CSIR-Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India.
CSIR-Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India.
Biochim Biophys Acta Mol Cell Biol Lipids. 2021 Apr;1866(4):158882. doi: 10.1016/j.bbalip.2021.158882. Epub 2021 Jan 9.
Endocytosis and intracellular trafficking constitute important regulatory features associated with G protein-coupled receptor (GPCR) function. GPCR endocytosis involves several remodeling events at the plasma membrane orchestrated by a concerted interplay of a large number of proteins and membrane lipids. Although considerable literature exists on the protein framework underlying GPCR endocytosis, the role of membrane lipids in this process remains largely unexplored. In order to explore the role of membrane cholesterol (an essential and important lipid in higher eukaryotes) in GPCR endocytosis, we monitored the effect of acute cholesterol depletion using methyl-β-cyclodextrin (MβCD) on endocytosis and intracellular trafficking of the serotonin receptor, an important neurotransmitter GPCR. Our results show that the serotonin receptor exhibits agonist-induced clathrin-mediated endocytosis with a concentration-dependent inhibition in internalization with increasing concentrations of MβCD, which was restored upon cholesterol replenishment. Interestingly, subsequent to internalization under these conditions, serotonin receptors were re-routed toward lysosomal degradation, instead of endosomal recycling observed under normal conditions, thereby implicating membrane cholesterol in modulation of intracellular trafficking of the receptor. This raises the possibility of a novel cholesterol-dependent role of intracellular sorting proteins in GPCR trafficking. These results differ from our previous observations on the endocytosis of the serotonin receptor upon statin-induced chronic cholesterol depletion, in terms of endocytic pathway. We conclude that analysis of complex cellular trafficking events such as GPCR endocytosis under acute and chronic cholesterol depletion conditions should be carried out with caution due to fundamental differences underlying these processes.
内吞作用和细胞内转运是与 G 蛋白偶联受体 (GPCR) 功能相关的重要调节特征。GPCR 的内吞作用涉及到质膜上的几个重塑事件，这些事件由大量蛋白质和膜脂的协同相互作用来协调。尽管有大量关于 GPCR 内吞作用的蛋白质结构基础的文献，但膜脂在这个过程中的作用在很大程度上仍未得到探索。为了探讨膜胆固醇（高等真核生物中一种必需和重要的脂质）在 GPCR 内吞作用中的作用，我们使用甲基-β-环糊精（MβCD）监测了胆固醇急性耗竭对血清素受体（一种重要的神经递质 GPCR）内吞作用和细胞内转运的影响。我们的结果表明，血清素受体表现出激动剂诱导的网格蛋白介导的内吞作用，随着 MβCD 浓度的增加，内吞作用呈浓度依赖性抑制，胆固醇补充后可恢复。有趣的是，在这些条件下内化后，血清素受体被重新定向到溶酶体降解，而不是在正常条件下观察到的内体循环，从而表明膜胆固醇参与了受体的细胞内转运的调节。这提出了细胞内分拣蛋白在 GPCR 运输中可能存在一种新的胆固醇依赖性作用的可能性。这些结果与我们之前关于他汀类药物诱导的慢性胆固醇耗竭后血清素受体内吞作用的观察结果不同，在胞吞途径方面存在差异。我们得出结论，由于这些过程的基础存在根本差异，因此应该谨慎地对急性和慢性胆固醇耗竭条件下的复杂细胞内运输事件（如 GPCR 内吞作用）进行分析。
