Ekaterina Posokhova

The R7 RGS Protein Family: Multi-Subunit Regulators of Neuronal G Protein Signaling

G protein-coupled receptor signaling pathways mediate the transmission of signals from the extracellular environment to the generation of cellular responses, a process that is critically important for neurons and neurotransmitter action. 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 their pivotal roles in the regulation of a range of crucial neuronal processes such as vision, motor control, reward behavior, and nociception in mammals. Four proteins in this group, RGS6, RGS7, RGS9, and RGS11, share a common molecular organization of three modules: (i) the catalytic RGS domain, (ii) a GGL domain that recruits Gβ5, an outlying member of the G protein beta subunit family, and (iii) a DEP/DHEX domain that mediates interactions with the membrane anchor proteins R7BP and R9AP. As heterotrimeric complexes, R7 RGS proteins not only associate with and regulate a number of G protein signaling pathway components, but have also been found to form complexes with proteins that are not traditionally associated with G protein signaling. This review summarizes our current understanding of the biology of the R7 RGS complexes including their structure/functional organization, protein–protein interactions, and physiological roles.

RGS6/Gβ5 complex accelerates IKACh gating kinetics in atrial myocytes and modulates parasympathetic regulation of heart rate

Rationale: The parasympathetic reduction in heart rate involves the sequential activation of m2 muscarinic cholinergic receptors (m2Rs), pertussis toxin–sensitive (Gi/o) heterotrimeric G proteins, and the atrial potassium channel IKACh. Molecular mechanisms regulating this critical signal transduction pathway are not fully understood. Objective: To determine whether the G protein signaling regulator Rgs6/G&bgr;5 modulates m2R-IKACh signaling and cardiac physiology. Methods and Results: Cardiac expression of Rgs6, and its interaction with G&bgr;5, was demonstrated by immunoblotting and immunoprecipitation. Rgs6−/− mice were generated by gene targeting, and the cardiac effects of Rgs6 ablation were analyzed by whole-cell recordings in isolated cardiomyocytes and ECG telemetry. Loss of Rgs6 yielded profound delays in m2R-IKACh deactivation kinetics in both neonatal atrial myocytes and adult sinoatrial nodal cells. Rgs6−/− mice exhibited mild resting bradycardia and altered heart rate responses to pharmacological manipulations that were consistent with enhanced m2R-IKACh signaling. Conclusions: The cardiac Rgs6/G&bgr;5 complex modulates the timing of parasympathetic influence on atrial myocytes and heart rate in mice.

Expression and Localization of RGS9-2/Gβ5/R7BP Complex In Vivo Is Set by Dynamic Control of Its Constitutive Degradation by Cellular Cysteine Proteases

A member of regulator of G-protein signaling family, RGS9-2, is an essential modulator of signaling through neuronal dopamine and opioid G-protein-coupled receptors. Recent findings indicate that the abundance of RGS9-2 determines sensitivity of signaling in the locomotor and reward systems in the striatum. In this study we report the mechanism that sets the concentration of RGS9-2 in vivo, thus controlling G-protein signaling sensitivity in the region. We found that RGS9-2 possesses specific degradation determinants which target it for constitutive destruction by lysosomal cysteine proteases. Shielding of these determinants by the binding partner R7 binding-protein (R7BP) controls RGS9-2 expression at the posttranslational level. In addition, binding to R7BP in neurons targets RGS9-2 to the specific intracellular compartment, the postsynaptic density. Implementation of this mechanism throughout ontogenetic development ensures expression of RGS9-2/type 5 G-protein β subunit/R7BP complexes at postsynaptic sites in unison with increased signaling demands at mature synapses.

GPR158/179 regulate G protein signaling by controlling localization and activity of the RGS7 complexes.

Interaction of RGS proteins with orphan GPCRs promotes signaling compartmentalization and specificity.

TRPM1 Forms Complexes with Nyctalopin In Vivo and Accumulates in Postsynaptic Compartment of ON-Bipolar Neurons in mGluR6-Dependent Manner

Synaptic transmission between light-sensory photoreceptor cells and downstream ON-bipolar neurons plays an important role for vertebrate vision. This process is mediated by the G-protein-coupled receptor pathway involving glutamate receptor mGluR6 and effector channel TRPM1. The signal transmission occurs on a rapid timescale; however, the molecular organization that ensures timely signaling in this cascade is unknown. Genetic studies in human patients and animal models reveal that ON-bipolar cell signaling depends on the synaptic protein nyctalopin. We have conducted a proteomic search for proteins associated with nyctalopin in the mouse retina and identified TRPM1 as the binding partner. We further demonstrate that nyctalopin additionally interacts with mGluR6 receptor. Disruption of mGluR6 prevented targeting of TRPM1 to the postsynaptic compartment of ON-bipolar neurons. These results reveal a unique macromolecular organization of the mGluR6 cascade, where principal signaling components are scaffolded by nyctalopin, creating an organization essential for the correct localization of the signaling ensemble and ultimately intact transmission of the signal at the first visual synapse.

Essential Role of the m2R-RGS6-IKACh Pathway in Controlling Intrinsic Heart Rate Variability

Normal heart function requires generation of a regular rhythm by sinoatrial pacemaker cells and the alteration of this spontaneous heart rate by the autonomic input to match physiological demand. However, the molecular mechanisms that ensure consistent periodicity of cardiac contractions and fine tuning of this process by autonomic system are not completely understood. Here we examined the contribution of the m2R-IKACh intracellular signaling pathway, which mediates the negative chronotropic effect of parasympathetic stimulation, to the regulation of the cardiac pacemaking rhythm. Using isolated heart preparations and single-cell recordings we show that the m2R-IKACh signaling pathway controls the excitability and firing pattern of the sinoatrial cardiomyocytes and determines variability of cardiac rhythm in a manner independent from the autonomic input. Ablation of the major regulator of this pathway, Rgs6, in mice results in irregular cardiac rhythmicity and increases susceptibility to atrial fibrillation. We further identify several human subjects with variants in the RGS6 gene and show that the loss of function in RGS6 correlates with increased heart rate variability. These findings identify the essential role of the m2R-IKACh signaling pathway in the regulation of cardiac sinus rhythm and implicate RGS6 in arrhythmia pathogenesis.

Disruption of the Chaperonin Containing TCP-1 Function Affects Protein Networks Essential for Rod Outer Segment Morphogenesis and Survival

Type II Chaperonin Containing TCP-1 (CCT, also known as TCP-1 Ring Complex, TRiC) is a multi-subunit molecular machine thought to assist in the folding of ∼10% of newly translated cytosolic proteins in eukaryotes. A number of proteins folded by CCT have been identified in yeast and cultured mammalian cells, however, the function of this chaperonin in vivo has never been addressed. Here we demonstrate that suppressing the CCT activity in mouse photoreceptors by transgenic expression of a dominant-negative mutant of the CCT cofactor, phosducin-like protein (PhLP), results in the malformation of the outer segment, a cellular compartment responsible for light detection, and triggers rapid retinal degeneration. Investigation of the underlying causes by quantitative proteomics identified distinct protein networks, encompassing ∼200 proteins, which were significantly affected by the chaperonin deficiency. Notably among those were several essential proteins crucially engaged in structural support and visual signaling of the outer segment such as peripherin 2, Rom1, rhodopsin, transducin, and PDE6. These data for the first time demonstrate that normal CCT function is ultimately required for the morphogenesis and survival of sensory neurons of the retina, and suggest the chaperonin CCT deficiency as a potential, yet unexplored, cause of neurodegenerative diseases.

RGS6, but Not RGS4, Is the Dominant Regulator of G Protein Signaling (RGS) Modulator of the Parasympathetic Regulation of Mouse Heart Rate

Background: RGS4 and RGS6 are regulator of G protein signaling (RGS) proteins, and both have been proposed to modulate parasympathetic regulation of heart rate (HR). Results: RGS6 ablation enhances parasympathetic influence on the heart; RGS4 ablation does not. Conclusion: RGS6 is the primary RGS modulator of parasympathetic influence on the heart. Significance: Understanding the parasympathetic regulation of HR will improve treatment of arrhythmias. Parasympathetic activity decreases heart rate (HR) by inhibiting pacemaker cells in the sinoatrial node (SAN). Dysregulation of parasympathetic influence has been linked to sinus node dysfunction and arrhythmia. RGS (regulator of G protein signaling) proteins are negative modulators of the parasympathetic regulation of HR and the prototypical M2 muscarinic receptor (M2R)-dependent signaling pathway in the SAN that involves the muscarinic-gated atrial K+ channel IKACh. Both RGS4 and RGS6-Gβ5 have been implicated in these processes. Here, we used Rgs4−/−, Rgs6−/−, and Rgs4−/−:Rgs6−/− mice to compare the relative influence of RGS4 and RGS6 on parasympathetic regulation of HR and M2R-IKACh-dependent signaling in the SAN. In retrogradely perfused hearts, ablation of RGS6, but not RGS4, correlated with decreased resting HR, increased heart rate variability, and enhanced sensitivity to the negative chronotropic effects of the muscarinic agonist carbachol. Similarly, loss of RGS6, but not RGS4, correlated with enhanced sensitivity of the M2R-IKACh signaling pathway in SAN cells to carbachol and a significant slowing of M2R-IKACh deactivation rate. Surprisingly, concurrent genetic ablation of RGS4 partially rescued some deficits observed in Rgs6−/− mice. These findings, together with those from an acute pharmacologic approach in SAN cells from Rgs6−/− and Gβ5−/− mice, suggest that the partial rescue of phenotypes in Rgs4−/−:Rgs6−/− mice is attributable to another R7 RGS protein whose influence on M2R-IKACh signaling is masked by RGS4. Thus, RGS6-Gβ5, but not RGS4, is the primary RGS modulator of parasympathetic HR regulation and SAN M2R-IKACh signaling in mice.

Type 5 G Protein β Subunit (Gβ5) Controls the Interaction of Regulator of G Protein Signaling 9 (RGS9) with Membrane Anchors

The R7 family of regulators of G protein signaling (RGS) proteins, comprising RGS6, RGS7, RGS9, and RGS11, regulate neuronal G protein signaling pathways. All members of the R7 RGS form trimeric complexes with the atypical G protein β subunit, Gβ5, and membrane anchor R7BP or R9AP. Association with Gβ5 and membrane anchors has been shown to be critical for maintaining proteolytic stability of the R7 RGS proteins. However, despite its functional importance, the mechanism of how R7 RGS forms complexes with Gβ5 and membrane anchors remains poorly understood. Here, we used protein-protein interaction, co-localization, and protein stability assays to show that association of RGS9 with membrane anchors requires Gβ5. We further establish that the recruitment of R7BP to the complex requires an intact interface between the N-terminal lobe of RGS9 and protein interaction surface of Gβ5. Site-directed mutational analysis reveals that distinct molecular determinants in the interface between Gβ5 and N-terminal Dishevelled, EGL-10, Pleckstrin/DEP Helical Extension (DEP/DHEY) domains are differentially involved in R7BP binding and proteolytic stabilization. On the basis of these findings, we conclude that Gβ5 contributes to the formation of the binding site to the membrane anchors and thus is playing a central role in the assembly of the proteolytically stable trimeric complex and its correct localization in the cell.

Nuclear localization of the G protein β5/R7–regulator of G protein signaling protein complex is dependent on R7 binding protein

J. Neurochem. (2010) 113, 1101–1112.