Department of Zoology, University of Otago, Dunedin, New Zealand.
PLoS One. 2011;6(11):e26809. doi: 10.1371/journal.pone.0026809. Epub 2011 Nov 11.
Three dopamine receptor genes have been identified that are highly conserved among arthropod species. One of these genes, referred to in honey bees as Amdop2, shows a close phylogenetic relationship to the a-adrenergic-like octopamine receptor family. In this study we examined in parallel the functional and pharmacological properties of AmDOP2 and the honey bee octopamine receptor, AmOA1. For comparison, pharmacological properties of the honey bee dopamine receptors AmDOP1 and AmDOP3, and the tyramine receptor AmTYR1, were also examined.
METHODOLOGY/PRINCIPAL FINDINGS: Using HEK293 cells heterologously expressing honey bee biogenic amine receptors, we found that activation of AmDOP2 receptors, like AmOA1 receptors, initiates a rapid increase in intracellular calcium levels. We found no evidence of calcium signaling via AmDOP1, AmDOP3 or AmTYR1 receptors. AmDOP2- and AmOA1-mediated increases in intracellular calcium were inhibited by 10 µM edelfosine indicating a requirement for phospholipase C-β activity in this signaling pathway. Edelfosine treatment had no effect on AmDOP2- or AmOA1-mediated increases in intracellular cAMP. The synthetic compounds mianserin and epinastine, like cis-(Z)-flupentixol and spiperone, were found to have significant antagonist activity on AmDOP2 receptors. All 4 compounds were effective antagonists also on AmOA1 receptors. Analysis of putative ligand binding sites offers a possible explanation for why epinastine acts as an antagonist at AmDOP2 receptors, but fails to block responses mediated via AmDOP1.
CONCLUSIONS/SIGNIFICANCE: Our results indicate that AmDOP2, like AmOA1, is coupled not only to cAMP, but also to calcium-signalling and moreover, that the two signalling pathways are independent upstream of phospholipase C-β activity. The striking similarity between the pharmacological properties of these 2 receptors suggests an underlying conservation of structural properties related to receptor function. Taken together, these results strongly support phylogenetic analyses indicating that the AmDOP2 and AmOA1 receptor genes are immediate paralogs.
已鉴定出三种多巴胺受体基因，它们在节肢动物物种中高度保守。这些基因之一，在蜜蜂中称为 Amdop2，与α-肾上腺素样章鱼胺受体家族具有密切的系统发育关系。在这项研究中，我们同时研究了 AmDOP2 和蜜蜂章鱼胺受体 AmOA1 的功能和药理学特性。为了进行比较，还检查了蜜蜂多巴胺受体 AmDOP1 和 AmDOP3 以及 tyramine 受体 AmTYR1 的药理学特性。
方法/主要发现：使用异源表达蜜蜂生物胺受体的 HEK293 细胞，我们发现 AmDOP2 受体的激活，与 AmOA1 受体一样，会引发细胞内钙离子水平的快速增加。我们没有发现 AmDOP1、AmDOP3 或 AmTYR1 受体通过钙信号传导的证据。10µM 埃德尔福辛表明该信号通路需要磷脂酶 C-β 活性，可抑制 AmDOP2 和 AmOA1 介导的细胞内钙离子增加。埃德尔福辛处理对 AmDOP2 或 AmOA1 介导的细胞内 cAMP 增加没有影响。合成化合物米氮平和埃皮那定，与顺式（Z）-氟哌啶醇和螺哌隆一样，对 AmDOP2 受体具有显著的拮抗活性。这 4 种化合物对 AmOA1 受体也都是有效的拮抗剂。对假定的配体结合位点的分析提供了一个可能的解释，即为什么埃皮那定作为 AmDOP2 受体的拮抗剂起作用，但不能阻断通过 AmDOP1 介导的反应。
结论/意义：我们的结果表明，AmDOP2 与 AmOA1 一样，不仅与 cAMP 偶联，而且与钙信号偶联，而且这两种信号通路在磷脂酶 C-β 活性的上游是独立的。这两个受体的药理学特性惊人相似，表明与受体功能相关的结构特性存在潜在的保守性。综上所述，这些结果强烈支持表明 AmDOP2 和 AmOA1 受体基因是直系同源的系统发育分析。
