Current Research Projects
Enzymology and structural/functional biology of Ric-8 G protein guanine nucleotide exchange factors (GEFs)
Ric-8 proteins are enzyme. They stimulate the rate-limiting step of G protein alpha catalysis, GDP release, by stabilizing a nucleotide-free form of the G protein. GTP binding to the open G alpha dissociates the complex releasing (activated) G alpha-GTP. Using enzyme kinetic assays, structural biology, and mutagenesis, we are investigating the mechanism of Ric-8-stimulated nucleotide exchange. Projects include ascertaining and measuring the putative GEF activity of Ric-8B proteins towards G protein alpha s family members. We are also working with collaborators to prepare and crystallize nucleotide free G alpha:Ric-8 protein complex for the purpose of obtaining the structure of a G protein not bound to nucleotide and to reveal the mechanism of GEF action catalyzed by Ric-8 proteins. Rational and random mutagenesis is being used to ascertain and map the catalytic core of Ric-8 enzymes, and to prepare impaired function mutants of Ric-8 for use in cellular signaling assays.
Asymmetric cell division (ACD) produces two distinct cell types. For many adult stem cells, ACD produces a daughter cell committed to a particular cell-fate and a daughter cell that remains a progenitor cell. Ric-8, Gi class G proteins, and G protein-binding GoLoco proteins are essential regulators of ACD in lower organisms (flies, worms). This G protein regulation of ACD is thought to occur without the direct influence of GPCR stimuli. We have provided a biochemical demonstration of the G protein regulation that may occur during ACD. G alpha i subunits bound in the GDP-bound state to the GoLoco-domain proteins LGN or AGS3 are activated by Ric-8A. This process may generate the signal that enables microtubule-dependent forces to mechanically demarcate an asymmetric cell cleavage plane prior to ACD. Currently, we are working to prove this biochemical model in mammalian cells. Work for this project includes the use of mouse models, live cell microscopic imaging of mitotic events and structures, and models of cell development and differentiation.
Ric-8A or Ric-8B knockout mice die early during embryogenesis. To develop cultured cell models that we will use to determine the function of Ric-8 proteins, we have cultured embryonic stem cell lines from viable, early Ric-8 knockout embryos. These invaluable cell lines will be used to test the influence of the absence of either Ric-8 homolog on all aspects of G protein function including: traditional G protein signaling pathways, G protein membrane localization/trafficking, and G protein-directed (asymmetric) cell division and cellular differentiation.
Plasma membrane (PM) localization of G proteins is essential for GPCR signaling. Two basic events are required for the biosynthetic targeting of G proteins to the PM: 1. The attachment of lipid (membrane anchoring) molecules to G protein alpha and gamma subunits -and- 2. The pre-PM assembly of G protein alpha beta gamma heterotrimers. Less is known of how G proteins are turned over, or removed from the PM. Cyclical G protein movements among the PM and intracellular compartments may be a means by which G proteins are regulated in unique fashion and lipidated dynamically.
In Drosophila ric-8 mutants, G protein alpha and G beta(gamma) subunits are not on the plasma membrane, but reside at undescribed locations in the cytoplasm. The overall expression of many G protein subunits are reduced in the absence of Drosophila RIC-8. Our lab is investigating whether these observations apply in mammals, and if so; what is the mechanism of Ric-8-mediated G protein PM localization? The mammalian system is more complicated. There are two Ric-8 homologs (A and B) with unique G alpha binding preferences, and four times the number of distinct G alpha (~20) and G beta (5) subunits. We have created mammalian reporter cell lines to stably express fluorescent-protein-tagged versions of G protein alpha subunits. These lines will be used as models to unveil and understand the potential role Ric-8 proteins might have in directing G protein membrane localization and/or trafficking. These investigations employ the use of RNAi and overexpression, fluorescence microscopy, biochemical fractionation, metabolic pulse/chase labeling, and lipid pulse/chase labeling.
Procedures to purify recombinant Heterotrimeric G protein subunits have been established over many years. Despite high homology, there are loose rules as to which subunits are susceptible to purification in large quantity. As an example, ~50 mg of recombinant Gas can routinely be purified from 6L of E. coli culture. The highly identical G alpha olf subunit (an olfactory/brain specific Gas homolog) has not been purified from E. coli successfully, and roughly 100 µg of impure protein can be obtained using a similar quantity of Sf9 insect cell culture. We have found that co-expression of tagged Ric-8 proteins and G alphas stabilizes the expression of all tested G alphas, permitting the co-purification of Ric-8:G alpha complexes. The G alpha is chemically dissociated from column-bound Ric-8 and eluted in reasonably pure form, and in some cases unprecedented quantity (i.e. G(alpha)q, G(alpha)13, G(alpha)olf). Work on this technology project is undergoing standardization. Our goal is to provide a reliable, and simplified technique of G alpha purification for our own enzymological and structural/functional pursuits, and to the research community as a whole.
Gregory G. Tall, Ph.D.
University of Rochester
School of Medicine and Dentistry
601 Elmwood Avenue
Rochester, NY 14642