R4 and R12 subfamily RGS proteins: structures, functions, and emerging chemical biology

It is essential that cells respond to their extracellular environment with appropriate intracellular changes. Many environmental cues are received at the cell membrane, by a family of G-protein coupled receptors (GPCRs) and their heterotrimeric G-proteins, composed of Gα, Gβ and Gγ subunits. Upon binding of a hormone, neurotransmitter, tastant, or small molecule agonist at the membrane-bound GPCR, the receptor catalyzes the exchange of GDP for GTP on the heterotrimeric Gα subunit. This change results in the release of Gβγ from the Gα subunit. The dissociated Gα and Gβγ dimer can each signal to downstream effectors until the Gα hydrolyzes GTP, resulting in the reassociation of the Gαβγ heterotrimer. The duration of effector activation is therefore controlled by the duration of the Gα subunit in its GTP-bound state. The state of Gα as a GTP-bound protein is short-lived, however, given that the protein has an intrinsic ability to hydrolyze GTP to GDP and inorganic phosphate - an activity that can be greatly accelerated by Regulator of G-protein Signaling (RGS) proteins, which are known to act as GTPase-accelerating proteins (GAPs) for Gα subunits. The work described herein represents series of studies aimed at furthering our understanding of the molecular determinants of RGS protein/Gα interaction specificity, facilitating the identification of small molecule modulators of RGS protein activity, and understanding the biochemical function and physiological roles of RGS21. Toward the first aim, I performed mutagenesis on residues predicted to change the Gα specificity of RGS2 and extensively characterized these mutants using GTP hydrolysis assays and Gα interaction assays employing surface plasmon resonance and in vitro FRET. To comprehensively understand the role that each mutation was playing in allowing RGS2 to bind to a non-native Gα binding partner, I solved a crystal structure of a mutant RGS2 in complex with Gαi. Toward the second aim, facilitating the identification of small molecule modulators of RGS protein function, I used a variety of biophysical tools to determine the mechanism of action of the first commercially available RGS protein inhibitor - which was ultimately determined to be a non-specific, thiol-reactive compound. In order to identify new small molecule modulators of RGS protein function, I developed and validated a high-throughput screen for the RGS12/Gαi1 interaction. This screen was run against several compound libraries, both locally and at the NIH Chemical Genomics Center (NCGC); however, no hits were subsequently validated as in vivo inhibitors of the RGS12/Gαi1 interaction. Given these setbacks, we rethought how we were screening for RGS protein inhibitors and developed a completely novel, enzymatic-based assay that can be used for high-throughput screening. Toward the final aim, we confirmed the disputed report by von Buchholtz et al. that RGS21 is expressed only in chemosensory cells; however, we were also able to identify RGS21 transcripts in sensory digestive and pulmonary epithelia. Using biochemical methods, we demonstrated that RGS21 exhibits high affinity binding toward a variety of Gα substrates and that it can accelerate their GTP hydrolysis in vitro. We also present data that endogenous RGS21 expression serves to negatively regulate tastant receptor signal transduction in a cellular model of gustation.