Cell Surface Protein Detection to Assess Receptor Internalization.
Cell Surface Protein Detection to Assess Receptor Internalization.
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DOI：
10.21769/bioprotoc.1968
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发表时间：
2016-10-20
期刊：
影响因子：
0.8
通讯作者：
Kitlinska, Joanna
中科院分区：
文献类型：
--
作者：
Czarnecka, Magdalena;Kitlinska, Joanna
膜受体在不同刺激剂/抑制剂作用下的迁移是非常重要的。其中，膜受体的内化影响它们对配体的可及性和细胞对环境提示的反应。在实验上，受体内化可以用来衡量它们的激活。在我们的研究中，我们使用这种方法来探索神经肽Y(NPY)的七个跨膜区受体Y5R和脑源性神经营养因子(BDNF)的酪氨酸激酶受体TrkB之间的相互作用。为此，我们测量了在TrkB配体BDNF刺激下Y5R的内化。在用BDNF处理后，细胞暴露在一种膜不渗透的生物素化试剂中，这种试剂选择性地标记表面蛋白。随后，生物素化的膜蛋白在亲和素树脂柱上进行亲和纯化，并用Western印迹进行分析。对照组和配基处理组细胞表面受体比例的差异可以作为它们内化和对特定刺激反应的衡量标准。
The migration of membrane receptors upon exposure to different stimulants/inhibitors is of great importance. Among others, the internalization of membrane receptors affects their accessibility to ligands and cell responsiveness to environmental cues. Experimentally, receptor internalization can be used as a measure of their activation. In our studies, we employed this approach to explore cross-talk between a seven transmembrane domain receptor for neuropeptide Y (NPY), Y5R, and a tyrosine kinase receptor for brain-derived neurotrophic factor (BDNF), TrkB. To this end, we measured the internalization of Y5R upon stimulation with the TrkB ligand, BDNF. Upon treatment with BDNF, the cells were exposed to a membrane impermeable, biotinylation reagent that selectively labels surface proteins. Subsequently, the biotinylated membrane proteins were affinity-purified on columns with avidin resins and analyzed by Western blot. Differences in the fraction of receptors present on the cell surface of control and ligand-treated cells served as a measure of their internalization and response to particular stimuli.