Unitat de Farmacologia, Departament de Patologia i Terapèutica Experimental, Facultat de Medicina, Universitat de Barcelona, L'Hospitalet de Llobregat, 08907 Barcelona, Spain.
Methods. 2012 Aug;57(4):467-72. doi: 10.1016/j.ymeth.2012.05.007. Epub 2012 Jun 6.
Understanding of the molecular mechanisms of protein-protein interactions (PPIs) at the cell surface of living cells is fundamental to comprehend the functional meaning of a large number of cellular processes. Here we discuss how new methodological strategies derived from non-invasive fluorescence-based approaches (i.e. fluorescence resonance energy transfer, FRET) have been successfully developed to characterize plasma membrane PPIs. Importantly, these technologies alone - or in concert with complementary methods (i.e. SNAP-tag/TR-FRET, TIRF/FRET) - can become extremely powerful approaches for visualizing cell surface PPIs, even between more than two proteins and also in native tissues. Interestingly, these methods would also be relevant in drug discovery in order to develop new high-throughput screening approaches or to identify new therapeutic targets. Accordingly, herein we provide a thorough assessment on all biotechnological aspects, including strengths and weaknesses, of these fluorescence-based methodologies when applied in the study of PPIs occurring at the cell surface of living cells.
了解活细胞表面蛋白质-蛋白质相互作用（PPIs）的分子机制对于理解大量细胞过程的功能意义至关重要。在这里，我们讨论了如何成功开发源自非侵入性荧光方法（即荧光共振能量转移，FRET）的新方法策略，以表征质膜 PPIs。重要的是，这些技术单独使用——或与互补方法（即 SNAP 标签/TR-FRET、TIRF/FRET）结合使用——可以成为可视化细胞表面 PPIs 的极其强大的方法，甚至可以在两个以上蛋白质之间，也可以在天然组织中使用。有趣的是，这些方法在药物发现中也很重要，以便开发新的高通量筛选方法或识别新的治疗靶点。因此，本文全面评估了这些基于荧光的方法在研究活细胞表面发生的 PPIs 时的所有生物技术方面，包括优势和劣势。
