Overview
The main emphasis of research in our laboratory is on the fundamental principles that regulate signal transmission in G protein pathways. G protein signaling mediates a vast variety of critical biological processes ranging from proliferation and motility to cellular reception and excitability. As such, unraveling the mechanisms involved in the regulation of G protein signaling is the key for understanding the basic principles of biological organization as well as origins of dysfunction in pathological conditions. In a simplistic model, all G protein systems consist of three major types of elements where (1) receptors at the surface of the cell triggered by ligands transmit their activation to, (2) the effectors that generate cellular responses via shuttle molecules, and (3) G proteins. G proteins serve as molecular switches that oscillate between ON and OFF states by dissociating into signal-transducing α and βγ subunits and re-assembling back into the inactive heterotrimer. While this elementary model accounts for main events involved in relaying the signal, it falls short of explaining how the duration and extent of signaling is controlled as most of processes relying on G protein signaling impose exquisite and unique timing requirements. Neither does it offer insights into the mechanisms involved in establishing the selectivity of the signaling as cells contain hundreds of G proteins coupled receptors that are linked to myriads of effectors. The underlying premise of our research efforts is that that the regulation of the G protein signaling extent and specificity is achieved at universal focal “control points”. Indeed, growing evidence suggests that one of such central control points is provided by the Regulator of G protein Signaling (RGS) proteins that serve as integrators of G protein signaling and are increasingly viewed as the forth key component of the G protein pathways. Biochemically, RGS proteins act to facilitate G protein inactivation thus providing an opposing balancing force to the receptor mediated activation in the pathways. Furthermore, growing data indicate that RGS proteins act as scaffolds uniquely compartmentalizing the classical components of the pathway. This brings the major focus of the research in my laboratory to understanding the roles and mechanisms of RGS proteins in the cellular signaling in addition to the efforts to uncover novel regulatory principles.
In addressing these research questions we use a multi-disciplinary platform that combines biochemical techniques with methods of cell biology, proteomics, and behavioral analysis in genetic mouse models. My laboratory also maintains active collaborations with several laboratories employing electrophysiological level of analysis.
RESEARCH DIRECTIONS
The role of RGS proteins in Opioid and Dopamine Signaling in Striatum
One of the central functions of the G protein signaling in the nervous system is to mediate the processing of the reward signals to reinforce behaviors important for survival and well-being. Anatomically positioned in the neurons of the basal ganglia, the reward system relies on signaling through two tightly integrated G protein pathways signaling via D2 dopamine and m-opioid receptors. Work by us and others have revealed that much of the sensitivity of these receptor signaling in the region is controlled by a three way balancing between two RGS proteins (RGS7 and RGS9) regulated by common subunit R7BP. Remarkably, it appears that the receptor stimulation acts to adjust the signaling throughput by differentially regulating the concentration of RGS complexes via proteolytic mechanisms. This introduces a new plasticity mechanism of G protein signaling in the nervous system that we are actively investigating by analyzing mechanisms mediating GPCR and G protein selectivity of RGS action, delineating signaling pathways controlled by dopamine and opioid receptors imaging optical sensors in live neurons to probe intracellular signaling cascades, and testing how deficiencies at the level of individual RGS components are translated into specific behavioral alterations by evaluating mouse genetic models.
Signal transmission at the first visual synapse
It is important to understand the types of neuronal activity that occur in living animals during the process of sensing and learning about the environment. Since it is difficult to probe neuronal activity in flies using standard electrophysiological techniques, we have turned to optical imaging of neuronal activity. We have introduced GFP-based transgenes into the fly that report changes in intracellular calcium concentration, synaptic transmission, and cAMP. We have used these to monitor calcium influx during neuronal depolarization and to visualize synaptic activity as the animals are exposed to odorants. The latter experiments have shown that specific antennal lobe glomeruli respond to different classes of odorants. We have used synaptic transmission reporters to visualize the changes in synaptic transmission that occur with conditioning and have discovered that new populations of synapses, defined by antennal lobe glomeruli, become activated after the flies are trained. In other words, we have visualized a olfactory memory trace for the first time. In addition to this antennal lobe memory trace, we have now identified three other memory traces that form after olfactory conditioning in the fly. These occur in different sets of neurons and form and exist across different intervals after training. The dorsal paired medial neurons form a delayed memory trace that forms at 30 min after training and persists for about 2 hours. It seems to correlate with medium-term memory. The alpha/beta neurons of the mushroom bodies form a memory trace that forms by nine hours after training and persists for at least 24 hours. This memory trace depends upon the activity of CREB and upon normal protein synthesis at the time of training. Finally, we have identified a memory trace that forms in GABAergic neurons that innervate the mushroom bodies. This memory trace is registered as a decreased calcium influx (decreased activity of the GABAergic, inhibitory neurons) immediately after learning. We are searching for other memory traces that may account for behavior at different times after training and are using reporter molecules to monitor other physiological events.
Dissection of the molecular landscape of GPCR signaling by proteomics.
Multiple interlinked signaling pathways operate at synapses and contribute to complex processing of information flow. G protein signaling pathways are integral to synapses modulating virtually every aspect of their function. In recent years, multiple new effectors and regulators of G proteins were described, yet the information about their expression and interactions at synapses is largely missing. Furthermore, dynamic changes in the interactions between components of the signaling pathways are thought to be directly responsible for many molecular changes associated with synaptic plasticity. We are interested in identifying novel regulatory mechanisms and molecular components of G protein signaling pathways in the central nervous system and retina. Towards this end, we have recently developed a quantitative iTRAQ-based mass-spectrometric approach to probe changes in protein-protein interactions in the nervous system of genetic mouse models. Using this approach we plan to build a quantitative database of synaptic proteins that undergo remodeling in their interactions in response to changes in signaling status and study changes in the formation of the macromolecular complexes triggered by neurodegenerative and neuropsychiatric conditions.
G protein signaling to GIRK channel in parasympathetic control of the heart rate.
Cardiac output adjusts on a beat-to-beat basis due to the changing balance of parasympathetic and sympathetic input to the heart. G protein signaling pathways are fundamental to this important process. Too much or too little parasympathetic influence on the heart can trigger arrhythmias, often with fatal consequences. In the prototypical signaling pathway that mediates the inhibitory effects of parasympathetic activity on the heart, activation of the M2 muscarinic acetylcholine receptor leads to cellular hyperpolarization by opening the G protein gated Inwardly Rectifying Potassium Channel (GIRK). The GIRK channel is directly activated by G protein βγ subunits. We have recently found that GIRK channel is associated with multi-modular RGS6-Gβ5 complex that acts to provide high temporal fidelity of the channel gating. Embarking on a new direction for our laboratory, we are moving ahead to characterize this novel signaling compartmentalization concept and study the roles of RGS6-GIRK complex in cardiovascular signaling.
