Michael J. Rizzo
Published: 2019
Total Pages: 132
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G protein coupled receptors (GPCRs) are a superfamily of transmembrane proteins responsible for transducing extracellular stimuli into intracellular responses. GPCRs are indispensable to a vast variety of distinct physiologies and behaviors and represent approximately 50% of all human drug targets. However, considerable debate exists as to the structural basis for GPCR activation, with a classical monomeric (two state model) conflicting with a growing number of reports indicating that these receptors form higher order functional oligomers. These receptor-receptor interactions can impact receptor trafficking, ligand sensitivity, desensitization, and strength of effector response. As such, an understanding of GPCR oligomerization is indispensable to our overall understanding of receptor dynamics. Additionally, the specific molecular events underlying receptor activation and signaling remain incompletely understood. Since the initial discovery of the GPCR receptor family, a number of conserved amino acid motifs have been identified that have been shown to play specific and critical roles in GPCR activation, intracellular G-protein coupling, and receptor desensitization. Still, many of these motifs remain incompletely described, with some motifs having only been evaluated in a small subset of receptors, and experimental evidence suggests that in some cases, these conserved motifs may have divergent roles in specific receptor subfamilies. As such, the conservation of these motifs throughout GPCR evolution represents and interesting and unresolved aspect of GPCR function. The goal of this research was two-fold. In one study, I utilized a combination of bioinformatics, site-directed mutagenesis, signaling assays, and fluorescent microscopy techniques to evaluate the functional role and evolutionary conservation of a specific amino acid motif, the WxFG motif, which is present in approximately 90% of all Class A receptors. Our investigation showed that, in contrast to previous studies of this motif, disruption of the WxFG motif results in trafficking defects across a range of GPCRs representing multiple Class A GPCR subfamilies, regardless of taxa. A second study evaluated whether Drosophila GPCRs, specifically a subset of neuropeptide receptors, assembled as higher order structures at the plasma membrane. While there have been many receptors shown to assemble as dimers or oligomers at the plasma membrane since the phenomenon was first recognized over two decades ago, the majority of these studies focused on vertebrate GPCRs, and the question of whether invertebrate GPCRs show similar phenotypes has been poorly evaluated, and to date, no Drosophila GPCR has been empirically demonstrated to assemble as a dimer. To gain a deeper understanding of GPCR molecular assembly, I evaluated multiple Drosophila receptors utilizing FRET microscopy to determine both the prevalence of GPCR dimerization among Drosophila neuropeptide receptors, and determine whether dimerization is conserved across taxa in specific receptor subfamilies. This investigation showed that all Drosophila GPCRs tested were able to assemble as homodimers when expressed in a heterologous expression system, suggesting that not only do Drosophila GPCRs likely assemble as higher order structures at the plasma membrane, but also that the phenomenon of receptor dimerization is an ancient property of the receptor superfamily that has been conserved throughout GPCR evolution. Taken together, these investigations further our understanding of the molecular events underlying GPCR signaling, and suggest that many aspects of receptor function are not taxa specific, and are likely fundamental features of GPCR function that have been conserved throughout the evolution of this receptor superfamily.