Li Xuezhi, Rosciglione Stéphanie, Laniel Andréanne, Lavoie Christine
Département de Pharmacologie-Physiologie, Faculté de Médecine et des Sciences de la Santé, Institut de Pharmacologie de Sherbrooke, Université de Sherbrooke, Sherbrooke, QC, Canada.
Methods Mol Biol. 2019;1947:303-322. doi: 10.1007/978-1-4939-9121-1_17.
Following stimulation, G protein-coupled receptors (GPCRs) are internalized and transported to early endosomes where they are either recycled back to the plasma membrane for another round of activation or targeted to the lysosomes for degradation and long-term signal termination. This latter requires internalization of receptors into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs) for complete degradation following fusion with lysosomes. This endosomal sorting step is highly regulated and has profound functional consequences. This chapter describes how RNAi and confocal microscopy methods can be combined to evaluate whether a protein of interest (herein Gαs) is involved in GPCR sorting into ILVs of MVBs.
受到刺激后，G蛋白偶联受体（GPCRs）会被内化并转运至早期内体，在那里它们要么被循环回到质膜进行新一轮激活，要么被靶向溶酶体进行降解以及长期信号终止。后者需要受体内化到多泡体（MVBs）的腔内小泡（ILVs）中，以便在与溶酶体融合后进行完全降解。这个内体分选步骤受到高度调控并具有深远的功能影响。本章描述了如何将RNA干扰（RNAi）和共聚焦显微镜方法结合起来，以评估感兴趣的蛋白质（此处为Gαs）是否参与GPCR分选至MVBs的ILVs中。
