Constitutive oligomerization of human D2 dopamine receptors expressed in Spodoptera frugiperda 9 (Sf9) and in HEK293 cells -: Analysis using co-immunoprecipitation and time-resolved fluorescence resonance energy transfer
Constitutive oligomerization of human D2 dopamine receptors expressed in Spodoptera frugiperda 9 (Sf9) and in HEK293 cells -: Analysis using co-immunoprecipitation and time-resolved fluorescence resonance energy transfer
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DOI：
10.1046/j.1432-1033.2003.03773.x
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发表时间：
2003-10-01
期刊：
影响因子：
--
通讯作者：
Strange, PG
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文献类型：
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作者：
Gazi, L;López-Giménez, JF;Strange, PG
通过添加人类免疫缺陷病毒 (HIV) 或 FLAG 表位标签，对人 D-2Long (D-2L) 和 D-2Short (D-2S) 多巴胺受体亚型的 N 末端进行了修饰。然后使用杆状病毒系统在草地贪夜蛾9（Sf9）细胞中表达受体，并通过免疫共沉淀和时间分辨荧光共振能量转移（FRET）研究它们的寡聚化。 [H-3]由表达表位标记的 D-2L 或 D-2S 受体的 Sf9 细胞制备的膜中螺哌酮标记的 D-2 受体，pK(d) 值接近 10。使用标记特异性抗体的免疫共沉淀显示 Sf9 细胞中 D-2L 和 D-2S 受体的组成型同源寡聚化。当FLAG标记的D-2S和HIV标记的D-2L受体共表达时，免疫共沉淀显示这两种亚型也可以在Sf9细胞中形成异源寡聚体。将使用铕和 XL665 标记抗体的时间分辨 FRET 应用于整个 Sf9 细胞和表达表位标记的 D-2 受体的 Sf9 细胞膜。在这两种情况下，都揭示了 D-2L 和 D-2S 亚型的组成型同源寡聚物。时间分辨 FRET 还揭示了表达 FLAG 标记的 D-2S 受体的 HEK293 细胞中的组成型同源寡聚物。 D-2受体配体多巴胺、R-(-)丙基去甲吗啡和雷氯必利不影响Sf9和HEK293细胞中D-2L和D-2S的寡聚化。因此，人 D-2 多巴胺受体可以在 Sf9 细胞和 HEK293 细胞中形成组成型寡聚体，可以通过不同的方法检测到，并且这些细胞中的 D-2 寡聚化不受配体调节。
Human D-2Long (D-2L) and D-2Short (D-2S) dopamine receptor isoforms were modified at their N-terminus by the addition of a human immunodeficiency virus (HIV) or a FLAG epitope tag. The receptors were then expressed in Spodoptera frugiperda 9 (Sf9) cells using the baculovirus system, and their oligomerization was investigated by means of co-immunoprecipitation and time-resolved fluorescence resonance energy transfer (FRET). [H-3] Spiperone labelled D-2 receptors in membranes prepared from Sf9 cells expressing epitope-tagged D-2L or D-2S receptors, with a pK(d) value of approximate to 10. Co-immunoprecipitation using antibodies specific for the tags showed constitutive homo-oligomerization of D-2L and D-2S receptors in Sf9 cells. When the FLAG-tagged D-2S and HIV-tagged D-2L receptors were co-expressed, co-immunoprecipitation showed that the two isoforms can also form hetero-oligomers in Sf9 cells. Time-resolved FRET with europium and XL665-labelled antibodies was applied to whole Sf9 cells and to membranes from Sf9 cells expressing epitope-tagged D-2 receptors. In both cases, constitutive homo-oligomers were revealed for D-2L and D-2S isoforms. Time-resolved FRET also revealed constitutive homo-oligomers in HEK293 cells expressing FLAG-tagged D-2S receptors. The D-2 receptor ligands dopamine, R-(-) propylnorapomorphine, and raclopride did not affect oligomerization of D-2L and D-2S in Sf9 and HEK293 cells. Human D-2 dopamine receptors can therefore form constitutive oligomers in Sf9 cells and in HEK293 cells that can be detected by different approaches, and D-2 oligomerization in these cells is not regulated by ligands.