Mechanisms of Regulation of Neuronal GPCRs by KCTDs

Abstract Neuromodulatory GPCRs are critical regulators of neuronal activity that function within complex networks of accessory proteins which shape their signaling across space and time. Proteins in the potassium channel tetramerization domain (KCTD) family are important accessory subunits of the GABAB receptor, serving to regulate the intensity and duration of signaling in response to the inhibitory neurotransmitter, GABA. However, a mechanistic understanding of KCTD regulation of GABAB receptors is lacking and whether KCTDs regulate other neuromodulatory GPCRs is unknown. In aim 1, we will investigate GABAB receptor/KCTD coupling at the molecular level, using a combination of structural studies by cryo-electron microscopy and biophysical analysis in vitro. In aim 2, we will probe the cellular mechanisms of KCTD regulation of GABAB receptor signaling with functional studies by electrophysiology and fluorescence microscopy and proximity labeling mass spectrometry both in cell lines and primary neurons. In aim 3, we will build on preliminary data identifying the M5 muscarinic acetylcholine receptor as a novel KCTD target by characterizing this novel signaling complex using in vitro biochemical and biophysical techniques and live cell functional assays. Collectively, the results of this study will serve to develop a detailed mechanistic understanding of KCTD modulation of GPCR signaling as well as laying a foundation for investigation of newly discovered regulatory proteins and signaling components. Proposed technology development efforts may also find broader utility in other areas of molecular neurobiology, further enhancing the potential impact of this work.