Multicolor time‐resolved Förster resonance energy transfer microscopy reveals the impact of GPCR oligomerization on internalization processes

Abstract

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 ...

Pharmacological evidence for a metabotropic glutamate receptor heterodimer in neuronal cells

“…In primary cultures of hippocampal neurons, co-expression of CLIP-mGlu2 and SNAP-mGlu4 subunits could be detected at the cell surface through labeling with CLIP and SNAP substrates carrying either Lumi4-Tb or Red. In these neurons, using a lanthanide-based time-resolved FRET microscope that we recently developed ( Faklaris et al, 2015 ), we detected a TR-FRET signal between the CLIP and SNAP subunits equivalent to that measured for homodimeric mGlu2 receptors. This observation is consistent with the formation of mGlu2-4 heterodimers in transfected neurons ( Figure 5 ).…”

“…Cells were imaged in imaging buffer. Images were acquired with a homebuilt TR-FRET microscope ( Faklaris et al, 2015 ). Briefly, the donor was excited with a 349 nm Nd:YLF pulsed laser at 300 Hz with ~68 µJ/pulse followed by collection of either the donor signal using a 550/32 nm bandpass filter or the TR-FRET signal using a 700/75 nm bandpass filter.…”

“…In the present study, we developed two different innovative approaches to characterize the pharmacological and functional properties of mGlu2-4 heterodimers specifically, without any influence of the co-existing mGlu2 and mGlu4 homodimers. We also used an innovative lanthanide-based time-resolved FRET microscopy approach ( Faklaris et al, 2015 ), to demonstrate mGlu2 and mGlu4 can form heterodimers in transfected neurons. Using our heterodimer selective assays, we identified three pharmacological fingerprints that can be used to identify mGlu2-4 heterodimers in native cells.…”

G Protein–Coupled Receptor Multimers: A Question Still Open Despite the Use of Novel Approaches

“…Subsequent coimmunoprecipitation and time-resolved FRET reveals the physical association and close proximity of GPCRs in heterologous cells (Fig. 1, A-C) (Milligan and Bouvier, 2005;Maurel et al, 2008;Faklaris et al, 2015). Moreover, fusion of bioluminescent, fluorescent proteins, or nonfunctional fragments of these proteins to the C-terminal tail of GPCRs allows close-proximity detection of GPCR dimers and/or oligomers in living cells using bioluminescence resonance energy transfer (BRET), fluorescence resonance energy transfer (FRET), or bimolecular complementation, respectively (Fig.…”

Evaluating the Performance of Time-Gated Live-Cell Microscopy with Lanthanide Probes

“…Various systems have employed flashlamps, UV lasers, or LEDs for pulsed, epi-illumination, and mechanically or electronically gated CCD cameras for wide-field image acquisition (12,13,(15)(16)(17)(18). Time-resolved confocal microscopy has also been explored (19,20).…”