Characterization of the Interaction of the Regulator of G-protein Signaling 10 (RGS10) and the Calcium Signaling Protein Calmodulin

Characterization of the interaction of the regulator of G-protein signaling 10 (RGS10) and the calcium signaling protein calmodulin G-Protein Coupled Receptors (GPCRs) are of high therapeutic importance, being the targets of approximately 35% of all drugs currently in clinical use. G-Protein activation upon agonist binding to a GPCR is mediated by the exchange of GDP to GTP in the G𝛼 subunit of the heterotrimeric G-Protein, which causes the dissociation of the individual subunits (G𝛼 and the G𝛽γ dimer), initiating different signaling pathways. Signaling is terminated upon hydrolysis of the GTP to GDP by the intrinsic GTPase activity of the G𝛼 subunit, promoting the return to the resting state. The RGS (Regulators of G-Protein Signaling) family of proteins are GTPase accelerating proteins (GAP) that stimulate the intrinsic GTPase catalytic activity of G𝛼, providing regulation to G-protein signaling. Proteins involved in regulating signaling initiated by GPCRs are considered potential drug targets, as is the case for RGS family members. RGS proteins are regulated by the competitive binding to the membrane lipid phosphatidylinositol (3,4,5)-triphosphate (PIP3) and the ubiquitous calcium-signaling protein, calmodulin (CaM). However, the nature of the interaction between RGS proteins and CaM remains poorly understood. The focus of our studies is the interaction of CaM with RGS10, a protein with a key role in neuroinflammation that is associated with several neurodegenerative diseases. We are studying the affinity of the RGS10-CaM complex and binding epitopes of their interaction. We have thus far recombinantly produced and purified full-length RGS10 and the RGS domain only (S31-L165), as well as two mutants. By monitoring the intrinsic tryptophan fluorescence of these RGS10 constructs, we have measured the affinities of the RGS10 proteins for CaM. The different RGS10 constructs studied bind to CaM with a low micromolar affinity, which is Ca²⁺ dependent. Using NMR spectroscopy, we confirmed this interaction and identified the residues of RGS10 that interact with CaM, based on NMR chemical shift changes. Using the individual C-terminal (M76-K148) and N-terminal (A1-D80) domains of CaM, we revealed that RGS10 binds preferentially to the C-terminal domain of CaM. Our results represent the critical first steps towards a comprehensive understanding of calmodulin regulation of G-protein signaling.