The PH Domain as a Mediator of G?? Effector Interactions

G protein βγ subunits regulate the activity, via direct interaction, of a large number of downstream effectors in GPCR signaling pathways. Whereas much is known about how these regulatory interactions impact cellular processes, less is understood about the structural determinants that mediate these interactions. Among the most extensively investigated Gβγ effectors are members of the PLCβ subfamily. PLC enzymes are a class of multi-domain phosphodiesterases that catalyze the hydrolysis of phosphatidylinositol 4,5-biphosphate to form two important second messengers, inositol 1,4,5-triphosphate and diacylglycerol. The activity of PLCβ isozymes is altered to varying extents by G protein α and βγ subunits as well as small GTPases. Several lines of biochemical and structural data have contributed to our understanding of the regulatory mechanisms that govern the activation PLCβ isozymes by Gαq subunits and Rac. However, despite several reports suggesting that regions of the PH domain and the catalytic core contain critical residues for Gβγ binding, the manner by which Gβγ subunits stimulate the activity of PLCβ remains an unsolved problem. The work presented in this dissertation aims to examine the role of specific structural elements of the PH domain of PLCβ isozymes in mediating the response of this PLC subfamily to Gβγ. To that end, we used a combination of peptide array analysis, site directed mutagenesis and activity assays in both cell-based and reconstituted systems to map the structural determinants of the Gβγ interaction. Our results suggest that, although the flexible loops along the PH/Rac1 interface, observed in the crystal structure of PLCβ2-Rac1 complex, do not participate in direct interactions with Gβγ they nonetheless contribute to activation. This work highlights the complexity of the Gβγ interaction and the importance of the PH domain towards activation of PLCβ by Gβγ. GRK2, best known for its ability to phosphorylate active GPCRs, is another well -known Gβγ effector. Owing to its role in normal and diseased heart function there is great interest in the development of inhibitors of GRK2. Based on the structural analysis of two potent GRK2 inhibitors bound to the GRK2-Gβγ complex our lab previously proposed that the potency and selectivity of less potent GRK2 inhibitors, with improved pharmacological properties, could be enhanced using a structure-activity relationship guided design strategy. As part of this effort, I determined the co-crystal structure of the GRK2-Gβγ complex bound to a small molecule inhibitor of GRK2 based on a paroxetine scaffold. This novel inhibitor exhibited increased potency and selectivity towards GRK2 as well as improved contractility of cardiac myocytes relative to its parent compounds. Additional work presented details my contribution to a study aimed at understanding the functional consequences of membrane lipid composition in relation to the orientation of the GRK2-Gβγ complex on lipid bilayers. This study revealed that the concerted binding of GRK2, via its PH domain, to Gβγ and PIP2 likely position the receptor docking site of GRK2 in orientation that optimizes its interaction with active GPCRs.PhDChemical BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/140945/1/osvcruz_1.pd