Grant: Discovery Grants Program - Individual

In the adaptation of cells to dynamic milieu, G protein-coupled receptors (GPCRs) serve as the primary sensors of external cues, which enact signal transduction cascades to regulate cellular behavior. To achieve this, cue-bound GPCRs recruit intracellular transducers/adaptors (e.g. heterotrimeric G proteins, beta-arrestins) that regulate downstream effectors (e.g. adenylyl cyclases; AC1-9) to prompt specific signaling outcomes. Among prototypical cues, dopamine (DA) uses this modus operandi to modulate a wide range of physiological actions in the brain and periphery. To do so, DA relies classically on stimulatory G protein-linked D1-class (D1R and D5R) and inhibitory G protein-linked D2-class (D2R, D3R and D4R) GPCR subtypes, which stimulate and inhibit AC activity, respectively. Intriguingly, the natural human D3R variants Ser9 and Gly9 selectively inhibit AC5 while other DA receptors couple to most AC isoforms. Building on my expertise on DA receptors and NSERC-funded HQP, we demonstrated that human Ser9-D3R and Gly9-D3R via their third intracellular loop (IL3) physically associate with AC5. In contrast to other DA receptor subtypes, Ser9-D3R and Gly9-D3R do not undergo DA-induced endocytosis in cells lacking AC5. Interestingly, while DA-activated Ser9-D3R and Gly9-D3R inhibit AC5 activity, beta-arrestin2 recruitment to both D3R�AC5 complexes only enables Ser9-D3R endocytosis. As polymorphic variations occur on the extracellular amino terminus (NT) of D3R, we hypothesize a functional interplay between the D3R-NT region and AC5, which regulates signaling and proteomic properties of D3R in a polymorphism-dependent fashion. To test this hypothesis, we have outlined the following two short-term objectives. Aim 1. Foundational Mechanisms of the Interplay between D3R variants and AC5 Aim 2. Proteomic Modalities of D3R�AC5 Signaling Complexes To address these objectives, we will develop a training framework, whereby HQP will acquire a wide range of experimental skills using molecular and cell biology, confocal microscopy and mass spectrometry. To assess the structural mechanisms underlying the formation of AC5 complexes with Ser9-D3R and Gly9-D3R variants and how these impinge on the regulation of signaling pathways, HQPs will use native brain preparations, cell co-transfections, site-directed mutagenesis, GST-fusion protein pulldowns, immunoprecipitation (IP), bioluminescence resonance energy transfer, ELISA, cAMP and kinase assays. To interrogate the proteomic profiles involved in the assembly of signaling complexes enacted by D3R variants and AC5, HQP will employ a Turbo-ID approach with IP and mass spectrometry. Overall, we trust that our research program will shed new light on the impact of D3R polymorphic variants on DA signaling function. This research will give impetus for the development of humanized mice harboring Ser9-hD3R and Gly9-hD3R and design of cell-penetrating peptides to target this complex in neurons co-expressing D3R and AC5.