Our laboratory studies signal transduction systems controlled by heterotrimeric G proteins as well as Ras-related GTPases. The superfamily of GTPases control numerous signaling cascades based on the regulated binding, hydrolysis, and exchange of guanine nucleotides; GTP-bound GTPases are active in downstream signaling while those bound to GDP are inactive. Mutant GTPases with abnormal GDP/GTP cycling are implicated in numerous human diseases, including cancer. It is our desire to better understand the regulation of heterotrimeric G proteins and Ras-related GTPases at the molecular level with the ultimate goal of using this information to design therapies to correct abnormal signaling mediated by these proteins and thereby treat associated pathologies.
Current major initiatives in the laboratory include:
Defining the regulation of phospholipase C isozymes by heterotrimeric G proteins and small GTPase
Phospholipase C (PLC) isozymes catalyze the hydrolysis of the minor phospholipid, phosphatidylinositol 4,5-bisphophate into the second messengers diacylglycerol and inositol 1,4,5-trisphosphate (see our review). These two second messengers act in concert to increase intracellular calcium concentrations and activate protein kinase C leading to the regulation of numerous cellular processes, including fertilization, division, differentiation, and chemotaxis. As such, the critical role of PLC isozymes in various signaling processes has been appreciated since the 1980’s. Yet surprisingly, very little is know about how these proteins are regulated at the molecular level. In collaboration with the laboratory of Dr. Ken Harden (Pharmacology, UNC Chapel Hill) we have determined a series of crystal structures of PLC-β isozymes and are using this information along with complementary biochemical and cellular studies to understand the regulation of PLC isozymes at atomic resolution and within the context of various signaling cascades. This work is part of a long-term and extensive collaboration with Dr. Harden that also includes similar studies of other PLC isozymes (e.g. PLC-γ, -ε and –η) and has led to our recent description of a general framework for understanding the regulated auto-inhibition of the entire PLC family.
Understanding the activation of Rho GTPases by guanine nucleotide exchange factors
We have published extensive crystallographic and supporting biophysical analyses on the activation of Rho GTPases by Dbl-family guanine nucleotide exchange factors (GEFs). These GEFs are characterized by a distinct Dbl-homology (DH) domain invariably followed by a pleckstrin homology domain. Much of our work has been focused on substantiating our hypothesis that these two domains work in concert to integrate information on the local concentrations of specific GTPases and phosphoinositides and to convert this information into highly localized pools of active Rho GTPases dictated by specific GEF/GTPase pairings. More recently, we have extended these studies to define how other signaling components directly impinge on these GEFs to regulate the activation of Rho GTPases. Particularly exciting research involves defining the direct activation of these GEFs by various kinases and heterotrimeric G proteins. Through such crosstalk, diverse Dbl-family GEFs function as nodes to integrate numerous signaling cascades leading ultimately to the activation of Rho GTPases and divergent cellular responses.
In related work, we are part of a National Collaborative Drug Discovery Group seeking to identify small molecule modulators that perturb the activation of Rho GTPases. These reagents will be used to probe associated signaling events and might ultimately provide lead hits for therapies to treat diseases driven by dysregulated GTPases.
Studies to elucidate the functions of RGS proteins
Gβ5 is an unusual Gβ subunit. It is about 65% identical to the other four Gβ subunits (Gβ1-4) which share greater than 90% identity; Gβ5 also does not normally form a tight, non-dissociable complex with individual Gγ subunits similar to other Gβs. Instead, Gβ5 tightly interacts with a subfamily of regulator of G proteins signaling (RGS) proteins that contain a Gγ-like motif. Relative to the more classical Gβγ dimers, these RGS/Gβ5 complexes dictate dramatically different signaling properties downstream of G protein-coupled receptors. With very little understood about how these complexes operate, we have invested ourselves in understanding the molecular regulation of these proteins. In recent exciting developments, we have determined the high-resolution crystal structure of RGS9/Gβ5 and are using this structure to guide studies to understand the relationship of RGS/Gβ5 complexes within larger signaling cascades including G protein-coupled receptors.
High throughput structural proteomics
We have a long-standing collaboration with the laboratory of Dr. Jeff Dangl (Dept. of Biology, UNC Chapel Hill) to understand the pathogenicity of bacteria that use type III secretion systems to inject avirulence factors into host cell to promote bacterial colonization. These avirulence factors typical interdict host signaling systems and we have been using structural proteomics techniques to determine crystal structures of the suite of avirulence factors injected into host plants by Pseudomonas syringae.
In related news, the crystallographic community has been exceptionally successful in obtaining funds from the University of North Carolina, the National Institutes of Health, and the North Carolina Biotechnology Center to revamp its crystallographic facilities. We now have a state-of-the-art crystallography core that is playing a central role in the integration of structural biology, chemical biology, and biophysics on campus.