Navarro Gemma, McCormick Peter J, Mallol Josefa, Lluís Carme, Franco Rafael, Cortés Antoni, Casadó Vicent, Canela Enric I, Ferré Sergi
Department of Biochemistry and Molecular Biology, University of Barcelona, Barcelona, Spain.
Methods Mol Biol. 2013;964:95-105. doi: 10.1007/978-1-62703-251-3_7.
Until very recently, dopamine receptors, like other G-protein-coupled receptors, were believed to function as individual units on the cell surface. Now it has been described by several groups including ours that dopamine receptors not only function as homomers but also form heteromers with other receptors at the membrane level. Bioluminescence and fluorescence resonance energy transfer (BRET and FRET) based techniques have been very useful to determine the interaction between two receptors, but to demonstrate the existence of higher-order complexes involving more than two molecules requires more sophisticated techniques. Combining BRET and FRET in the Sequential BRET-FRET (SRET) technique permits heteromers formed by three different proteins to be identified. In SRET experiments, the oxidation of a Renilla Luciferase substrate triggers acceptor excitation by BRET and subsequent energy transfer to a FRET acceptor. Using this methodology here we describe the heteromerization between adenosine A(2A), dopamine D(2), and cannabinoids CB(1) receptors in living cells.
直到最近，多巴胺受体与其他G蛋白偶联受体一样，被认为在细胞表面作为单个单元发挥作用。现在，包括我们小组在内的几个研究团队已经表明，多巴胺受体不仅以同聚体的形式发挥作用，还能在膜水平与其他受体形成异聚体。基于生物发光和荧光共振能量转移（BRET和FRET）的技术对于确定两个受体之间的相互作用非常有用，但要证明涉及两个以上分子的高阶复合物的存在则需要更复杂的技术。在顺序BRET-FRET（SRET）技术中结合BRET和FRET，可以识别由三种不同蛋白质形成的异聚体。在SRET实验中，海肾荧光素酶底物的氧化通过BRET触发受体激发，随后能量转移到FRET受体。在这里，我们使用这种方法描述了活细胞中腺苷A(2A)、多巴胺D(2)和大麻素CB(1)受体之间的异聚化。
