Current work focuses on mechanisms of G protein activation by an intracellular guanine nucleotide exchange factor called Ric-8A. This protein appears to carry out multiple functions in cells: it is essential for asymmetric cell division and abscission, regulates neurotransmitter secretion and assists in the biogenesis of G protein alpha subunits, in addition to its role as a G protein activator. Ric-8A functions as a guanine nucleotide exchange factor by catalyzing the release of GDP from G alpha subunits. Ric-8A forms a nucleotide-free complex with G alpha, which dissociates only in the presence of GTP, leading to the formation of active GTP-bound alpha. In this regard, Ric-8A is functionally analogous to the well-characterized family of trans-membrane G Protein-Coupled Receptors (GPCR), which, upon agonist binding, catalyze nucleotide exchange from G protein heterotrimers.

We have shown that Ric-8A catalyzes nucleotide exchange by inducing large conformational changes within G alpha, possibly by inducing local unfolding at the nucleotide binding site and in surrounding elements of secondary structure. In analogy to the action of GPCRs, Ric-8A also induces separation of the two major domains of G alpha. We have employed a variety of biophysical methods to characterize Ric-8A-induced structural changes in G alpha, including scanning calorimetry, Double Electron-Electron spectroscopy (in collaboration with Wayne Hubbell's laboratory at UCLA), Hydrogen-Deuterium Mass Spectrometry (in collaboration with Brian Bothner's group at Montana State University), single-molecule fluorescence spectroscopy (with Sandy Ross at UM), and most recently using camelid nanobodies as functional and structural probes (in collaboration with Jan Staeyert's laboratory at VIB, the Netherlands). We make extensive use of site-directed mutagenesis, in conjunction with enzymological methods to understand the mechanism of Ric-8A action. Our laboratory studies other aspects of G protein function, as described in our publications, as well as the mechanism of adenylyl cyclase regulation.

Our work is funded by grants from the National Institutes of Health (R01GM105993)