Sequence, characterization and pharmacological analyses of the adipokinetic hormone receptor in the stick insect, Carausius morosus

Abstract

Background: Adipokinetic/hypertrehalosaemic hormone (AKH/HrTH), corazonin (Crz) and the AKH/Crz-related peptide (ACP) are neuropeptides considered homologous to the vertebrate gonadotropin-releasing hormone (GnRH). AKH/HrTH are important peptidergic metabolic regulators in insects that are crucial to provide energy during periods of high output mobility or when large amounts of energy-rich substrates are synthesized (for example, during vitellogenesis). AKH functions via a G protein-coupled receptor. Understanding which residue of the peptide (the ligand), activates the receptor with high efficacy is an important step to get insights into the ligand-receptor interaction, which is essential for further research on creating a model of how the ligand behaves in the binding pocket of the receptor. Such data are necessary for the search of non-peptidic mimetic agonists or antagonists in pesticide design.

Methods: Using bioinformatics and cloning techniques, the complete coding sequence of an AKH receptor was cloned and sequenced from fat body tissues and nervous tissues from the Indian stick insect, Carausius morosus. The resulting Carmo-AKHR was then expressed in a mammalian cell line where it could couple with a Gq protein to mediate calcium mobilization in vitro and cause bioluminescence when activated by a ligand. This receptor assay was used not only with the natural AKH ligands of the stick insect, but also with AKHs from other species and analogs with targeted modifications. A phylogenetic analysis of Carmo-AKHR with the AKH receptors and related receptors from other insects was also carried out.

Results: The stick insect AKH receptor was successfully cloned and sequenced from fat body and, separately, from nervous tissues. Comparison with known insect AKH, Crz and ACP receptors clearly put the stick insect receptor in the AKH clade and as sister group to other putative Phasmatodean AKH receptors. Moreover, the receptor expressed in mammalian cells was only activated by AKH and not by Crz or ACP indicating a true AKH receptor. Structure-activity studies in an Ala replacement series revealed the ligand residues that are absolutely essential for activating the AKHR: the N-terminal pGlu, Phe⁴, Trp⁸ and the C-terminal carboxyamide. Almost as important are Thr³ and Thr⁵ since their replacement reduced the efficacy more than a 100-fold, whereas Thr¹⁰ can be replaced without any real loss of activity. When substituted by Ala at positions 2, 6, 7 and 9, the ligand is somewhat affected with the loss of receptor activation being between 5- to 20-fold. Chain length of the ligand is important for the receptor: an octa- or nonapeptide with the same sequence otherwise as the endogenous stick insect ligand, display a 5- to 10 fold reduced activity. Carefully selected naturally occurring AKH analogs from other insects support the above results.

Conclusions: The AKH receptor from stick insects (Phasmatodea) cluster together in one clade distinct from other insect AKHRs, although still similar enough to be an insect AKHR, as opposed to the other GnRH-related receptors of insects, such as ACP and Crz receptors. The phylogenetic analyses support the data obtained from other studies involving receptors for AKH, Crz and ACP peptides. The receptor assay results with AKH analogs corroborated most of the results obtained previously using in vivo studies, thus emphasizing that the endogenous AKHs operate through this receptor to cause hypertrehalosemia in the stick insect. It is also clear that certain residues of the AKH peptides are consistently important in their interaction with the cognate AKH receptor, while other amino acid residues are of different importance to AKH receptors on a broad species- or group-specific manner. The previously observed peculiarity that hypertrehalosemia, in response to AKH injection, is only measurable in stick insects ligated below the head is discussed. No explanations for this, however, can be inferred from the current study.

Keywords: G protein-coupled receptor; adipokinetic hormone receptor; alanine replacement series; in vitro receptor activation; metabolism; pharmacological analyses; stick insect; structure-activity relationship.

Figure 1

Predicted topology and post-translational modifications of the complete and functional C. morosus AKH receptor. The deduced 528 amino acid Carmo-AKH receptor protein sequence was used to predict membrane topology (residues in membrane), N-linked glycosylation sites (residues denoted by green squares) along with potential phosphorylation sites within cytoplasmic domains of the receptor (residues denoted by purple diamonds). See the methods section for full description of steps in the analysis and prediction of post-translational modifications along with references for the web-based applications. Protter, an open-source tool, was used prepare the schematic of the receptor topology as well as label the predicted post-translational modifications.

Figure 2

Phylogenetic analysis of Carausius morosus AKH receptor using maximum likelihood method, supports its classification as an arthropod AKH receptor. The bootstrap consensus tree is shown inferred from 1000 replicates representing the relationship of the three evolutionarily related GnRH-like peptide receptor families present in arthropods. Branches corresponding to partitions with less than 50% bootstrap support are collapsed. The Carausius morosus AKH receptor (Carmo-AKHR) is highlighted in blue text within the clade containing other AKH receptors (denoted by blue circles). The ACP receptors (denoted by black squares) are clustered in a sister clade to the AKH receptors while the Crz receptors (denoted by red triangles) cluster in a separate sister clade to the monophyletic group comprised of AKH and ACP receptors. Receptor protein sequences are labelled by species name from which they originate and their GenBank accession numbers. The human gonadotropin releasing hormone receptor isoform 1 (GenBank: NP000397) was included in the analysis and designated as the outgroup.

Figure 3

The identified receptor is a true adipokinetic hormone receptor with no sensitivity to other GnRH-related peptides found in insects. (A) Raw luminescent response to three GnRH-related peptide family members from A. aegypti, specifically Aedae-AKH, Aedae-ACP and Aedae-Crz tested at 10 µM concentration. Only Aedae-AKH was able to activate the C. morosus receptor demonstrating a significantly elevated luminescent response. (B) Dose-response analysis of three GnRH-related peptides from A. aegypti against the C. morosus AKH receptor with only Aedae-AKH resulting in dose-dependent receptor activation. In (A), different letters denote bars that are significantly different from one another as determined by one-way ANOVA and Tukey’s post-hoc test; in (B), data normalized to the maximum luminescent response using ATP (mean +/- SEM, n = 3).

Figure 4

The effect of endogenous stick insect hypertrehalosemic peptides on stick insect AKH receptor activation. Dose-response analysis of two endogenous HrTH peptides, Carmo-HrTH-I and Carmo-HrTH-II, against the C. morosus AKH receptor. The primary structure of the endogenous HrTH peptides is identical, except that HrTH-I has an unusually modified Trp at position 8, with a C-bonded alpha-mannopyranose (see Table 1 ). Data normalized to the maximum luminescent response using ATP (mean +/- SEM, n = 3).

Figure 5

Activity of synthetic modified analogs of Carmo-HrTH-II on the stick insect AKH receptor. (A) Dose-response analysis of select synthetic analogs of Carmo-HrTH-II against the C. morosus AKH receptor, including alanine-substituted analogs in positions 1, 2, 4, 6 and 9 along with the non-amidated analog of Carmo-HrTH-II. (B) Dose-response analysis of distinct synthetic analogs of Carmo-HrTH-II against the C. morosus AKH receptor, including alanine-substituted analogs in positions 3, 5, 7, 8 and 10 along with an analog of Carmo-HrTH-II lacking the N-terminal pyroglutamic acid. Data normalized to the maximum luminescent response using ATP (mean +/- SEM, n = 3).

Figure 6

Activity of naturally occurring arthropod AKH analogs on the stick insect AKH receptor. (A) Natural AKH analogs with one, two or three non-alanine amino acid substitutions or different chain length compared to Carmo-HrTH-II. (B) Two naturally occurring AKH analogs from different insects (bioanalogs), Triin-AKH and Peram-CAH-II, which share the identical nine and eight, respectively, N-terminal residues to Carmo-HrTH-II and offer insights on the effect of shortened C-terminal chain length on the receptor selectivity for its endogenous ligands. Data normalized to the maximum luminescent response using ATP (mean +/- SEM, n = 3).