Elucidation of Drosophila melanogaster G protein coupled receptor interactions through heterodimerization and chimeric receptor studies. Michael J. Rizzo, Erik C. Johnson. Wake Forest University, Department of Biology, Winston Salem, NC 27109.

   G protein coupled receptors (GPCRs) represent a well-characterized protein superfamily present in most Eukaryotic taxa. GPCRs are critically important to a multitude of physiological and behavioral processes, and currently represent a target for approximately 50% of all pharmaceutical drugs. Canonical GPCR signaling relies on conformational changes upon ligand binding to the receptor, which results in activation of a heterotrimeric G protein and liberation of a second messenger pathway. Additionally, activation of a GPCR results in the recruitment of specific kinases which phosphorylate the GPCR and cause the subsequent recruitment of arrestins, which lead to the termination of GPCR signaling and internalization. To identify the structural aspects that underlie ligand binding domains and specific G protein and arrestin interactions, we are generating a series of receptor chimeras focusing on the proctolin and FMRF receptor pairs. Such structural-functional studies have not been evaluated for Drosophila receptors. Furthermore, recent research indicates that many GPCRs exist as homodimers or heterodimers in the cell, and this dimerization has broad ramifications on signaling mechanisms, trafficking, expression, and internalization. To this date, no Drosophila GPCRs have been shown to assemble as heterodimers. To determine which GPCRs are able to form heterodimers, we are currently tagging the entire cohort of peptide and amine GPCRs with both a fluorophore (CFP/YFP) and Hemoagglutinin (HA) epitope tag and expressing these modified receptors in HEK-293 cells. Through FRET (Forster Resonance Energy Transfer) and co-immunoprecipitation (Co-IP) studies, we will evaluate a matrix of all Drosophila GPCRs to determine heterodimer pairs, and aim to determine the impact of such events on signaling. We suspect the results of both studies to dissect peptide receptor function will offer insight into the specific mechanisms underlying receptor signaling and inform future experiments into how GPCRs act as points of integration of different endocrine signals.