Molecular mechanisms regulating G protein signaling in brain and heart: role of R7 RGS proteins and their binding partners

Abstract

G Protein Coupled Receptor (GPCR) signaling pathways convert signals from the extracellular environment into cellular responses, which is critically important for neurotransmitter action both in central and peripheral nervous systems. The ability to promptly respond to rapidly changing stimulation requires timely inactivation of G proteins, a process controlled by a family of specialized proteins known as Regulators of G protein Signaling (RGS). The R7 group of RGS proteins (R7 RGS) has received special attention due to pivotal roles in the regulation of a range of crucial neuronal processes such as vision, motor control, reward behavior, and nociception in mammals. One member of R7 RGS family, RGS9-2 has been previously implicated as a key regulator of dopamine and opioid signaling pathways in the basal ganglia of the brain, where it mediates motor control and reward behavior. Dynamic association of RGS9-2 with R7BP (R7 family Binding Protein) is critically important for the regulation of RGS9-2 expression level by proteolytic mechanisms. Changes in RGS9-2 expression are observed in response to a number of signaling events and are thought to contribute to the plasticity of the neurotransmitter action. To unravel the molecular mechanisms regulating levels of RGS9-2 upon its dissociation from R7BP we developed a novel application of the quantitative proteomics approach to monitor interactome dynamics of RGS9-2 in mice. We show that a molecular chaperone HSC70 (Heat Shock Cognate protein 70) identified by this approach is a critical regulator of RGS9-2 expression. HSC70 binds the intrinsically disordered C-terminal domain of RGS9-2 upon the dissociation of R7BP/RGS9-2 complex, and targets the complex to degradation. In addition to their critical role in shaping neurotransmitter response in the brain, RGS proteins can regulate function of peripheral organs by modulating their responses to the influences of autonomic nervous system. The role of RGS proteins in the regulation of cardiac function and heart rate has received significant attention in the recent years. With over 30 RGS proteins identified, their specific roles in heart physiology remain to be established. Parasympathetic autonomic influence plays an important role in shaping cardiac output acting to decrease heart rate and counteract the pro-arrhythmic effects of sympathetic activation. Acetylcholine (ACh) released from post-ganglionic parasympathetic neurons activates M2 muscarinic receptor (M2R) and its downstream effector, potassium channel IKACh, in pacemaker cells and atrial myocytes. This leads to cell hyperpolarization and ultimately, decreased heart rate (HR). The second part of the dissertation demonstrates cardiac expression of RGS6 member of R7 RGS family, which has been previously thought to be a neuron-specific regulator. Elimination of RGS6 in mice results in potentiated M2R-IKACh signaling, as evidenced by prolonged deactivation kinetics of IKACh in cardiomyocytes, mild resting bradycardia, and augmented HR deceleration in response to M2R activation. Furthermore, RGS6 specifically co-precipitates with one of the two subunits of IKACh, GIRK4 in transfected HEK293 cells. Direct binding to the effector channel might serve to facilitate RGS6-mediated modulation of parasympathetic influence on atrial myocytes and in mice. Altogether, the findings comprising this dissertation demonstrate a novel role of RGS6 in regulation of cardiac function, as well as two novel protein-protein interactions of R7 RGS proteins. Identified protein complexes influence G protein signaling by either (i) altering the availability of the regulator (RGS9-2/HSC70), or (ii) by serving to co-localize the major pathway components (RGS6/GIRK4).