Figure 1.
Phylogram of selected arthropod amine receptor sequences.
A phylogenetic analysis showing known invertebrate-type DA receptors and α-adrenergic-like OA receptors and their relationship to other DA, OA and TA receptors from Apis mellifera and Drosophila melanogaster. The honey bee rhodopsin protein is used as an outlier. Conserved regions of receptor protein sequences were aligned using ClustalW2 software (version 2.0.10) using the default settings (http://ww.ebi.ac.uk/Tools/clustalw2/). Phylograms were prepared as described in [30] by a 1000 trial N-J bootstrap analysis, and using ClustalX software (version 2.0). Resulting bootstrap scores are displayed on selected nodes as percentages values, if greater than 50%.
Figure 2.
Agonist-induced changes in intracellular Ca2+ levels in HEK293 cells expressing either AmDOP2 receptors or AmOA1 receptors.
Cells were preloaded with the [Ca2+]I reporter dye Fluo-4 and monitored for agonist-induced changes in fluorescence signal. The data shown are from a single trial and are representative of results obtained from 3 independent trials at each agonist concentration (with two internal replicates per trial), and over 30 trials using 1 µM. Changes in fluorescence examined in the following: (A) AmDOP2-expressing HEK293 cells exposed to 1 µM DA, OA or TA; (B) AmOA1-expressing HEK293 cells exposed to 1 µM DA, OA or TA; (C) AmDOP2-expressing HEK293 cells exposed to DA at the concentrations indicated to the right of Figure 1C; (D) AmOA1-expressing HEK293 cells exposed to OA at the concentrations indicated to the right of Figure 1D.
Figure 3.
PLCβ activity is required for AmDOP2- and AmOA1-mediated intracellular Ca2+ signaling.
Treatment with edelfosine (edlf) was found to inhibit AmDOP2 (P<0.0001) and AmOA1 (P = 0.0002) mediated Ca2+ signaling. HEK293 cells expressing either AmDOP2 receptors (A) or AmOA1 receptors (B) were loaded with the intracellular Ca2+ reporter dye Fluo-4, with or without the inclusion of 10 µM edelfosine (Edlf) in the loading buffer. Cells were subsequently exposed to a 1 µM concentration of agonist and the maximal ΔF/Fb in following 50 s period determined. Data are normalized to the percentage response observed in cells not treated with edelfosine, and are the result of three independent experiments, with two internal replicates per experiment. Error bars represent the SEM. Statistical significance was determined using Student's two-tailed t tests.
Figure 4.
PLCβ activity is not required for AmDOP2- or AmOA1-mediated intracellular cAMP signaling.
Treatment with edelfosine (edlf) had no significant effect on [cAMP]i signaling mediated via AmDOP2 receptors (P = 0.9751), or via AmOA1 receptors (P = 0.2224). HEK293 cells were co-transfected with expression constructs for either the AmDOP2 receptor (A) or AmOA1 receptor (B), and a CRE-luciferase reporter construct. Cells were treated with either 1 µM agonist (DA and OA respectively) or 1 µM agonist and 10 µM edelfosine (edlf). Data are normalized to the response observed in cells not treated with edelfosine, and the mean of three independent experiments within which, each treatment was tested twice. Error bars represent the SEM. Statistical significance was determined using Student's two-tailed t tests.
Figure 5.
Effects of amine-receptor antagonists on cAMP responses mediated via AmDOP2 and AmOA1 receptors.
HEK293 cells were co-transfected with expression constructs for either the AmDOP2 receptor (blue symbols) or AmOA1 receptor (red symbols) and a CRE-luciferase reporter construct. Cells were treated with 1 µM agonist (DA or OA respectively) or 1 µM agonist and either cis-(Z)-flupentixol (A), spiperone (B), mianserin (C) or epinastine (D), at a range of concentrations indicated in the figure. Due to evidence of significant cell toxicity, cis-(Z)-flupentixol was not tested at a concentration higher than 10 µM. Data are normalized to the response observed in cells treated with agonist alone (not shown), and are the result of two independent experiments within which, each treatment was tested twice. Error bars (estimated SEM) are included to provide an indication of consistency between experiments. Dose response curves were determined by non-linear regression using GraphPad Prism software for Macintosh version 5.0b.
Figure 6.
Effect of antagonists on AmDOP2- and AmOA1-mediated intracellular Ca2+ signaling.
HEK293 cells expressing either AmDOP2 receptors (A) or AmOA1 receptors (B) were loaded with the intracellular Ca2+ reporter dye Fluo-4 and exposed to 1 µM agonist (DA and OA respectively), or 1 µM agonist and either 10 µM cis-(Z)-flupentixol, spiperone, mianserin or epinastine and the maximal ΔF/Fb over following 50 second period determined. Data are normalized to the response observed in cells treated with agonist alone, and are the result of three independent experiments with two internal replicates per experiment. Error bars represent the SEM. Statistical significance was determined using one-way ANOVA and Dunnett's multiple comparison test, with treatment with agonist alone used as the control column. F4,10 = 239.1, P<0.0001 for (A); F4,10 = 35.95, P<0.0001 for (B).
Figure 7.
Pharmacological profile of AmDOP1, AmDOP3 and AmTYR1 receptors.
HEK293 cells were co-transfected with expression constructs for one of the following amine receptors, the AmDOP1 receptor (A, D, G, J), AmDOP3 receptor (B, E, H, K), or AmTYR1 receptor (C, F, I, L) and a CRE-luciferase reporter construct. Cells were treated with 1 µM agonist, or with 1 µM agonist together with one of the following antagonists: cis-(Z)-flupentixol (A, B, C), spiperone (D, E, F), mianserin (G, H, I) or epinastine (J, K, L) at concentrations indicated in each figure. For assaying the activity of AmDOP3 and AmTYR1 receptors, both of which reduce intracellular cAMP levels, basal cAMP levels in test cells were elevated by inclusion of a nonsaturating concentration of the adenylyl cyclase stimulant, forskolin (100 nM). Data are normalized to the percentage response observed in cells treated with agonist alone (not shown), and are the result of two independent experiments within which, each treatment was tested twice. Error bars represent the estimated SEM. Dose response curves were determined by non-linear regression using GraphPad Prism software for Macintosh version 5.0b, and displayed when the resulting curves were unambiguous.
Table 1.
Estimated IC50 values of antagonists on cAMP responses mediated via honeybee biogenic amine receptors.
Figure 8.
Sequence alignment of AmDOP1, AmDOP2, AmOA1, AmTYR1, human H1 histamine receptor (NP_001091683.1), human β-adrenergic receptor (NP_000015.1).
Residues highlighted in gray represent the transmembrane helices from the structure of the human β-adrenergic receptor (pdb2rh1), residues highlighted in cyan are those conserved in the GPCR family [43], the red aspartic acid is the highly conserved D107 (hHis numbering: Asp113 - HmB-Adr) on helix 3 (TMIII; residues not shown for I3).