<p>Recent advances in structural biology and biochemistry have identified proteins downstream of G protein-coupled receptors (GPCRs) as promising drug targets. These proteins are highly regulated to ensure proper physiological responses from extracellular stimuli. Dysregulation of these signaling enzymes can have detrimental consequences, including cardiovascular disease and cancer. Understanding how these proteins are regulated from a structural and biochemical standpoint can therefore be exploited to develop new therapeutics.</p> <p>In this work, the molecular mechanism of regulation of two different proteins downstream of GPCRs is investigated. The first protein, Regulator of G Protein Signaling 17 (RGS17), is involved in numerous processes throughout the body, including the development and progression of lung cancer. This work presents the crystal structure of RGS17 bound to Ca²⁺. Ca²⁺ was found to bind to the same site as the predicted Ga binding surface and increases interactions between RGS17 and Ga_o. Therefore, Ca²⁺ positively regulates RGS17, supporting a mechanism in which Ca²⁺ increases the GTPase activating function of the RZ-family of RGS proteins to ultimately downregulate Ca²⁺ signaling.</p> <p>The second protein, phospholipase Ce (PLCe), has been implicated in cardiac hypertrophy through its production of second messengers. This process is regulated by the small GTPase Rap1A. This work presents insight into the molecular mechanism of Rap1A-dependent activation of PLCe, in which four conserved, hydrophobic residues on the surface of the RA2 domain of PLCe play an essential role. Furthermore, small angle X-ray scattering studies show that binding of Rap1A induces conformational changes in PLCe, resulting in a more compact activated complex. This supports a mechanism in which Rap1A is an allosteric activator of PLCe, inducing conformational changes in PLCe that increase lipid hydrolysis by relieving autoinhibitory interactions and/or by promoting interactions with the cell membrane.</p>

<p dir="ltr">Phospholipase Cε (PLCε) is required for normal cardiovascular function, and dysregulation of its expression or activity has been shown to cause cardiac hypertrophy and heart failure. However, regulation of PLCε by the RhoA small GTPase protects the heart against ischemia-reperfusion injury, particularly downstream of G_12/13-coupled receptors. Despite the role of RhoA and PLCε in driving the cardioprotective response, little is known about how these proteins interact to increase lipase activity.<b> </b>RhoA was initially thought to bind to PLCε through one of its C-terminal Ras association (RA) domains, which are essential for its regulation by other GTPases. However, the RA domains are dispensable for both RhoA binding and activation, and further truncations of PLCε narrowed its binding site to the highly conserved PLC catalytic core. Functional studies implicated an insertion within the catalytic TIM barrel domain, known as the Y-box, as a requirement for RhoA-dependent activation of PLCε. However, the Y-box does not bind the GTPase. The goal of this dissertation is to identify the molecular mechanism by which RhoA binds to PLCε and increases its activity using structural and functional studies. The successful completion of these studies will map the interaction between these two critical signaling proteins, as well as identify elements in PLCε required for activation at the membrane. Ultimately, this knowledge can be exploited to develop lead therapeutic compounds that modulate this interaction to improve cardiovascular health.</p>