Homodimerization of Drosophila Class A neuropeptide GPCRs: Evidence for conservation of GPCR dimerization throughout metazoan evolution

While many instances of GPCR dimerization have been reported for vertebrate receptors, invertebrate GPCR dimerization remains poorly investigated, with few invertebrate GPCRs having been shown to assemble as dimers. To date, no Drosophila GPCRs have been shown to assemble as dimers. To explore the evolutionary conservation of GPCR dimerization, we employed an acceptor-photobleaching FRET methodology to evaluate whether multiple subclasses of Drosophila GPCRs assembled as homodimers when heterologously expressed in HEK-293 T cells. We C-terminally tagged multiple Drosophila neuropeptide GPCRs that exhibited structural homology with a vertebrate GPCR family member previously shown to assemble as a dimer with CFP and YFP fluorophores and visualized these receptors through confocal microscopy. FRET responses were determined based on the increase in CFP emission intensity following YFP photobleaching for each receptor pair tested. A significant FRET response was observed for each receptor expressed as a homodimer pair, while non-significant FRET responses were displayed by both cytosolic CFP and YFP expressed alone, and a heterodimeric pair of receptors from unrelated families. These findings suggest that receptors exhibiting positive FRET responses assemble as homodimers at the plasma membrane and are the first to suggest that Drosophila GPCRs assemble as homodimeric complexes. We propose that GPCR dimerization arose early in metazoan evolution and likely plays an important and underappreciated role in the cellular signaling of all animals.