Herrick-Davis Katharine, Grinde Ellinor, Mazurkiewicz Joseph E
Center for Neuropharmacology and Neuroscience, Albany Medical College, Albany, New York 12208, USA.
Biochemistry. 2004 Nov 9;43(44):13963-71. doi: 10.1021/bi048398p.
While many studies have provided evidence of homodimerization and heterodimerization of G-protein-coupled receptors (GPCRs), few studies have used fluorescence resonance energy transfer (FRET) combined with confocal microscopy to visualize receptor dimerization on the plasma membrane, and there have been no reports demonstrating the expression of serotonin receptor dimers/oligomers on the plasma membrane of living cells. In the study presented here, biochemical and biophysical techniques were used to determine if 5-HT(2C) receptors exist as homodimers on the plasma membrane of living cells. Immunoprecipitation followed by Western blotting revealed the presence of immunoreactive bands the predicted size of 5-HT(2C) receptor monomers and homodimers that were detergent and cross-linker sensitive. Bioluminescence resonance energy transfer (BRET) was assessed in HEK293 cells expressing 5-HT(2C) receptors labeled with Renilla luciferase and yellow fluorescent protein. BRET levels were not altered by pretreatment with serotonin. Confocal microscopy provided direct visualization of FRET on the plasma membrane of live cells expressing 5-HT(2C) receptors labeled with cyan (donor) and yellow (acceptor) fluorescent proteins. FRET, assessed by acceptor photobleaching, was dependent on the donor/acceptor ratio and independent of acceptor expression levels, indicating that FRET resulted from receptor clustering and not from overexpression of randomly distributed receptors, providing evidence for GPCR dimers/oligomers in a clustered distribution on the plasma membrane. The results of this study suggest that 5-HT(2C) receptors exist as constitutive homodimers on the plasma membrane of living cells. In addition, a confocal-based FRET method for monitoring receptor dimerization directly on the plasma membrane of living cells is described.
虽然许多研究已提供了G蛋白偶联受体（GPCRs）同二聚化和异二聚化的证据，但很少有研究使用荧光共振能量转移（FRET）结合共聚焦显微镜来观察质膜上的受体二聚化，并且尚无报道证明5-羟色胺受体二聚体/寡聚体在活细胞质膜上的表达。在本文介绍的研究中，采用生化和生物物理技术来确定5-HT(2C)受体在活细胞质膜上是否以同二聚体形式存在。免疫沉淀后进行蛋白质印迹分析，结果显示存在免疫反应条带，其大小与5-HT(2C)受体单体和同二聚体预测的大小一致，且对去污剂和交联剂敏感。在表达用海肾荧光素酶和黄色荧光蛋白标记的5-HT(2C)受体的HEK293细胞中评估生物发光共振能量转移（BRET）。用5-羟色胺预处理后，BRET水平未改变。共聚焦显微镜直接观察到在表达用青色（供体）和黄色（受体）荧光蛋白标记的5-HT(2C)受体的活细胞质膜上的FRET。通过受体光漂白评估的FRET取决于供体/受体比例，且与受体表达水平无关，这表明FRET是由受体聚集引起的，而非随机分布的受体过度表达所致，为质膜上呈聚集分布的GPCR二聚体/寡聚体提供了证据。本研究结果表明，5-HT(2C)受体在活细胞质膜上以组成型同二聚体形式存在。此外，还描述了一种基于共聚焦的FRET方法，用于直接监测活细胞质膜上的受体二聚化。
