Determining the Molecular Arrangement of GABABR-GIRK Signaling Cascades

Project Summary/Abstract Drug addiction is marked by functional changes to once healthy cell signaling pathways. Drugs of abuse such as morphine, cannabinoids, cocaine, and gamma-hydroxybutyrate (GHB) disrupt inhibitory neurotransmission signaling through G protein-coupled inward rectifying potassium (GIRK) channels. In fact, while some substances like alcohol bind directly to GIRK, the channels are also directly downstream of G protein coupled receptors (GPCRs) which make up the largest class of drug targets for FDA approved pharmaceuticals. GPCRs, heterotrimeric G proteins of the Gi/o family, and GIRK channels are linked by a series of distinct protein-protein interactions, and a substantial proportion of inhibitory neuromodulation is mediated by this pathway. One major inhibitory GPCR found in the hippocampus called GABAB receptor (GABABR) initiates a series of conformational changes that propagates through the signaling cascade resulting in GIRK channel opening. While individual subunit interactions have been studied, no consensus on the comprehensive macromolecular organization of this signaling pathway has been reached. GHB, a drug of abuse, acts as a weak agonist at GABABR and in high doses induces sedation and euphoric effects. Understanding how GABABR and GIRK channel proteins interact and are organized as a signaling cascade will provide valuable insight into how inhibitory signals are sensed, propagated, and transmitted. Significantly, GABABRs and GIRK channels have been shown to co-localize and interact in neurons, and both GABABR and GIRK channels form complexes with inhibitory G protein subunits. Thus, this proposal plans to test the hypothesis that GIRK channel activation consists of a preformed complex that is rearranged upon GPCR/G protein activation. To test this hypothesis, I will use optical biosensors and GIRK signaling assays in Gα null cells to define the functional signalosome. Additionally, I will define the protein-protein binding interfaces within the complex using crosslinking-mass spectrometry. Finally, I will determine how the protein-protein interactions change from the inactive to the fully active signaling state. Taken together, this project will provide a clear understanding of how inhibitory GABAB-G protein-GIRK signaling occurs and more broadly, how drugs such as GHB hijack GPCR- ion channel dynamics.