Search

The GPR147, also known as Neuropeptide FF1 Receptor (Bonini et al., 2000), is a seven-transmembrane G protein-coupled receptor (GpCR) that binds the Gonadotropin inhibitory hormone (GnIH) or the RFamide-related peptide-3 (RfRP3) peptide (Smith & Clarke, 2010).These ligands play a role in the control of reproduction in mammals and birds. The GPR147 is expressed in neurons (i.e GnRH neurons) and takes part with other GpCR, as GnRHr and GPR54 (GnRH and Kiss peptide receptor respectively), in the regulation of the hypothalamic-pituitary-gonadal axe. We have generated plasmids to express all these receptors as fusion fluorescent proteins and studied the homodimerization of GPR147 and the heterodimerization with GPR54 and GnRHr receptors, by FRET, BiFC and in the current approach by Fluorescence anisotropy.

The investigation of the physical and functional interaction of the G protein-coupled receptors (GPCRs) with their cognate heterotrimeric G proteins constitutes one of the most evolved aspects in this family of cell surface receptors. The development of the energy transfer-based approaches, bioluminescence or fluorescence resonance energy transfer (BRET or FRET, respectively), has strongly contributed to such advance. Using protease-activated receptors 1 and 2 (PAR1 and 2) as model for their multiple G protein coupling profiles, we revealed a preassembly between the two receptors and Gαi1 and Gαo proteins whereas their association with Gα12 occurred only upon receptor activation. Kinetic analysis in real-time and live cells showed important differences in the activation between these two modes of coupling which may be consistent with their implication in the physiological responses of PARs. Together, our finding indicate that preassembly or agonist-promoted association depend on receptor-G protein pair representing the key mechanisms of temporal and spatial regulation and integration of the multiple G protein coupling and signaling of GPCRs.