PaOctβ2R: Identification and Functional Characterization of an Octopamine Receptor Activating Adenylyl Cyclase Activity in the American Cockroach Periplaneta americana

Abstract

Biogenic amines constitute an important group of neuroactive substances that control and modulate various neural circuits. These small organic compounds engage members of the guanine nucleotide-binding protein coupled receptor (GPCR) superfamily to evoke specific cellular responses. In addition to dopamine- and 5-hydroxytryptamine (serotonin) receptors, arthropods express receptors that are activated exclusively by tyramine and octopamine. These phenolamines functionally substitute the noradrenergic system of vertebrates Octopamine receptors that are the focus of this study are classified as either α- or β-adrenergic-like. Knowledge on these receptors is scarce for the American cockroach (Periplaneta americana). So far, only an α-adrenergic-like octopamine receptor that primarily causes Ca²⁺ release from intracellular stores has been studied from the cockroach (PaOctα1R). Here we succeeded in cloning a gene from cockroach brain tissue that encodes a β-adrenergic-like receptor and leads to cAMP production upon activation. Notably, the receptor is 100-fold more selective for octopamine than for tyramine. A series of synthetic antagonists selectively block receptor activity with epinastine being the most potent. Bioinformatics allowed us to identify a total of 19 receptor sequences that build the framework of the biogenic amine receptor clade in the American cockroach. Phylogenetic analyses using these sequences and receptor sequences from model organisms showed that the newly cloned gene is an β2-adrenergic-like octopamine receptor. The functional characterization of PaOctβ2R and the bioinformatics data uncovered that the monoaminergic receptor family in the hemimetabolic P. americana is similarly complex as in holometabolic model insects like Drosophila melanogaster and the honeybee, Apis mellifera. Thus, investigating these receptors in detail may contribute to a better understanding of monoaminergic signaling in insect behavior and physiology.

Keywords: GPCR; biogenic amines; cellular signaling; cockroach; gene annotation; gene family; second messenger.

Figure 1

Structural characteristics of the amino acid sequence deduced for PaOctβ2R. (A) Hydrophobicity profile of PaOctβ2R. The profile was calculated according to Kyte and Doolittle algorithm [32] using a window size of 19 amino acids. Peaks with scores greater than 1.6 (dashed line) indicate possible transmembrane regions. (B) Prediction of transmembrane domains with TMHMM server v. 2.0 [33]. Putative transmembrane domains are indicated in red. Extracellular regions are shown with a purple line, intracellular regions with a blue line. (C) Color-coded (rainbow) 3D model of the receptor as predicted by Phyre2 [34]. The extracellular N-terminus (N) and the intracellular C-terminus (C) are labeled.

Figure 2

Phylogenetic relationships of monoaminergic receptors. Alignments were performed with BioEdit [40] by using the core amino-acid sequences of TM 1-4, TM 5, TM 6, and TM 7. The evolutionary history was inferred by using the Maximum Likelihood method based on the Poisson correction model. The tree with the highest log likelihood (−28600.17) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 93 amino acid sequences. Human rhodopsin (HsRHOD) was used to root the tree. Receptor subclasses are given on the right. Abbreviations of species in alphabetical order are: Am Apis mellifera, Dm Drosophila melanogaster, Hs Homo sapiens, Pa Periplaneta americana, Pc Priapulus caudatus, Pd Platynereis dumerilii, Sk Saccoglossus kowalevskii. Protostomian species names are highlighted in red, whereas deuterostomian species names are given in blue. Accession numbers and annotations of all sequences used in the phylogenetic analysis can be found in Supplementary Table S1.

Figure 3

Tissue distribution of PaOctβ2R mRNA. A 100 bp DNA ladder is shown on the left. Detection of PCR products amplified on total RNA isolated from brain, salivary glands, Malpighian tubules, midgut, and leg muscle. Amplification failed when samples were digested with an RNAse cocktail prior to RT-PCR (data not shown). The lower panel shows RT-PCR products amplified with actin-specific primers (Accession No. AY116670) as a control.

Figure 4

Confocal microscopy of flpTM and PaOctβ2R-HA-expressing flpTM cells. FlpTM (A–D) and PaOctβ2R-HA-expressing flpTM cells (E–H) were co-immunostained with rat anti-HA antibodies and specific antibodies against the CNG channel. (A,E) Samples were incubated with primary rat anti-HA antibodies (dilution 1:100) and secondary goat anti-rat-Alexa488 (dilution 1:500) antibodies. Non-transfected flpTM cells do not show fluorescent signals (A). In PaOctβ2R-expressing flpTM cells (E), the PaOctβ2R-HA protein was detected. (B,F) The same samples were incubated with specific antibodies directed against the C-terminus of the CNG channel (dilution 1:200) and secondary donkey anti-mouse-Cy3 (dilution 1:400) antibodies. In both cell lines, the CNG channel was detected. (C,G) Nuclei were stained with TOPRO-3 and are clearly differentiated from the cytosol. (D,H) Composite images.

Figure 5

Biogenic amine evoked responses in flpTM and PaOctβ2R-HA-expressing cells. The relative change in fluorescence (corresponding to the amount of cAMP produced) in (A,B) is given as the percentage of the value obtained in the presence of 0.1 µM octopamine in flpTM + PaOctβ2R-HA cells (=100%). All measurements were performed in the presence of 100 µM 3-isobutyl-1-methylxanthine (IBMX). Biogenic amines were applied at two concentrations (0.1 µM (black bar) and 1 µM (white bar)). (A) Control measurements performed on flpTM cells did not result in increases in [cAMP]_i. (B) Only octopamine and tyramine evoked an increase in [cAMP]_i in cells expressing PaOctβ2R-HA. A representative of four independent measurements is shown. Mean values ± SD of four-fold determinations is displayed.

Figure 6

Concentration-dependent effects of octopamine and tyramine on [cAMP]_i in PaOctβ2R-HA-expressing and flpTM (control) cells. Relative change in fluorescence (corresponding to the amount of cAMP) is given as the percentage of the value obtained with the highest octopamine or tyramine concentration (=100%). All measurements were performed in the presence of 100 µM IBMX. Octopamine (•) and tyramine (▲) activation of PaOctβ2R-HA led to a concentration-dependent increase in the fluorescence signal. No change in the fluorescence signal was observed in flpTM cells (octopamine ♦; tyramine ■). A total of five independent measurements were performed. Data points represent the mean ± SD of a representative eight-fold determination.

Figure 7

Effects of putative antagonists on octopamine-activated PaOctβ2R-HA. Concentration series of (A) epinastine, (B) mianserin, (C) phentolamine, and (D) ketanserin were applied in the presence of 1 × 10⁻⁹ M octopamine and 1 × 10⁻⁴ M IBMX. Relative change in fluorescence (corresponding to the amount of cAMP) is given as the percentage of the value obtained in the exclusive presence of 1 × 10⁻⁹ M octopamine (=100%). Data represent the mean ± SD of eight values from a typical experiment. All determinations were independently repeated at least three times.

Figure 8

Effects of putative antagonists on tyramine-activated PaOctβ2R-HA. Concentration series of (A) epinastine, (B) mianserin, (C) phentolamine, and (D) ketanserin were applied in the presence of 1 × 10⁷ M tyramine and 1 × 10⁻⁴ M IBMX. Relative change in fluorescence (corresponding to the amount of cAMP) is given as the percentage of the value obtained in the exclusive presence of 1 × 10⁻⁷ M tyramine (=100%). Data represent the mean ± SD of eight values from a typical experiment. All determinations were independently repeated at least three times.