Research Overview

The main research direction of my laboratory is to explore the molecular and cellular mechanisms of G protein signal transduction. G protein signaling pathways are ubiquitous systems mediating a variety of processes such as perception of light and odor or responses to the action of hormones and neurotransmitters. In these pathways the flow of information between the surface receptors (GPCR) and intracellular effectors is mediated by the heterotrimeric G proteins. G proteins act as molecular switches: after being turned on by receptor-triggered GTP binding they modulate the activity of their intracellular effectors until the hydrolysis of the GTP turns them off. The process of G protein inactivation is a key step that determines the duration of the signaling and therefore the extent of the cellular response. It is controlled by a specialized protein family – Regulators of G protein Signaling (RGS) that act to speed up the inactivation of the signaling in the pathway by accelerating the GTP hydrolysis of the G proteins. In our laboratory we are particularly interested in how the function of the RGS proteins contributes to the regulation of the signaling duration in neurons. Currently, our attention is focused on the R7 subfamily of the neuronal specific RGS proteins: RGS6, RGS7, RGS9 and RGS11. RGS proteins of this subfamily are involved in the regulation of a variety of critical neuronal functions such as vision, locomotion and behavior in almost all eukaryotic organisms from C.elegans to humans.

Model Systems for Studying R7 RGS Function

The vertebrate retina offers many advantages as a model system to study fundamental principles in cell signaling. It contains 5 major types of neurons: photoreceptor, bipolar, amacrine, horizontal and ganglion cells. These neurons form a neural network which has a well defined physiological function. Retinal neurons utilize all four R7 RGS proteins to regulate their G protein signaling. However, except for photoreceptors, the organization of G protein pathways in the retina neurons is far from being understood. An illustration of the important role the R7 RGS proteins play for the signaling in the retina comes from the studies on RGS9-1 function in photoreceptors. RGS9-1 acts to speed up the rate of the photoreceptor G protein - transducin inactivation providing the high temporal resolution of our vision. Mutations in RGS9-1 gene result in the human vision disease called bradyopsia, which affects the ability of patients to adapt to changes in luminance and to recognize moving objects. The goal of our future studies is to investigate the roles of the R7 RGS proteins in mediating signal transduction in the retinal neurons downstream from the photoreceptors.

G protein signaling in basal ganglia mediates the effects of a number of neurotransmitters which control behavior, locomotion and underlie drug abuse and addiction. The alteration of the G protein signaling in basal ganglia leads to many psychiatric conditions and neurological diseases such as chronic depression, obsessive compulsive disorder, Parkinson's disease, Huntington's disease and many others. Recent reports indicate that G protein signaling in the medium spiny neurons of the striatum is regulated by R7 RGS proteins. Studies with knockout animals indicated that the long splice isoform of RGS9, RGS9-2, regulates the reward-seeking and locomotor responses mediated by the dopamine and opioid pathways in the striatum. The main focus of our current efforts is to study the molecular organization of the G protein signaling cascade regulated by RGS9-2 in the medium spiny neurons.

Research Projects

We use a multidisciplinary approach to address a variety of fundamental questions pertaining to the physiological function of R7 RGS proteins. Specifically, we are interested in:

Intracellular trafficking of RGS proteins. The function of a signaling protein within the cell to the large extent is determined by its localization. The proper function of R7 RGS proteins within the context of the specific G protein pathway requires its delivery to sites of its function. Using a combination of the transgenic approaches with the laser confocal imaging we have recently found the conserved DEP domain represents a specific targeting element in the R7 RGS protein. We have shown that the DEP domain of RGS9-1 mediates its specific delivery to the place of its function – outer segment of the photoreceptor. We are currently examining the molecular mechanisms of this phenomenon.

Specificity of RGS- G protein recognition. A single neuron contains several G protein pathways that mediate its responses to the action of different stimulants. For the precise regulation of signaling in each individual pathway the action of RGS protein has to be extraordinarily specific. We use a combination of macromolecular structure analysis with site directed mutagenesis and enzyme kinetics to delineate the elements in R7 RGS proteins that confer the specificity of their action. The action of such elements, which we call “affinity adapters”, was found to determine the specific interaction between RGS9 and its cognate G proteins. We currently study the structure/functional organization of other members of R7 RGS proteins to further explore the “affinity adaptor” concept.

Identification of the molecular components of the G protein pathways regulated by RGS proteins. Thorough understanding of R7 RGS function requires obtaining the precise information about the molecular organization of the G protein pathways regulated by these RGS proteins. Our approach is to use a proteomics screen for the interaction partners of R7 RGS proteins in the striatum and retina. Immunoprecipitation and mass-spectrometry. Using this approach we have recently identified a novel neuronal protein, R7BP (R7 Binding Protein), that serves as the universal interaction partner for all R7 RGS proteins. Analyzing the role of R7BP in regulation of G protein signaling is a subject of current investigations in the laboratory.

Regulation of RGS protein function. R7 RGS proteins possess a complex modular organization. Data obtained by us and others indicate that the multiple domains of R7 RGS proteins differentially contribute to the regulation of their catalytic activity and substrate specificity. One of the most conserved features of all R7 proteins is their constitutive association with the collateral subunit Gβ5 (type 5 G protein β subunit). It is established that Gβ5 is essential for the folding and stability of R7 proteins but its role in the functioning of these proteins remains poorly understood. We use a combination of the site directed mutagenesis with detailed in vitro kinetic analysis and protein/protein interaction assays to address the role of Gβ5 in the function of R7 RGS proteins.