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首页> 外文期刊>Journal of receptor and signal transduction research >Functional reconstitution of human neuropeptide y (NPY) Y2 and Y4 receptors in Sf9 insect cells
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Functional reconstitution of human neuropeptide y (NPY) Y2 and Y4 receptors in Sf9 insect cells

机译：Sf9昆虫细胞中人神经肽y（NPY）Y2和Y4受体的功能重建

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摘要

The four functionally expressed human neuropeptide Y receptor subtypes (hY1R, hY_2R, hY_4R, hY5R) belong to class A of the G-protein-coupled receptors (GPCRs) and interact with pertussis toxin-sensitive Gi/o-proteins. The number of small molecules described as ligands for hY1R and hY5R exceeds by far those for hY_2R. Potent non-peptidergic ligands for the hY_4R are not available so far. Here, we report on the functional reconstitution of the hY_2R and the hY_4R in Sf9 insect cells using the baculovirus system. Sf9 cells were genetically engineered by infection with up to four different baculoviruses, combining the receptors with G-proteins of the Gi/o family and regulators of G-protein signaling (RGS) proteins to improve signal-to-noise ratio. In steady-state GTPase assays, using pNPY (Y_2) and hPP (Y_4), the GPCRs coupled to various Gi/Go-proteins and both, RGS4 and GAIP, enhanced the signals. Co-expression systems hY_2R + Gα_(i2) and hY_4R + Gα_(i2)/Gα_o + RGS4, combined with Gβ_1γ_2, yielded best signal-to-noise ratios. hY_2R function was validated using both agonistic peptides (NPY, PYY, NPY1336) and selective non-peptidergic antagonists (BIIE0246 and derivatives), whereas the hY_4R model was characterized with peptidergic agonists (PP, NPY, GW1229, and BW1911U90). Tunicamycin inhibited receptor N-glycosylation diminished NPY signals at hY_2R and abolished hY_4R function. Investigations with monovalent salts showed sensitivity of hY _4R toward Na~+, revealing moderate constitutive activity. After validation, an acylguanidine (UR-PI284) was identified as a weak non-peptide Y_4R antagonist. In summary, the established steady-state GTPase assays provide sensitive test systems for the characterization of Y _2 and Y_4 receptor ligands.
机译：四种功能性表达的人神经肽Y受体亚型（hY1R，hY_2R，hY_4R，hY5R）属于G蛋白偶联受体（GPCR）的A类，并与百日咳毒素敏感的Gi / o蛋白相互作用。被描述为hY1R和hY5R配体的小分子的数量远远超过了hY_2R的数量。到目前为止，尚无有效的hY_4R非肽能配体。在这里，我们报告使用杆状病毒系统的Sf9昆虫细胞中的hY_2R和hY_4R的功能重建。通过感染多达4种不同的杆状病毒对Sf9细胞进行基因工程改造，将受体与Gi / o家族的G蛋白和G蛋白信号（RGS）蛋白的调节剂结合起来，以提高信噪比。在使用pNPY（Y_2）和hPP（Y_4）的稳态GTPase检测中，与各种Gi / Go蛋白以及RGS4和GAIP偶联的GPCR增强了信号。共表达系统hY_2R +Gα_（i2）和hY_4R +Gα_（i2）/Gα_o+ RGS4，与Gβ_1γ_2组合，产生了最佳的信噪比。 hY_2R功能已通过激动肽（NPY，PYY，NPY1336）和选择性非肽能拮抗剂（BIIE0246及其衍生物）进行了验证，而hY_4R模型则由肽能激动剂（PP，NPY，GW1229和BW1911U90）表征。衣霉素抑制受体N-糖基化减少了hY_2R处的NPY信号并废除了hY_4R功能。单价盐的研究表明hY _4R对Na〜+的敏感性，显示出适度的本构活性。验证后，酰基胍（UR-PI284）被鉴定为弱非肽Y_4R拮抗剂。总而言之，已建立的稳态GTPase测定法为表征Y _2和Y_4受体配体提供了灵敏的测试系统。

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