Identifying the interacting partners and the dynamics of the molecular networks constitutes the key point in understanding cellular processes. Different methods often based on energy transfer strategies have been developed to examine the molecular dynamics of protein complexes. However, these methods suffer a couple of drawbacks: a single complex can be studied at a time, and its localization and tracking cannot generally be investigated. Here, we report a multicolor time-resolved Förster resonance energy transfer microscopy method that allows the identification of up to 3 different complexes simultaneously, their localization in cells, and their tracking after activation. Using this technique, we studied GPCR oligomerization and internalization in human embryonic kidney 293 cells. We definitively show that receptors can internalize as oligomers and that receptor coexpression deeply impacts oligomer internalization processes.-Faklaris, O., Cottet, M., Falco, A., Villier, B., Laget, M., Zwier, J. M., Trinquet, E., Mouillac, B., Pin, J.-P., Durroux, T. Multicolor time-resolved Förster resonance energy transfer microscopy reveals the impact of GPCR oligomerization on internalization processes. FASEB J. 29, 2235-2246 (2015). www.fasebj.org Key Words: oligomers • lanthanide • vasopressin V1a/V2 receptor • quantum dot • multigate mode MOLECULAR INTERACTIONS constitute the starting point of all cellular processes. Interactions lead to conformational modification of molecules inducing their activation or their inhibition and result in modifications of spatiotemporal molecular networks. Identifying interacting partners and studying spatiotemporal interaction dynamics are the key points in explaining the function of the molecules. This is particularly well illustrated for GPCRs, which play key roles in the regulation of physiologic functions. Interacting with numerous molecules-ligands or membrane or cytosolic proteins-they are considered as platform for signal integration. After their activation by agonists, GPCRs enter in desensitization and internalization processes. First considered as a simple termination of receptor signaling, internalization processes now appear as finely tuned and participate in the regulation of the GPCR functions. Receptor internalization is accompanied by dramatic protein network modifications, such as b-arrestins recruitment. In many cases, the b-arrestin/receptor complex is transient (receptor of type A) or very stable (type B), leading in the latter case to a slow receptor recycling or degradation (1, 2).GPCRs interact with many other partners, the role of which mostly remains elusive. Among these partners are GPCRs themselves through oligomerization, but the role of the second or other protomers is not well defined. Coexpression of GPCRs can dramatically impact internalization processes by increasing or decreasing endocytosis of one of the expressed receptors (3-7). These results, based on binding experiments or fluorescent imaging and receptor colocalization, have been interpreted ...

We report the design and evaluation of pH responsive luminescent europium(III) probes that allow conjugation to targeting vectors to monitor receptor internalisation in cells. The approach adopted here can be...

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