormia regina (Diptera: Calliphoridae)

    J. Insect Physiol.

    (2001)
  • K. Ukena et al.

    A novel gut tetradecapeptide isolated from the earthworm, Eisenia foetida

    Peptides

    (1995)
  • J. Vanden Broeck

    Neuropeptides and their precursors in the fruitfly, Drosophila melanogaster

    Peptides

    (2001)
  • J.A. Veenstra

    Isolation and structure of corazonin, a cardioactive peptide from the American cockroach

    FEBS Lett.

    (1989)
  • J.A. Veenstra et al.

    Isolation and identification of a peptide and its cDNA from the mosquito Aedes aegypti related to Manduca sexta allatotropin

    Peptides

    (1999)
  • R.J. Weaver et al.

    Neuropeptides of the beetle, Tenebrio molitor identified using MALDI-TOF mass spectrometry and deduced sequences from the Tribolium castaneum genome

    Peptides

    (2008)
  • G.R. Wyatt et al.

    Cell and molecular actions of juvenile hormone II. Roles of juvenile hormone in adult insects

    Adv. In Insect Phys.

    (1996)
  • N. Yamanaka et al.

    Identification of a novel prothoracicostatic hormone and its receptor in the silkworm, Bombyx mori

    J. Biol. Chem.

    (2005)
  • R. Ziegler et al.

    Amino acid sequence of Manduca sexta adipokinetic hormone elucidated by combined fast atom bombardment/tandem mass spectrometry

    Biochem. Biophys. Res. Commun.

    (1985)
  • M. Abdel-latief et al.

    Neuropeptide regulators of the juvenile hormone biosynthesis (in vitro) in the beetle, Tenebrio molitor (Coleoptera, Tenebrionidae)

    Arch. Insect Biochem. Physiol.

    (2010)
  • C.U. Allen et al.

    Fura-2 measurement of cytosolic free Ca2+ concentration in corpus allatum cells of larval Manduca sexta

    J. Exp. Biol.

    (1992)
  • S.F. Altschul et al.

    Gapped BLAST and PHI-BLAST: a new generation of protein database search programs

    Nucleic Acids Res.

    (1997)
  • R.A. Bell et al.

    Techniques for rearing laboratory colonies of tobacco hornworms and pink bollworms

    Ann. Entomol. Soc. Am.

    (1976)
  • R.H. Don et al.

    ‘Touchdown’ PCR to circumvent spurious priming during gene amplification

    Nucleic Acids Res.

    (1991)
  • H. Duve et al.

    Triple co-localization of two types of allatostatin and an allatotropin in the frontal ganglion of the lepidopteran Lacanobia oleracea (Noctuidae): innervation and action on the foregut

    Cell Tissue Res.

    (2000)
  • Cited by (46)

    • Allatotropin

      2021, Handbook of Hormones: Comparative Endocrinology for Basic and Clinical Research
    • G protein-coupled receptor signal transduction and Ca<sup>2+</sup> signaling pathways of the allatotropin/orexin system in Hydra

      2021, General and Comparative Endocrinology
      Citation Excerpt :

      The presence of an AT-like signaling system in Cnidaria and other groups, including the neuron-less phylum Placozoa, suggests that this system has been present in the common ancestor of Metazoa, and that myoregulation is the ancestral function of the peptide, while the stimulation of JHs in insects would be a derived function (Alzugaray et al., 2013, 2019b; Alzugaray and Ronderos, 2018; Elekonich and Horodyski, 2003). AT exerts its functions by binding to a plasma membrane receptor that belongs to the G protein-coupled receptors (GPCRs) family (Yamanaka et al., 2008; Horodyski et al., 2011; Alzugaray and Ronderos, 2018; Alzugaray et al., 2019b). Like other GPCRs, the AT receptor (ATR) is composed by seven transmembrane domains and belongs to the rhodopsin subfamily, which is characterized by the presence of an E/DRY motif associated to the third transmembrane domain (TM3) (i.e. second intracellular loop), which seems to be relevant for the activity of the associated G proteins (Fredriksson et al., 2003; Millar and Newton, 2010).

    View all citing articles on Scopus
    View full text