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Abstract
In order for a cell to adapt to changes in its environment, it must be able to translate an extracellular cue into an intracellular signal. One way in which this can be done is through the use of G-protein coupled receptors (GPCRs). This class of transmembrane receptors promotes the exchange of GTP for GDP in the G subunits of associated heterotrimeric G-proteins. Once GTP is bound, the G subunit dissociates from the G heterodimer and these now activated subunits are able to interact with downfield effectors. A class of proteins, known as regulators of G-protein signaling (RGS) proteins, act as GTPase-accelerating proteins (GAPs), hastening the hydrolysis of GTP to GDP by G¬. This hydrolysis promotes the re-association of the subunits, effectively terminating G-protein signaling induced by GPCRs. The binding of RGS proteins to G subunits is known to occur via the RGS domain, a structure conserved among all RGS proteins. Studies have shown that the GAP activity of RGS proteins can be regulated through the competitive binding of the membrane phospholipid dipalmitoylphosphatidylinositol 3,4,5-triphosphate (PIP3), and the calcium-binding protein, calmodulin (CaM). When calcium concentrations are low, PIP3 binds to RGS proteins, inhibiting their GAP activity. Once calcium concentrations increase, calcium-bound CaM then displaces the PIP3 and binds to the RGS protein, restoring their GAP activity, and thus deactivating G-protein signaling. However, the binding epitopes for CaM on RGS proteins are not known.
To date, there are no studies to determine the CaM binding epitope on RGS10 nor to determine the structural implications of CaM binding on the interaction of RGS10 and Gαi3. My research has explored the interaction of CaM with both the RGS10 and Gαi3 proteins and determined some structural implications for the binding of CaM to RGS10 for the Gαi3 binding site on RGS10. These interactions are important to understand due to their mediation of the pharmacologically important GPCR signaling. This dissertation covers my research using physical methods, such as NMR spectroscopy and intrinsic tryptophan fluorescence, to determine how CaM binds to RGS and Gα proteins and what implications these interactions may have on G-protein signaling.