| Cellular communication is an important phenomenon for the maintenance of cellular homeostasis. Recently, important progress has been made in the cell signalling research field concerning the identification of the major actors and the cellular pathways engaged in response to these extracellular factors. However, in spite of this new information, the interrelationships at the molecular level between the various cellular actors and the different signalling pathways remain badly understood.;Combining these new BRET techniques reveals for the first time the formation of a complex between the alpha2A adrenergic receptor (alpha 2AAR), Galphai1, the Gbetagamma dimer and G protein-receptor kinase (GRK2) following receptor activation. Moreover, only the entry of GRK2 into the receptor complex is required for the alpha2AAR desensitization, by inserting between Galphai1 and Gbetagamma. On the other hand, the stabilization of the interaction between alpha2AAR and beta-arrestin2 requires the kinase activity of GRK2.;Another study revealed the importance of multiple Galpha subunits for calcium mobilization induced upon activation of the delta opioid receptor (DOR). Galphaq subfamily member overexpression altered the DOR-induced calcium mobilization, but this Galphaq calcium mobilization remained sensitive to pre-treatement pertussis toxin, through selective inhibition of the activity of Galphai members. Moreover, Galphai and Galphaq co-expression potentiated calcium mobilization, suggesting an interrelationship between these two Galpha families in DOR signaling. This Galphai and Galphaq interrelationship could result from the formation of a complex close to the receptor. In order to test this hypothesis, BRET experiments were performed, with the aim of measuring the presence of complexes between different Galpha. In addition to demonstrating complex formation between Galpha subunits, the BRET experiments in parallel with sequence analysis, also revealed a selective interaction site between the Galpha, the alpha4 helix. By swapping the a4 helix of Galphai with the alpha4 helix of Galpha12, which doesn't normally interact with Galpha12, it was possible to force the interaction between Galpha12 and Galphai to confirm that this alpha helix contains information concerning the selectivity of interactions between Galpha subunits.;During this thesis, new methods were to detect protein interactions and multiplexing these methods allowed the detection of novel interactions between signalling effectors of GPCRs.;Bioluminescence resonance energy transfer (BRET) monitors interactions between proteins and can be used in two configurations, the BRET480-YFP (also known as BRET1) and the BRET400-GFP (also known as BRET2). Following oxidation of its substrate, renilla luciferase transfers its energy to a fluorescent protein, only if they are in close proximity (≤100A). By combining the BRET 480-YFP and BRET400-GFP in one assay, it is possible to follow three pair-wise interactions in the same cellular population. However, using two bioluminescence reaction substrates limits the possibility of measuring the different BRET signals simultaneously. In order to measure multiple BRET signals simultaneously, three new BRET configurations, based on the BRET 400-GFP, were developed using fluorescent proteins with different emission wavelengths. Two of the new BRET colors which have resolved emission wavelengths, the BRET400-BFP and BRET400mAmetrine, were combined for measuring the heterotrimeric G protein engagement by the vasopressin V2 receptor, as well as the accumulation of the second messenger.;Keywords : G protein coupled receptor (GPCR), Heterotrimeric G protein, G protein coupled receptor kinase (GRK), beta-arresitn, bioluminescence resonance energy transfer (BRET), fluorescence resonance energy transfer (FRET), obelin, calcium mobilization, multiplexing, luciferase, fluorescent protein (FP), opioid, adrenergic, vasopressin. |
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