Regulation of G Protein-Coupled Receptor Kinase 5 by Calmodulin

G protein-coupled receptors (GPCRs) are critical in the transduction of extracellular stimuli into an intracellular signaling response. GPCRs are regulated by G-protein coupled receptor kinases (GRKs), which phosphorylate the active receptor to initiate desensitization and arrestin-mediated signaling. Calmodulin(CaM) is a ubiquitously expressed calcium-binding protein that has been shown to bind GRK5 to inhibit receptor phosphorylation. In this thesis work, we reveal the crystal structure of the GRK5-CaM complex. We utilized biophysical techniques to characterize complex stoichiometry and molecular dynamic simulations to determine CaM-mediated stabilization of the kinase active state. In addition, we utilized mutagenesis, lipid binding assays and kinase assays to assess how CaM binds GRK5 to regulate function. We determined that CaM binding to the GRK5αN-helix stabilizes the secondary structure for kinase activation, similar to GPCRs. Additionally, CaM binds the GRK5 αC-helix site to inhibit GRK5 lipid binding. Together, we applied this in vitro characterization to determine how these mechanisms of regulation impact GRK5 function in the cell in response to PAR1-mediated calcium activation and calcium ionophore treatment. We observed acytoplasmic accumulation of GRK5 that was dependent on the GRK5 αC-helix binding site. Additionally, GRK5 phosphorylation of α-synuclein was dependent on CaM-mediated kinase activation. We also utilized the proximity labeling technique BioID miniTurbo to identify potential novel GRK5 interactors, many of which were localized at cell junctions. We also applied BioID to determine how the GRK5proximal proteome changes in response to PAR1 activation. The majority of proteins identified were attributed to the cytoplasm. This supports the notion that CaM-mediated GRK5 translocation targets the kinase to a novel pool of protein interactors and substrates. Together, this thesis work illustrates the structural mechanism in which CaM regulates GRK5 function and the implications for GRK5non-receptor signaling within the cell