The association of GNB5 with Alzheimer disease revealed by genomic analysis restricted to variants impacting gene function.

Disease-associated variants identified from genome-wide association studies (GWASs) frequently map to non-coding areas of the genome such as introns and intergenic regions. An exclusive reliance on gene-agnostic methods of genomic investigation could limit the identification of relevant genes associated with polygenic diseases such as Alzheimer disease (AD). To overcome such potential restriction, we developed a gene-constrained analytical method that considers only moderate- and high-risk variants that affect gene coding sequences. We report here the application of this approach to publicly available datasets containing 181,388 individuals without and with AD and the resulting identification of 660 genes potentially linked to the higher AD prevalence among Africans/African Americans. By integration with transcriptome analysis of 23 brain regions from 2,728 AD case-control samples, we concentrated on nine genes that potentially enhance the risk of AD: AACS, GNB5, GNS, HIPK3, MED13, SHC2, SLC22A5, VPS35, and ZNF398. GNB5, the fifth member of the heterotrimeric G protein beta family encoding Gß5, is primarily expressed in neurons and is essential for normal neuronal development in mouse brain. Homozygous or compound heterozygous loss of function of GNB5 in humans has previously been associated with a syndrome of developmental delay, cognitive impairment, and cardiac arrhythmia. In validation experiments, we confirmed that Gnb5 heterozygosity enhanced the formation of both amyloid plaques and neurofibrillary tangles in the brains of AD model mice. These results suggest that gene-constrained analysis can complement the power of GWASs in the identification of AD-associated genes and may be more broadly applicable to other polygenic diseases.

A RGS7-CaMKII complex drives myocyte-intrinsic and myocyte-extrinsic mechanisms of chemotherapy-induced cardiotoxicity.

Dose-limiting cardiotoxicity remains a major limitation in the clinical use of cancer chemotherapeutics. Here, we describe a role for Regulator of G protein Signaling 7 (RGS7) in chemotherapy-dependent heart damage, the demonstration for a functional role of RGS7 outside of the nervous system and retina. Though expressed at low levels basally, we observed robust up-regulation of RGS7 in the human and murine myocardium following chemotherapy exposure. In ventricular cardiomyocytes (VCM), RGS7 forms a complex with Ca2+/calmodulin-dependent protein kinase (CaMKII) supported by key residues (K412 and P391) in the RGS domain of RGS7. In VCM treated with chemotherapeutic drugs, RGS7 facilitates CaMKII oxidation and phosphorylation and CaMKII-dependent oxidative stress, mitochondrial dysfunction, and apoptosis. Cardiac-specific RGS7 knockdown protected the heart against chemotherapy-dependent oxidative stress, fibrosis, and myocyte loss and improved left ventricular function in mice treated with doxorubicin. Conversely, RGS7 overexpression induced fibrosis, reactive oxygen species generation, and cell death in the murine myocardium that were mitigated following CaMKII inhibition. RGS7 also drives production and release of the cardiokine neuregulin-1, which facilitates paracrine communication between VCM and neighboring vascular endothelial cells (EC), a maladaptive mechanism contributing to VCM dysfunction in the failing heart. Importantly, while RGS7 was both necessary and sufficient to facilitate chemotherapy-dependent cytotoxicity in VCM, RGS7 is dispensable for the cancer-killing actions of these same drugs. These selective myocyte-intrinsic and myocyte-extrinsic actions of RGS7 in heart identify RGS7 as an attractive therapeutic target in the mitigation of chemotherapy-driven cardiotoxicity.

Two regulators of G-protein signaling (RGS) proteins FlbA1 and FlbA2 differentially regulate fumonisin B1 biosynthesis in Fusarium verticillioides.

Fumonisins are a group of mycotoxins produced by maize pathogen Fusarium verticillioides that pose health concerns to humans and animals. Yet we still lack a clear understanding of the mechanism of fumonisins regulation during pathogenesis. The heterotrimeric G protein complex, which consists of canonical subunits and various regulators of G-protein signaling (RGS) proteins, plays an important role in transducing signals under environmental stress. Earlier studies demonstrated that Gα and Gß subunits are positive regulators of fumonisin B1 (FB1) biosynthesis and that two RGS genes, FvFlbA1 and FvFlbA2, were highly upregulated in Gß deletion mutant ∆Fvgbb1. Notably, FvFlbA2 has a negative role in FB1 regulation. While many fungi contain a single copy of FlbA, F. verticillioides harbors two putative FvFlbA paralogs, FvFlbA1 and FvFlbA2. In this study, we further characterized functional roles of FvFlbA1 and FvFlbA2. While ∆FvflbA1 deletion mutant exhibited no significant defects, ∆FvflbA2 and ∆FvflbA2/A1 mutants showed thinner aerial hyphal growth while promoting FB1 production. FvFlbA2 is required for proper expression of key conidia regulation genes, including putative FvBRLA, FvWETA, and FvABAA, while suppressing FUM21, FUM1, and FUM8 expression. Split luciferase assays determined that FvFlbA paralogs interact with key heterotrimeric G protein components, which in turn will lead altered G-protein-mediated signaling pathways that regulate FB1 production and asexual development in F. verticillioides.

RGS14 Regulation of Post-Synaptic Signaling and Spine Plasticity in Brain.

The regulator of G-protein signaling 14 (RGS14) is a multifunctional signaling protein that regulates post synaptic plasticity in neurons. RGS14 is expressed in the brain regions essential for learning, memory, emotion, and stimulus-induced behaviors, including the basal ganglia, limbic system, and cortex. Behaviorally, RGS14 regulates spatial and object memory, female-specific responses to cued fear conditioning, and environmental- and psychostimulant-induced locomotion. At the cellular level, RGS14 acts as a scaffolding protein that integrates G protein, Ras/ERK, and calcium/calmodulin signaling pathways essential for spine plasticity and cell signaling, allowing RGS14 to naturally suppress long-term potentiation (LTP) and structural plasticity in hippocampal area CA2 pyramidal cells. Recent proteomics findings indicate that RGS14 also engages the actomyosin system in the brain, perhaps to impact spine morphogenesis. Of note, RGS14 is also a nucleocytoplasmic shuttling protein, where its role in the nucleus remains uncertain. Balanced nuclear import/export and dendritic spine localization are likely essential for RGS14 neuronal functions as a regulator of synaptic plasticity. Supporting this idea, human genetic variants disrupting RGS14 localization also disrupt RGS14's effects on plasticity. This review will focus on the known and unexplored roles of RGS14 in cell signaling, physiology, disease and behavior.

Temporal control over the initiation of cell motility by a regulator of G-protein signaling.

The control over the acquisition of cell motility is central for a variety of biological processes in development, homeostasis, and disease. An attractive in vivo model for investigating the regulation of migration initiation is that of primordial germ cells (PGCs) in zebrafish embryos. In this study, we show that, following PGC specification, the cells can polarize but do not migrate before the time chemokine-encoded directional cues are established. We found that the regulator of G-protein signaling 14a protein, whose RNA is a newly identified germ plasm component, regulates the temporal relations between the appearance of the guidance molecules and the acquisition of cellular motility by regulating E-cadherin levels.

Potent inhibition of angiotensin AT1 receptor signaling by RGS8: importance of the C-terminal third exon part of its RGS domain.

R4/B subfamily RGS (regulator of G protein signaling) proteins play roles in regulation of many GPCR-mediated responses. Multiple RGS proteins are usually expressed in a cell, and it is difficult to point out which RGS protein species are functionally important in the cell. To evaluate intrinsic potency of these RGS proteins, we compared inhibitory effects of RGS1, RGS2, RGS3, RGS4, RGS5, RGS8 and RGS16 on AT1 receptor signaling. Intracellular Ca(2+) responses to angiotensin II were markedly attenuated by transiently expressed RGS2, RGS3 and RGS8, compared to weak inhibition by RGS1, RGS4, RGS5 and RGS16. N-terminally deleted RGS2 (RGS2 domain) lost this potent inhibitory effect, whereas RGS domains of RGS3 and RGS8 showed strong inhibition similar to those of the full-length proteins. To investigate key determinants that specify the differences in potency, we constructed chimeric domains by replacing one or two of three exon parts of RGS8 domain with the corresponding part of RGS5. The chimeric RGS8 domains containing the first or the second exon part of RGS5 showed strong inhibitory effects similar to that of wild type RGS8, but the chimeric domain with the third exon part of RGS5 lost its activity. On the contrary, replacement of the third exon part of RGS5 with the corresponding residues of RGS8 increased the inhibitory effect. The role of the third exon part of RGS8 domain was further confirmed with the chimeric RGS8/RGS4 domains. These results indicate the potent inhibitory activity of RGS8 among R4/B subfamily proteins and importance of the third exon.

Dissecting the molecular basis for the modulation of neurotransmitter GPCR signaling by GINIP.

It is well established that G-protein-coupled receptors (GPCRs) stimulated by neurotransmitters are critical for neuromodulation. Much less is known about how heterotrimeric G-protein (Gαßγ) regulation after receptor-mediated activation contributes to neuromodulation. Recent evidence indicates that the neuronal protein GINIP shapes GPCR inhibitory neuromodulation via a unique mechanism of G-protein regulation that controls pain and seizure susceptibility. However, the molecular basis of this mechanism remains ill-defined because the structural determinants of GINIP responsible for binding and regulating G proteins are not known. Here, we combined hydrogen-deuterium exchange mass spectrometry, computational structure predictions, biochemistry, and cell-based biophysical assays to demonstrate an effector-like binding mode of GINIP to Gαi. Specific amino acids of GINIP's PHD domain first loop are essential for G-protein binding and subsequent regulation of Gαi-GTP and Gßγ signaling upon neurotransmitter GPCR stimulation. In summary, these findings shed light onto the molecular basis for a post-receptor mechanism of G-protein regulation that fine-tunes inhibitory neuromodulation.

Common and selective signal transduction mechanisms of GPCRs.

G protein-coupled receptors (GPCRs) are coupled by four major subfamilies of G proteins. GPCR coupling is processed through a combination of common and selective activation mechanisms together. Common mechanisms are shared for a group of receptors. Recently, researchers managed to identify shared activation pathways for the GPCRs belonging to the same subfamilies. On the other hand, selective mechanisms are responsible for the variations within activation mechanisms. Selective processes can regulate subfamily-specific interactions between the receptor and the G proteins, and intermediate receptor conformations are required to couple particular G proteins through G protein-specific activation mechanisms. Moreover, G proteins can also selectively interact with RGS (regulators of G protein signaling) proteins as well. Selective processes modulate the signaling profile of the receptor and the tissue they are present. This chapter summarizes the recent research conducted on common and selective signal transduction mechanisms within GPCRs from an evolutionary perspective.

Unrestrained Gαi2 Signaling Disrupts Neutrophil Trafficking, Aging, and Clearance.

Neutrophil trafficking, homeostatic and pathogen elicited, depends upon chemoattractant receptors triggering heterotrimeric G-protein Gαißγ signaling, whose magnitude and kinetics are governed by RGS protein/Gαi interactions. RGS proteins typically limit Gαi signaling by reducing the duration that Gαi subunits remain GTP bound and able to activate downstream effectors. Yet how in totality RGS proteins shape neutrophil chemoattractant receptor activated responses remains unclear. Here, we show that C57Bl/6 mouse neutrophils containing a genomic knock-in of a mutation that disables all RGS protein-Gαi2 interactions (G184S) cannot properly balance chemoattractant receptor signaling, nor appropriately respond to inflammatory insults. Mutant neutrophils accumulate in mouse bone marrow, spleen, lung, and liver; despite neutropenia and an intrinsic inability to properly mobilize from the bone marrow. In vitro they rapidly adhere to ICAM-1 coated plates, but in vivo they poorly adhere to blood vessel endothelium. Those few neutrophils that cross blood vessels and enter tissues migrate haphazardly. Following Concanavalin-A administration fragmented G184S neutrophils accumulate in liver sinusoids leading to thrombo-inflammation and perivasculitis. Thus, neutrophil Gαi2/RGS protein interactions both limit and facilitate Gαi2 signaling thereby promoting normal neutrophil trafficking, aging, and clearance.

Emerging themes in heterotrimeric G-protein signaling in plants.

Heterotrimeric G-proteins are key signaling components involved during the regulation of a multitude of growth and developmental pathways in all eukaryotes. Although the core proteins (Gα, Gß, Gγ subunits) and their basic biochemistries are conserved between plants and non-plant systems, seemingly different inherent properties of specific components, altered wirings of G-protein network architectures, and the presence of novel receptors and effector proteins make plant G-protein signaling mechanisms somewhat distinct from the well-established animal paradigm. G-protein research in plants is getting a lot of attention recently due to the emerging roles of these proteins in controlling many agronomically important traits. New findings on both canonical and novel G-protein components and their conserved and unique signaling mechanisms are expected to improve our understanding of this important module in affecting critical plant growth and development pathways and eventually their utilization to produce plants for the future needs. In this review, we briefly summarize what is currently known in plant G-protein research, describe new findings and how they are changing our perceptions of the field, and discuss important issues that still need to be addressed.

RGS6 as a Novel Therapeutic Target in CNS Diseases and Cancer.

Regulator of G protein signaling (RGS) proteins are gatekeepers regulating the cellular responses induced by G protein-coupled receptor (GPCR)-mediated activation of heterotrimeric G proteins. Specifically, RGS proteins determine the magnitude and duration of GPCR signaling by acting as a GTPase-activating protein for Gα subunits, an activity facilitated by their semiconserved RGS domain. The R7 subfamily of RGS proteins is distinguished by two unique domains, DEP/DHEX and GGL, which mediate membrane targeting and stability of these proteins. RGS6, a member of the R7 subfamily, has been shown to specifically modulate Gαi/o protein activity which is critically important in the central nervous system (CNS) for neuronal responses to a wide array of neurotransmitters. As such, RGS6 has been implicated in several CNS pathologies associated with altered neurotransmission, including the following: alcoholism, anxiety/depression, and Parkinson's disease. In addition, unlike other members of the R7 subfamily, RGS6 has been shown to regulate G protein-independent signaling mechanisms which appear to promote both apoptotic and growth-suppressive pathways that are important in its tumor suppressor function in breast and possibly other tissues. Further highlighting the importance of RGS6 as a target in cancer, RGS6 mediates the chemotherapeutic actions of doxorubicin and blocks reticular activating system (Ras)-induced cellular transformation by promoting degradation of DNA (cytosine-5)-methyltransferase 1 (DNMT1) to prevent its silencing of pro-apoptotic and tumor suppressor genes. Together, these findings demonstrate the critical role of RGS6 in regulating both G protein-dependent CNS pathology and G protein-independent cancer pathology implicating RGS6 as a novel therapeutic target.

RGS14 regulates the lifetime of Gα-GTP signaling but does not prolong Gßγ signaling following receptor activation in live cells.

RGS14 is a multifunctional scaffolding protein possessing two distinct G protein interaction sites including a regulator of G protein signaling (RGS) domain that acts as a GTPase activating protein (GAP) to deactivate Gαi/o-GTP proteins, and a G protein regulatory (GPR) motif that binds inactive Gαi1/3-GDP proteins independent of Gßγ. GPR interactions with Gαi recruit RGS14 to the plasma membrane to interact with Gαi-linked GPCRs and regulate Gαi signaling. While RGS14 actions on Gα proteins are well characterized, consequent effects on Gßγ signaling remain unknown. Conventional RGS proteins act as dedicated GAPs to deactivate Gα and Gßγ signaling following receptor activation. RGS14 may do the same or, alternatively, may coordinate its actions to deactivate Gα-GTP with the RGS domain and then capture the same Gα-GDP via its GPR motif to prevent heterotrimer reassociation and prolong Gßγ signaling. To test this idea, we compared the regulation of G protein activation and deactivation kinetics by a conventional RGS protein, RGS4, and RGS14 in response to GPCR agonist/antagonist treatment utilizing bioluminescence resonance energy transfer (BRET). Co-expression of either RGS4 or RGS14 inhibited the release of free Gßγ after agonist stimulation and increased the deactivation rate of Gα, consistent with their roles as GTPase activating proteins (GAPs). Overexpression of inactive Gαi1 to recruit RGS14 to the plasma membrane did not alter RGS14's capacity to act as a GAP for a second Gαo protein. These results demonstrate the role of RGS14 as a dedicated GAP and suggest that the G protein regulatory (GPR) motif functions independently of the RGS domain and is silent in regulating GAP activity in a cellular context.

Decreased RGS6 expression is associated with poor prognosis in pancreatic cancer patients.

Regulator of G-protein signaling 6 (RGS6), a member of a family of RGS proteins, has been reported to involve in multiple processes during tumor development. However, its role in pancreatic cancer has not been studied yet. In this study, we aimed to investigate the expression of RGS6 in pancreatic cancer and its role in predicting outcomes of patients with pancreatic cancer. We first measured the expression of RGS6 mRNA in 20 cases of tumor tissues and matched adjacent non-tumorous tissues by quantitative real-time PCR and examined RGS6 protein by immunohistochemistry in tissue microarrays containing 90 tumor and 90 paired adjacent non-tumor tissues. Decreased RGS6 mRNA detected in primary tumor, compared with their non-tumor counterparts. In addition, decreased RGS6 protein expression was associated with tumor differentiation (P = 0.027), pT classification (P = 0.034), smoking status (P = 0.041) and a poor survival (P = 0.007). Cox proportional hazards regression modeling analysis revealed that lymph node metastasis (P = 0.001; hazard ratio, 2.347, 95% CI, 1.387-3.972), tumor differentiation (P = 0.015; hazard ratio, 0.505, 95% CI, 0.291-0.876) and RGS6 expression (P = 0.048; hazard ratio, 0.567, 95% CI, 0.324-0.994) were three independent prognostic factors. Taken together, these date demonstrate that RGS6 decreases in tumor tissue and may serve as a novel biomarker for outcomes in pancreatic cancer patients and be a potential therapeutic target potential therapeutic target.

The eIF2B-interacting domain of RGS2 protects against GPCR agonist-induced hypertrophy in neonatal rat cardiomyocytes.

The protective effect of Regulator of G protein Signaling 2 (RGS2) in cardiac hypertrophy is thought to occur through its ability to inhibit the chronic GPCR signaling that promotes pathogenic growth both in vivo and in cultured cardiomyocytes. However, RGS2 is known to have additional functions beyond its activity as a GTPase accelerating protein, such as the ability to bind to eukaryotic initiation factor, eIF2B, and inhibit protein synthesis. The RGS2 eIF2B-interacting domain (RGS2(eb)) was examined for its ability to regulate hypertrophy in neonatal ventricular myocytes. Both full-length RGS2 and RGS2(eb) were able to inhibit agonist-induced cardiomyocyte hypertrophy, but RGS2(eb) had no effect on receptor-mediated inositol phosphate production, cAMP production, or ERK 1/2 activation. These results suggest that the protective effects of RGS2 in cardiac hypertrophy may derive at least in part from its ability to govern protein synthesis.

Controlling Parasympathetic Regulation of Heart Rate: A Gatekeeper Role for RGS Proteins in the Sinoatrial Node.

Neurotransmitters released from sympathetic and parasympathetic nerve terminals in the sinoatrial node (SAN) exert their effects via G-protein-coupled receptors. Integration of these different G-protein signals within pacemaker cells of the SAN is critical for proper regulation of heart rate and function. For example, excessive parasympathetic signaling can be associated with sinus node dysfunction (SND) and supraventricular arrhythmias. Our previous work has shown that one member of the regulator of G-protein signaling (RGS) protein family, RGS4, is highly and selectively expressed in pacemaker cells of the SAN. Consistent with its role as an inhibitor of parasympathetic signaling, RGS4-knockout mice have reduced basal heart rates and enhanced negative chronotropic responses to parasympathetic agonists. Moreover, RGS4 appears to be an important part of SA nodal myocyte signaling pathways that mediate G-protein-coupled inwardly rectifying potassium channel (GIRK) channel activation/deactivation and desensitization. Since RGS4 acts immediately downstream of M2 muscarinic receptors, it is tempting to speculate that RGS4 functions as a master regulator of parasympathetic signaling upstream of GIRKs, HCNs, and L-type Ca(2+) channels in the SAN. Thus, loss of RGS4 function may lead to increased susceptibility to conditions associated with increased parasympathetic signaling, including bradyarrhythmia, SND, and atrial fibrillation.

A sweet cycle for Arabidopsis G-proteins: Recent discoveries and controversies in plant G-protein signal transduction.

Heterotrimeric G-proteins are a class of signal transduction proteins highly conserved throughout evolution that serve as dynamic molecular switches regulating the intracellular communication initiated by extracellular signals including sensory information. This property is achieved by a guanine nucleotide cycle wherein the inactive, signaling-incompetent Galpha subunit is normally bound to GDP; activation to signaling-competent Galpha occurs through the exchange of GDP for GTP (typically catalyzed via seven-transmembrane domain G-protein coupled receptors [GPCRs]), which dissociates the Gbetagamma dimer from Galpha-GTP and initiates signal transduction. The hydrolysis of GTP, greatly accelerated by "Regulator of G-protein Signaling" (RGS) proteins, returns Galpha to its inactive GDP-bound form and terminates signaling. Through extensive characterization of mammalian Galpha isoforms, the rate-limiting step in this cycle is currently considered to be the GDP/GTP exchange rate, which can be orders of magnitude slower than the GTP hydrolysis rate. However, we have recently demonstrated that, in Arabidopsis, the guanine nucleotide cycle appears to be limited by the rate of GTP hydrolysis rather than nucleotide exchange. This finding has important implications for the mechanism of sugar sensing in Arabidopsis. We also discuss these data on Arabidopsis G-protein nucleotide cycling in relation to recent reports of putative plant GPCRs and heterotrimeric G-protein effectors in Arabidopsis.