Department of Biomedical Engineering, Oregon Health and Science University, Portland, OR 97239, USA.
Proc Natl Acad Sci U S A. 2010 Oct 26;107(43):18658-63. doi: 10.1073/pnas.1013763107. Epub 2010 Oct 12.
G-protein-coupled receptors (GPCRs) are the largest protein superfamily in the human genome; they comprise 30% of current drug targets and regulate diverse cellular signaling responses. The role of endosomal trafficking in GPCR signaling regulation is gaining substantial consideration. However, this process remains difficult to study due to the inability to distinguish among many individual receptors, simultaneously trafficking within multiple endosomal pathways. Here we show accurate measurement of the internalization and endosomal trafficking of single groups of serotonin (5-hydroxytryptamine, 5-HT) receptors using single quantum dot (QD) probes and quantitative colocalization. We demonstrate that the presence of a QD tag does not interfere with 5-HT receptor internalization or endosomal recycling. Direct measurements show simultaneous trafficking of the 5-HT1A receptor in two distinct endosomal recycling pathways. Single-molecule imaging of endosomal trafficking will significantly impact the understanding of cellular signaling and provide powerful tools to elucidate the actions of GPCR-targeted therapeutics.
G 蛋白偶联受体（GPCRs）是人类基因组中最大的蛋白质超家族；它们占当前药物靶点的 30%，调节多种细胞信号反应。内体运输在 GPCR 信号调节中的作用正受到越来越多的关注。然而，由于无法同时区分许多单独的受体在多种内体途径中的运输，因此这个过程仍然难以研究。在这里，我们使用单量子点（QD）探针和定量共定位显示了对单群血清素（5-羟色胺，5-HT）受体的内化和内体运输的准确测量。我们证明 QD 标记的存在不会干扰 5-HT 受体的内化或内体再循环。直接测量显示 5-HT1A 受体同时在两种不同的内体再循环途径中运输。内体运输的单分子成像将极大地影响对细胞信号的理解，并提供有力的工具来阐明针对 GPCR 的治疗药物的作用。
