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.
[Background] Cell membrane receptor internalization in response to external stimuli can be measured using two major strategies – microscopic and biochemical. The most common approach is the use of microscopy – either in real-time or on fixed cells. In the first approach, the cells expressing receptors labelled with fluorescent tags (e.g., fused to the fluorescent proteins) are examined in live cells by time-lapse confocal microscopy. Alternatively, cells expressing fluorescently labeled receptors can be exposed to the desired stimuli and then fixed at a pre-defined time. Subsequently, sub-cellular localization of these receptors (i.e., membrane vs intracellular fraction) is examined by fluorescence microscopy and compared with the untreated control. The advantage of time-lapse microscopy is the ability to examine the same cells at different time points and directly assess changes in the receptor distribution upon stimulation (Czarnecka et al., 2015). However, since this assessment has to be performed under high magnification, the number of cells that can be analyzed is limited and the response is not always uniform among the cells. On the other hand, fixing the cells upon stimulation allows for examining a larger cell population and for analysis of the native, not-labeled receptors, if combined with fluorescently labeled ligands or immunocytochemistry (Bohme et al., 2008; Fabry et al., 2000). However, in this case, the analysis of the receptor sub-cellular localization is usually qualitative and the time of exposure may not be optimal, as the changes are not examined in real time.
The biochemical approach takes advantage of cell-impermeable biotinylation reagents that selectively cross-link extracellular domains of cell surface receptors. The biotin-labeled cell membrane proteins are then affinity-purified and the receptor of interest can be selectively detected by Western blot (Czarnecka et al., 2015). This approach allows for quantitative analysis of the cells as a whole population and does not require fusion with a fluorescent protein that may potentially change the behavior of the tested receptors. However, as with microscopic analysis of fixed cells upon treatment, the time of exposure to the ligand remains to be determined. Therefore, in our study, we combined time-lapse confocal microscopy, which allowed us to perform the initial assessment of the internalization rate and determine the time of ligand exposure allowing for detecting maximal changes in receptor sub-cellular localization, and the subsequent selective isolation of cell surface receptors at this time point to achieve quantitative results and confirm microscopic observations (Czarnecka et al., 2015). This strategy was successful in demonstrating neuropeptide Y (NPY) Y5R receptor internalization upon stimulation with non-cognate ligand, brain-derived neurotrophic factor (BDNF), and therefore proving the interactions between NPY and BDNF systems.
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