AmOctα2R: Functional Characterization of a Honeybee Octopamine Receptor Inhibiting Adenylyl Cyclase Activity

Abstract

The catecholamines norepinephrine and epinephrine are important regulators of vertebrate physiology. Insects such as honeybees do not synthesize these neuroactive substances. Instead, they use the phenolamines tyramine and octopamine for similar physiological functions. These biogenic amines activate specific members of the large protein family of G protein-coupled receptors (GPCRs). Based on molecular and pharmacological data, insect octopamine receptors were classified as either α- or β-adrenergic-like octopamine receptors. Currently, one α- and four β-receptors have been molecularly and pharmacologically characterized in the honeybee. Recently, an α₂-adrenergic-like octopamine receptor was identified in Drosophila melanogaster (DmOctα2R). This receptor is activated by octopamine and other biogenic amines and causes a decrease in intracellular cAMP ([cAMP]_i). Here, we show that the orthologous receptor of the honeybee (AmOctα2R), phylogenetically groups in a clade closely related to human α₂-adrenergic receptors. When heterologously expressed in an eukaryotic cell line, AmOctα2R causes a decrease in [cAMP]_i. The receptor displays a pronounced preference for octopamine over tyramine. In contrast to DmOctα2R, the honeybee receptor is not activated by serotonin. Its activity can be blocked efficiently by 5-carboxamidotryptamine and phentolamine. The functional characterization of AmOctα2R now adds a sixth member to this subfamily of monoaminergic receptors in the honeybee and is an important step towards understanding the actions of octopamine in honeybee behavior and physiology.

Keywords: GPCR; biogenic amines; cellular signaling; honeybee; second messenger.

Figure 1

Structural characteristics of the amino acid sequence deduced for AmOctα2R. (a) Hydrophobicity profile of AmOctα2R. The profile was calculated according to the algorithm of Kyte and Doolittle [36] using a window size of 19 amino acids. Peaks with scores greater than 1.6 (dashed line) indicate possible transmembrane (TM) regions; (b) prediction of TM domains with TMHMM server v. 2.0 [37]. Putative TM domains are indicated in red. Extracellular regions are shown with a purple line, and intracellular regions are shown with a blue line; (c) color-coded (rainbow) three-dimensional (3D) model of the receptor as predicted by Phyre2 [38]. The extracellular N-terminus (N) and the intracellular C-terminus (C) are labeled. Note that the first 216 amino acid residues of AmOctα2R were omitted in this simulation.

Figure 2

Phylogenetic relationships of monoaminergic receptors. Alignments were performed using Clustal W [46] by using the core amino-acid sequences of TM 1–4, TM 5, TM 6, and TM 7. The evolutionary history was inferred using the neighbor-joining method. The percentage of replicate trees, in which the associated taxa clustered together in the bootstrap test (10,000 replicates), are shown next to the branches. The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site. The analysis involved 76 amino acid sequences. Human rhodopsin (HsRHOD) was used to root the tree. Receptor subclasses are given on the right. The abbreviations of species are shown in alphabetical order: 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. The accession numbers and annotations of all sequences used in the phylogenetic analysis can be found in Supplementary Table S2.

Figure 3

Expression of AmOctα2R-hemagglutinin A (HA) in flpTM cells. (a) Western blot of membrane proteins (30 µg) from flpTM cells expressing AmOctα2R-HA receptors were not treated (lane 1) or treated with PNGaseF (lane 2). As a control, 30 µg of membrane proteins from nontransfected flpTM cells (lane 3) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted to a polyvinylidene difluoride (PVDF) membrane. The blot was probed with a rat anti-(hemagglutinin A) HA antibody. (b) The same blot as shown in (a) was subsequently probed with an antibody directed against the C-terminus of the cyclic nucleotide-gated (CNG) channel. The sizes of marker proteins in kDa are given on the left margin.

Figure 4

Concentration-dependent effects of octopamine on intracellular cAMP in AmOctα2R-HA-expressing flpTM cells. Relative fluorescence (corresponding to the amount of cAMP) is given as the percentage of the value obtained with 10 µM NKH 477 (=100%), a water-soluble forskolin analog. All measurements were performed in the presence of 100 µM isobutylmethylxanthine (IBMX). In the range from 10⁻⁹ M to 10⁻⁶ M, the octopamine activation of AmOctα2R-HA led to a concentration-dependent decrease in the fluorescence signal. Conversely, an increase in the fluorescence signal was observed with octopamine concentrations of 3 × 10⁻⁶ M and higher. Data points represent the mean ± SD of four-fold determinations.

Figure 5

Concentration-dependent effects of octopamine on relative fluorescence in nontransfected (control) flpTM cells. The concentration–response curves for octopamine were established in the absence (open circles) or presence (filled circles) of 10 µM NKH 477. Relative fluorescence is given as the percentage of the value obtained with 10 μM NKH 477 (=100%). All measurements were performed in the presence of 100 μM IBMX. In both conditions, higher octopamine concentrations led to an increase in fluorescence. Data points represent the mean ± SD of four values.

Figure 6

Concentration-response curves for agonists on intracellular cAMP level in AmOctα2R-HA-expressing flpTM cells. Relative fluorescence (corresponding to the amount of cAMP) is given as the percentage of the value obtained with 10 µM NKH 477 (=100%). All measurements were performed in the presence of 100 µM IBMX. Data points represent the mean ± SD of four values from a typical experiment.

Figure 7

Effects of putative antagonists on tyramine-activated AmOctα2R-HA. The concentration series of the substances were applied in the presence of 10 µM NKH 477, 10 µM tyramine, and 100 µM IBMX. Ligands used were (a) phnetolamine, (b) epinastine, (c) mainserin, and (d) yohimbine. Data represent the mean ± SD of four values from a typical experiment. All determinations were independently repeated at least three times.