McVey M, Ramsay D, Kellett E, Rees S, Wilson S, Pope A J, Milligan G
Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland.
J Biol Chem. 2001 Apr 27;276(17):14092-9. doi: 10.1074/jbc.M008902200. Epub 2001 Jan 22.
Oligomerization of the human delta-opioid receptor and its regulation by ligand occupancy were explored following expression in HEK293 cells using each of co-immunoprecipitation of differentially epitope-tagged forms of the receptor, bioluminescence resonance energy transfer and time-resolved fluorescence resonance energy transfer. All of the approaches identified constitutively formed receptor oligomers, and the time-resolved fluorescence studies confirmed the presence of such homo-oligomers at the cell surface. Neither the agonist ligand [d-Ala(2),d-Leu(5)]enkephalin nor the inverse agonist ligand ICI174864 were able to modulate the oligomerization status of this receptor. Interactions between co-expressed delta-opioid receptors and beta(2)-adrenoreceptors were observed in co-immunoprecipitation studies. Such hetero-oligomers could also be detected using bioluminescence resonance energy transfer although the signal obtained was substantially smaller than for homo-oligomers of either receptor type. Signal corresponding to the delta-opioid receptor-beta(2)-adrenoreceptor hetero-oligomer was increased in the presence of agonist for either receptor. However, substantial levels of this hetero-oligomer were not detected at the cell surface using time-resolved fluorescence resonance energy transfer. These studies demonstrate that, following transient transfection of HEK293 cells, constitutively formed oligomers of the human delta-opioid receptor can be detected by a variety of approaches. However, these are not regulated by ligand occupancy. They also indicate that time-resolved fluorescence resonance energy transfer represents a means to detect such oligomers at the cell surface in populations of intact cells.
在HEK293细胞中表达后，利用差异表位标记形式的受体共免疫沉淀、生物发光共振能量转移和时间分辨荧光共振能量转移，对人δ阿片受体的寡聚化及其受配体占据的调节进行了研究。所有方法均鉴定出组成型形成的受体寡聚体，时间分辨荧光研究证实细胞表面存在此类同型寡聚体。激动剂配体[d-Ala(2),d-Leu(5)]脑啡肽和反向激动剂配体ICI174864均不能调节该受体的寡聚化状态。在共免疫沉淀研究中观察到共表达的δ阿片受体与β(2)-肾上腺素能受体之间的相互作用。使用生物发光共振能量转移也可以检测到此类异型寡聚体，尽管获得的信号比两种受体类型的同型寡聚体小得多。在任一受体的激动剂存在下，与δ阿片受体-β(2)-肾上腺素能受体异型寡聚体对应的信号都会增加。然而，使用时间分辨荧光共振能量转移在细胞表面未检测到大量的这种异型寡聚体。这些研究表明，在HEK293细胞瞬时转染后，可以通过多种方法检测到人δ阿片受体组成型形成的寡聚体。然而，这些不受配体占据的调节。它们还表明，时间分辨荧光共振能量转移是在完整细胞群体中检测细胞表面此类寡聚体的一种手段。
