Regulators Of G Protein Signaling Proteins Research Articles

Structure-function analysis of plant G-protein regulatory mechanisms identifies key Gα-RGS protein interactions

Heterotrimeric GTP-binding protein alpha subunit (Gα) and its cognate regulator of G-protein signaling (RGS) protein transduce signals in eukaryotes spanning protists, amoeba, animals, fungi, and plants. The core catalytic mechanisms of the GTPase activity of Gα and the interaction interface with RGS for the acceleration of GTP hydrolysis seem to be conserved across these groups; however, the RGS gene is under low selective pressure in plants, resulting in its frequent loss. Our current understanding of the structural basis of Gα:RGS regulation in plants has been shaped by Arabidopsis Gα, (AtGPA1), which has a cognate RGS protein. To gain a comprehensive understanding of this regulation beyond Arabidopsis, we obtained the x-ray crystal structures of Oryza sativa Gα, which has no RGS, and Selaginella moellendorffi (a lycophyte) Gα that has low sequence similarity with AtGPA1 but has an RGS. We show that the three-dimensional structure, protein-protein interaction with RGS, and the dynamic features of these Gα are similar to AtGPA1 and metazoan Gα. Molecular dynamic simulation of the Gα-RGS interaction identifies the contacts established by specific residues of the switch regions of GTP-bound Gα, crucial for this interaction, but finds no significant difference due to specific amino acid substitutions. Together, our data provide valuable insights into the regulatory mechanisms of plant G-proteins but do not support the hypothesis of adaptive co-evolution of Gα:RGS proteins in plants.

A regulatory node involving Gαq, PLCβ, and RGS proteins modulates platelet reactivity to critical agonists

BackgroundMost platelet agonists work through G protein-coupled receptors, activating pathways that involve members of the Gq, Gi, and G12/G13 families of heterotrimeric G proteins. Gq signaling has been shown to be critical for efficient platelet activation. Growing evidence suggests that regulatory mechanisms converge on G protein-coupled receptors and Gq to prevent overly robust platelet reactivity. ObjectivesTo identify and characterize mechanisms by which Gq signaling is regulated in platelets. MethodsBased on our prior experience with a Gαi2 variant that escapes regulation by regulator of G protein signaling (RGS) proteins, a Gαq variant was designed with glycine 188 replaced with serine (G188S) and then incorporated into a mouse line so that its effects on platelet activation and thrombus formation could be studied in vitro and in vivo. Results and ConclusionsAs predicted, the G188S substitution in Gαq disrupted its interaction with RGS18. Unexpectedly, it also uncoupled PLCβ-3 from activation by platelet agonists as evidenced by a loss rather than a gain of platelet function in vitro and in vivo. Binding studies showed that in addition to preventing the binding of RGS18 to Gαq, the G188S substitution also prevented the binding of PLCβ-3 to Gαq. Structural analysis revealed that G188 resides in the region that is also important for Gαq binding to PLCβ-3 in platelets. We conclude that the Gαq signaling node is more complex than that has been previously understood, suggesting that there is cross-talk between RGS proteins and PLCβ-3 in the context of Gαq signaling.

The roles of RGS proteins in cardiometabolic disease.

G protein-coupled receptors (GPCRs) are the most prominent receptors on the surface of the cell and play a central role in the regulation of cardiac and metabolic functions. GPCRs transmit extracellular stimuli to the interior of the cells by activating one or more heterotrimeric G proteins. The duration and intensity of G protein-mediated signalling are tightly controlled by a large array of intracellular mediators, including regulators of G protein signalling (RGS) proteins. RGS proteins selectively promote the GTPase activity of a subset of Gα subunits, thus serving as negative regulators of G protein signalling in a pathway-dependent manner. In the current review, we summarise the involvement of RGS proteins in cardiometabolic function with a focus on their tissue distribution, mechanisms of action and dysregulation under various disease conditions. We also discuss the potential therapeutic applications for targeting RGS proteins in treating cardiometabolic conditions and current progress in developing RGS modulators.

Mechanisms of mGluR-dependent plasticity in hippocampal area CA2.

Pyramidal cells in hippocampal area CA2 have synaptic properties that are distinct from the other CA subregions. Notably, this includes a lack of typical long-term potentiation of stratum radiatum synapses. CA2 neurons express high levels of several known and potential regulators of metabotropic glutamate receptor (mGluR)-dependent signaling including Striatal-Enriched Tyrosine Phosphatase (STEP) and several Regulator of G-protein Signaling (RGS) proteins, yet the functions of these proteins in regulating mGluR-dependent synaptic plasticity in CA2 are completely unknown. Thus, the aim of this study was to examine mGluR-dependent synaptic depression and to determine whether STEP and the RGS proteins RGS4 and RGS14 are involved. Using whole cell voltage-clamp recordings from mouse pyramidal cells, we found that mGluR agonist-induced long-term depression (mGluR-LTD) is more pronounced in CA2 compared with that observed in CA1. This mGluR-LTD in CA2 was found to be protein synthesis and STEP dependent, suggesting that CA2 mGluR-LTD shares mechanistic processes with those seen in CA1, but in addition, RGS14, but not RGS4, was essential for mGluR-LTD in CA2. In addition, we found that exogenous application of STEP could rescue mGluR-LTD in RGS14 KO slices. Supporting a role for CA2 synaptic plasticity in social cognition, we found that RGS14 KO mice had impaired social recognition memory as assessed in a social discrimination task. These results highlight possible roles for mGluRs, RGS14, and STEP in CA2-dependent behaviors, perhaps by biasing the dominant form of synaptic plasticity away from LTP and toward LTD in CA2.

Cardiac RGS Proteins in Human Heart Failure and Atrial Fibrillation: Focus on RGS4.

The regulator of G protein signaling (RGS) proteins are crucial for the termination of G protein signals elicited by G protein-coupled receptors (GPCRs). This superfamily of cell membrane receptors, by far the largest and most versatile in mammals, including humans, play pivotal roles in the regulation of cardiac function and homeostasis. Perturbations in both the activation and termination of their G protein-mediated signaling underlie numerous heart pathologies, including heart failure (HF) and atrial fibrillation (AFib). Therefore, RGS proteins play important roles in the pathophysiology of these two devasting cardiac diseases, and several of them could be targeted therapeutically. Although close to 40 human RGS proteins have been identified, each RGS protein seems to interact only with a specific set of G protein subunits and GPCR types/subtypes in any given tissue or cell type. Numerous in vitro and in vivo studies in animal models, and also in diseased human heart tissue obtained from transplantations or tissue banks, have provided substantial evidence of the roles various cardiomyocyte RGS proteins play in cardiac normal homeostasis as well as pathophysiology. One RGS protein in particular, RGS4, has been reported in what are now decades-old studies to be selectively upregulated in human HF. It has also been implicated in protection against AFib via knockout mice studies. This review summarizes the current understanding of the functional roles of cardiac RGS proteins and their implications for the treatment of HF and AFib, with a specific focus on RGS4 for the aforementioned reasons but also because it can be targeted successfully with small organic molecule inhibitors.

The therapeutic potential of targeting cardiac RGS4.

G protein-coupled receptors (GPCRs) play pivotal roles in regulation of cardiac function and homeostasis. To function properly, every cell needs these receptors to be stimulated only when a specific extracellular stimulus is present, and to be silenced the moment that stimulus is removed. The regulator of G protein signaling (RGS) proteins are crucial for the latter to occur at the cell membrane, where the GPCR normally resides. Perturbations in both activation and termination of G protein signaling underlie numerous heart pathologies. Although more than 30 mammalian RGS proteins have been identified, each RGS protein seems to interact only with a specific set of G protein subunits and GPCR types/subtypes in any given tissue or cell type, and this applies to the myocardium as well. A large number of studies have provided substantial evidence for the roles various RGS proteins expressed in cardiomyocytes play in cardiac physiology and heart disease pathophysiology. This review summarizes the current understanding of the functional roles of cardiac RGS proteins and their implications for the treatment of specific heart diseases, such as heart failure and atrial fibrillation. We focus on cardiac RGS4 in particular, since this isoform appears to be selectively (among the RGS protein family) upregulated in human heart failure and is also the target of ongoing drug discovery efforts for the treatment of a variety of diseases.

Functional Assessment of Cancer-Linked Mutations in Sensitive Regions of Regulators of G Protein Signaling Predicted by Three-Dimensional Missense Tolerance Ratio Analysis.

Regulators of G protein signaling (RGS) proteins modulate G protein-coupled receptor (GPCR) signaling by acting as negative regulators of G proteins. Genetic variants in RGS proteins are associated with many diseases, including cancers, although the impact of these mutations on protein function is uncertain. Here we analyze the RGS domains of 15 RGS protein family members using a novel bioinformatic tool that measures the missense tolerance ratio (MTR) using a three-dimensional (3D) structure (3DMTR). Subsequent permutation analysis can define the protein regions that are most significantly intolerant (P < 0.05) in each dataset. We further focused on RGS14, RGS10, and RGS4. RGS14 exhibited seven significantly tolerant and seven significantly intolerant residues, RGS10 had six intolerant residues, and RGS4 had eight tolerant and six intolerant residues. Intolerant and tolerant-control residues that overlap with pathogenic cancer mutations reported in the COSMIC cancer database were selected to define the functional phenotype. Using complimentary cellular and biochemical approaches, proteins were tested for effects on GPCR-Gα activation, Gα binding properties, and downstream cAMP levels. Identified intolerant residues with reported cancer-linked mutations RGS14-R173C/H and RGS4-K125Q/E126K, and tolerant RGS14-S127P and RGS10-S64T resulted in a loss-of-function phenotype in GPCR-G protein signaling activity. In downstream cAMP measurement, tolerant RGS14-D137Y and RGS10-S64T and intolerant RGS10-K89M resulted in change of function phenotypes. These findings show that 3DMTR identified intolerant residues that overlap with cancer-linked mutations cause phenotypic changes that negatively impact GPCR-G protein signaling and suggests that 3DMTR is a potentially useful bioinformatics tool for predicting functionally important protein residues. SIGNIFICANCE STATEMENT: Human genetic variant/mutation information has expanded rapidly in recent years, including cancer-linked mutations in regulator of G protein signaling (RGS) proteins. However, experimental testing of the impact of this vast catalogue of mutations on protein function is not feasible. We used the novel bioinformatics tool three-dimensional missense tolerance ratio (3DMTR) to define regions of genetic intolerance in RGS proteins and prioritize which cancer-linked mutants to test. We found that 3DMTR more accurately classifies loss-of-function mutations in RGS proteins than other databases thereby offering a valuable new research tool.

Functions of regulators of G protein signaling 16 in immunity, inflammation, and other diseases.

Regulators of G protein signaling (RGS) act as guanosine triphosphatase activating proteins to accelerate guanosine triphosphate hydrolysis of the G protein α subunit, leading to the termination of the G protein-coupled receptor (GPCR) downstream signaling pathway. RGS16, which is expressed in a number of cells and tissues, belongs to one of the small B/R4 subfamilies of RGS proteins and consists of a conserved RGS structural domain with short, disordered amino- and carboxy-terminal extensions and an α-helix that classically binds and de-activates heterotrimeric G proteins. However, with the deepening of research, it has been revealed that RGS16 protein not only regulates the classical GPCR pathway, but also affects immune, inflammatory, tumor and metabolic processes through other signaling pathways including the mitogen-activated protein kinase, phosphoinositide 3-kinase/protein kinase B, Ras homolog family member A and stromal cell-derived factor 1/C-X-C motif chemokine receptor 4 pathways. Additionally, the RGS16 protein may be involved in the Hepatitis B Virus -induced inflammatory response. Therefore, given the continuous expansion of knowledge regarding its role and mechanism, the structure, characteristics, regulatory mechanisms and known functions of the small RGS proteinRGS16 are reviewed in this paper to prepare for diagnosis, treatment, and prognostic evaluation of different diseases such as inflammation, tumor, and metabolic disorders and to better study its function in other diseases.

Phylogeny of Regulators of G-Protein Signaling Genes in Leptographium qinlingensis and Expression Levels of Three RGSs in Response to Different Terpenoids.

Leptographium qinlingensis is a bark beetle-vectored pine pathogen in the Chinese white pine beetle (Dendroctonus armandi) epidemic in Northwest China. L. qinlingensis colonizes pines despite the trees’ massive oleoresin terpenoid defenses. Regulators of G-protein signaling (RGS) proteins modulate heterotrimeric G-protein signaling negatively and play multiple roles in the growth, asexual development, and pathogenicity of fungi. In this study, we have identified three L. qinlingensis RGS genes, and the phylogenetic analysis shows the highest homology with the regulators of G-protein signaling proteins sequence from Ophiostoma piceae and Grosmannia clavigera. The expression profiles of three RGSs in the mycelium of L. qinlingensis treated with six different terpenoids were detected, as well as their growth rates. Under six terpenoid treatments, the growth and reproduction in L. qinlingensis were significantly inhibited, and the growth inflection day was delayed from 8 days to 12–13 days. By analyzing the expression level of three RGS genes of L. qinlingensis with different treatments, results indicate that LqFlbA plays a crucial role in controlling fungal growth, and both LqRax1 and LqRgsA are involved in overcoming the host chemical resistances and successful colonization.

Regulator of G Protein Signaling 20 Correlates with Long Intergenic Non-Coding RNA (lincRNAs) Harboring Oncogenic Potential and Is Markedly Upregulated in Hepatocellular Carcinoma.

Simple SummaryClinical and molecular advances have improved knowledge and treatment prospects for cancer, yet hepatocellular carcinoma (HCC), the most common form of liver cancer, still ranks significantly higher in terms of the global cancer burden. Herein, we investigated the role of RGS20 as a potential prognostic marker in 28 different cancers with a particular focus on HCC.Hepatocellular carcinoma (HCC) is at the forefront of the global cancer burden, and biomarkers for HCC are constantly being sought. Interestingly, RGS (Regulators of G protein signaling) proteins, which negatively regulate GPCR signaling, have been associated with various cancers, with some members of the RGS family being associated with liver cancer as well. Considering this, we investigated the role of RGS20 as a potential prognostic marker in 28 different cancer types with special emphasis on HCC. By using the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) data, our analysis revealed that (a) RGS20 was strongly upregulated in tumor tissue compared with adjacent normal tissue of HCC patients; (b) RGS20 was strongly associated with some important clinical parameters such as alpha-fetoprotein and tumor grade in the HCC patients; (c) besides HCC (p < 0.001), RGS20 was found to be an important factor for survival in four other cancers (clear renal cell carcinoma: p < 0.001, lung adenocarcinoma: p = 0.004, mesothelioma: p = 0.039, ovarian serous cystadenocarcinoma: p = 0.048); (d) RGS20 was found to be significantly associated with some tumor-related signaling pathways and long intergenic non-coding RNAs (lincRNAs: LINC00511, PVT1, MIR4435-2HG, BCYRN1, and MAPKAPK5-AS1) that exhibit oncogenic potential. Taken together, we showed that RGS20 correlates with a few HCC-associated lincRNAs harboring oncogenic potential and is markedly upregulated in HCC patients. Our analysis further supports the putative function of RGS proteins, particularly RGS20, in cancer.

Interacting partners of Brassica juncea regulator of G-protein signaling protein suggest its role in cell wall metabolism and cellular signaling.

Heterotrimeric G-proteins interact with various upstream and downstream effectors to regulate various aspects of plant growth and development. G-protein effectors have been recently reported in Arabidopsis thaliana; however, less information is available from polyploid crop species having complex networks of G-protein components. Regulator of G-protein signaling (RGS) is a well-characterized GTPase accelerating protein, which plays an important role in the regulation of the G-protein cycle in plants. In the present study, four homologs encoding RGS proteins were isolated from the allotetraploid Brassica juncea, a globally important oilseed, vegetable, and condiment crop. The B. juncea RGS proteins were grouped into distinct BjuRGS1 and BjuRGS2 orthologous clades, and the expression of BjuRGS1 homologs was predominantly higher than BjuRGS2 homologs across the tested tissue types of B. juncea. Utilizing B. juncea Y2H library screening, a total of 30 nonredundant interacting proteins with the RGS-domain of the highly expressed BjuA.RGS1 was identified. Gene ontology analysis indicated that these effectors exerted various molecular, cellular, and physiological functions. Many of them were known to regulate cell wall metabolism (BjuEXP6, Bju-α-MAN, BjuPGU4, BjuRMS3) and phosphorylation-mediated cell signaling (BjuMEK4, BjuDGK3, and BjuKinase). Furthermore, transcript analysis indicated that the identified interacting proteins have a coexpression pattern with the BjuRGS homologs. These findings increase our knowledge about the novel targets of G-protein components from a globally cultivated Brassica crop and provide an important resource for developing a plant G-protein interactome network.

Self-activating G protein α subunits engage seven-transmembrane regulator of G protein signaling (RGS) proteins and a Rho guanine nucleotide exchange factor effector in the amoeba Naegleria fowleri

The free-living amoeba Naegleria fowleri is a causative agent of primary amoebic meningoencephalitis and is highly resistant to current therapies, resulting in mortality rates >97%. As many therapeutics target G protein–centered signal transduction pathways, further understanding the functional significance of G protein signaling within N. fowleri should aid future drug discovery against this pathogen. Here, we report that the N. fowleri genome encodes numerous transcribed G protein signaling components, including G protein–coupled receptors, heterotrimeric G protein subunits, regulator of G protein signaling (RGS) proteins, and candidate Gα effector proteins. We found N. fowleri Gα subunits have diverse nucleotide cycling kinetics; Nf Gα5 and Gα7 exhibit more rapid nucleotide exchange than GTP hydrolysis (i.e., “self-activating” behavior). A crystal structure of Nf Gα7 highlights the stability of its nucleotide-free state, consistent with its rapid nucleotide exchange. Variations in the phosphate binding loop also contribute to nucleotide cycling differences among Gα subunits. Similar to plant G protein signaling pathways, N. fowleri Gα subunits selectively engage members of a large seven-transmembrane RGS protein family, resulting in acceleration of GTP hydrolysis. We show Nf Gα2 and Gα3 directly interact with a candidate Gα effector protein, RGS-RhoGEF, similar to mammalian Gα12/13 signaling pathways. We demonstrate Nf Gα2 and Gα3 each engage RGS-RhoGEF through a canonical Gα/RGS domain interface, suggesting a shared evolutionary origin with G protein signaling in the enteric pathogen Entamoeba histolytica. These findings further illuminate the evolution of G protein signaling and identify potential targets of pharmacological manipulation in N. fowleri.

R4 RGS proteins as fine tuners of immature and mature hematopoietic cell trafficking.

G-protein-coupled receptors (GPCRs) are the largest and most diverse group of membrane receptors. They are involved in almost every physiologic process and consequently have a pivotal role in an extensive number of pathologies, including genetic, neurologic, and immune system disorders. Indeed, the vast array of GPCRs mechanisms have led to the development of a tremendous number of drug therapies and already account for about a third of marketed drugs. These receptors mediate their downstream signals primarily via G proteins. The regulators of G-protein signaling (RGS) proteins are now in the spotlight as the critical modulatory factors of active GTP-bound Gα subunits of heterotrimeric G proteins to fine-tune the biologic responses driven by the GPCRs. Also, they possess noncanonical functions by multiple mechanisms, such as protein-protein interactions. Essential roles and impacts of these RGS proteins have been revealed in physiology, including hematopoiesis and immunity, and pathologies, including asthma, cancers, and neurologic disorders. This review focuses on the largest subfamily of R4 RGS proteins and provides a brief overview of their structures and G-proteins selectivity. With particular interest, we explore and highlight, their expression in the hematopoietic system and the regulation in the engraftment of hematopoietic stem/progenitor cells (HSPCs). Distinct expression patterns of R4 RGS proteins in the hematopoietic system and their pivotal roles in stem cell trafficking pave the way for realizing new strategies for enhancing the clinical performance of hematopoietic stem cell transplantation. Finally, we discuss the exciting future trends in drug development by targeting RGS activity and expression with small molecules inhibitors and miRNA approaches.

Small molecule inhibitors of human RGS2 developed by structure based drug design approach

Regulators of G‐protein signaling (RGS) is a family of approximately 30 proteins that bind to the alpha subunits of G‐proteins (Galpha) and accelerate their GTP hydrolysis rates. This deactivates the G‐protein and terminates G‐Protein Coupled Receptors (GPCRs) signaling. RGS proteins, therefore, play important roles in regulating GPCR signaling and most members are implicated in human diseases such as hypertension, depression, and others. Regulator of G‐protein Signaling 2 (RGS2) is a regulator of angiotensin‐II receptor signaling and a modulator of oxytocin receptor signaling as well. More importantly, RGS2 was reported to be over expressed in the majority of solid breast cancers and in metastatic prostate cancer. For this reason, we sought to develop RGS2 inhibitors as potential chemotherapeutic agents. We utilized structure‐based drug design approaches to develop inhibitors of RGS2‐Galpha‐q interactions. Available structures of the RGS2‐Galpha complex were used to extract a pharmacophore model for searching of commercially available chemical databases. The identified hits were docked to different RGS structures to screen for compounds with the highest binding potential and most selectivity towards RGS2. We report the first group of inhibitors of RGS2‐Galpha‐q interactions developed through rational drug design that interfered with RGS2 signaling in cell‐based assays. In addition, inhibitor AJ‐3 inhibited the growth of MCF‐7 breast cancer cells in cell culture assays, which suggests that RGS2 inhibitors may have a potential to be a new class of chemotherapeutic agents.

Effect of Regulator of G Protein Signaling Proteins on Bone.

Regulator of G protein signaling (RGS) proteins are critical negative molecules of G protein-coupled receptor (GPCR) signaling, which mediates a variety of biological processes in bone homeostasis and diseases. The RGS proteins are divided into nine subfamilies with a conserved RGS domain which plays an important role in regulating the GTPase activity. Mutations of some RGS proteins change bone development and/or metabolism, causing osteopathy. In this review, we summarize the recent findings of RGS proteins in regulating osteoblasts, chondrocytes, and osteoclasts. We also highlight the impacts of RGS on bone development, bone remodeling, and bone-related diseases. Those studies demonstrate that RGS proteins might be potential drug targets for bone diseases.

Differential methamphetamine-induced behavioral effects in male and female mice lacking regulator of G Protein signaling 4

Methamphetamine-induced behavioral effects are mediated by several neurotransmitters that act via the G-protein coupled receptors (GPCRs). The functioning of GPCRs are negatively regulated by regulators of G-protein signaling (RGS) proteins. The goal of this study was to assess the role of two specific RGS proteins namely the RGS2 and the RGS4 proteins in methamphetamine-induced behaviors. The effects of methamphetamine (1 mg/kg; i.p.) on conditioned place preference (CPP) and locomotor activity were assessed in genetically modified male and female mice lacking either RGS2 or RGS4 and their wildtype littermates to achieve the above goal. Locomotor activity after methamphetamine administration was assessed in both methamphetamine-naïve and -experienced mice. Methamphetamine-induced CPP at the tested dose was blocked in male, but not female, mice lacking RGS4 compared to respective controls. Interestingly, methamphetamine-induced increase in locomotor activity at the tested dose was observed in methamphetamine-experienced, but not in the methamphetamine-naïve, male mice lacking RGS4. However, methamphetamine-induced increase in locomotor activity at the tested dose was blocked in both methamphetamine-naïve and -experienced female mice lacking RGS4. Interestingly, methamphetamine-induced rewarding effects and methamphetamine-induced increase in locomotor activity at the tested dose were observed in mice lacking RGS2, irrespective of sex and/or history of methamphetamine exposure. Together, the data suggest that RGS4 plays a role in methamphetamine-induced behaviors and could serve as a potential target for medications intended to treat the acute effects of methamphetamine.

Cardiovascular GPCR regulation by regulator of G protein signaling proteins.

G protein-coupled receptors (GPCRs) play pivotal roles in regulation of cardiovascular homeostasis across all vertebrate species, including humans. In terms of normal cellular function, termination of GPCR signaling via the heterotrimeric G proteins is equally (if not more) important to its stimulation. The Regulator of G protein Signaling (RGS) protein superfamily are indispensable for GPCR signaling cessation at the cell membrane, and thus, for cellular control of GPCR signaling and function. Perturbations in both activation and termination of G protein signaling underlie many examples of cardiovascular dysfunction and heart disease pathogenesis. Despite the plethora of over 30 members comprising the mammalian RGS protein superfamily, each member interacts with a specific set of second messenger pathways and GPCR types/subtypes in a tissue/cell type-specific manner. An increasing number of studies over the past two decades have provided compelling evidence for the involvement of various RGS proteins in physiological regulation of cardiovascular GPCRs and, consequently, also in the pathophysiology of several cardiovascular ailments. This chapter summarizes the current understanding of the functional roles of RGS proteins as they pertain to cardiovascular, i.e., heart, blood vessel, and platelet GPCR function, with a particular focus on their implications for chronic heart failure pathophysiology and therapy.

R4 RGS proteins suppress engraftment of human hematopoietic stem/progenitor cells by modulating SDF-1/CXCR4 signaling

AbstractHoming and engraftment of hematopoietic stem/progenitor cells (HSPCs) into the bone marrow (BM) microenvironment are tightly regulated by the chemokine stromal cell–derived factor-1 (SDF-1) and its G-protein–coupled receptor C-X-C motif chemokine receptor 4 (CXCR4), which on engagement with G-protein subunits, trigger downstream migratory signals. Regulators of G-protein signaling (RGS) are GTPase-accelerating protein of the Gα subunit and R4 subfamily members have been implicated in SDF-1–directed trafficking of mature hematopoietic cells, yet their expression and influence on HSPCs remain mostly unknown. Here, we demonstrated that human CD34+ cells expressed multiple R4 RGS genes, of which RGS1, RGS2, RGS13, and RGS16 were significantly upregulated by SDF-1 in a CXCR4-dependent fashion. Forced overexpression of RGS1, RGS13, or RGS16 in CD34+ cells not only inhibited SDF-1–directed migration, calcium mobilization, and phosphorylation of AKT, ERK, and STAT3 in vitro, but also markedly reduced BM engraftment in transplanted NOD/SCID mice. Genome-wide microarray analysis of RGS-overexpressing CD34+ cells detected downregulation of multiple effectors with established roles in stem cell trafficking/maintenance. Convincingly, gain-of-function of selected effectors or ex vivo priming with their ligands significantly enhanced HSPC engraftment. We also constructed an evidence-based network illustrating the overlapping mechanisms of RGS1, RGS13, and RGS16 downstream of SDF-1/CXCR4 and Gαi. This model shows that these RGS members mediate compromised kinase signaling and negative regulation of stem cell functions, complement activation, proteolysis, and cell migration. Collectively, this study uncovers an essential inhibitory role of specific R4 RGS proteins in stem cell engraftment, which could potentially be exploited to develop improved clinical HSPC transplantation protocols.

Regulators of G protein Signaling (RGS) proteins in GtoPdb v.2021.2

Regulator of G protein Signaling, or RGS, proteins serve an important regulatory role in signaling mediated by G protein-coupled receptors (GPCRs). They all share a common RGS domain that directly interacts with active, GTP-bound Gα subunits of heterotrimeric G proteins. RGS proteins stabilize the transition state for GTP hydrolysis on Gα and thus induce a conformational change in the Gα subunit that accelerates GTP hydrolysis, thereby effectively turning off signaling cascades mediated by GPCRs. This GTPase accelerating protein (GAP) activity is the canonical mechanism of action for RGS proteins, although many also possess additional functions and domains. RGS proteins are divided into four families, R4, R7, R12 and RZ based on sequence homology, domain structure as well as specificity towards Gα subunits. For reviews on RGS proteins and their potential as therapeutic targets, see e.g. [225, 529, 578, 583, 584, 742, 753, 444, 10].

Engraftment and SDF-1/CXCR4 Signaling in Human CD34+ Hematopoietic Stem/Progenitor Cells Are Regulated By R4 Rgs Subfamily Proteins

Regulator of G-protein Signaling (RGS) family proteins are known to negatively regulate G-protein-coupled receptor signaling through their GTPase-accelerating activity. In several types of mature hematopoietic cells (e.g., B lymphocytes and megakaryocytes), responses to chemokines are subjected to regulation by R4 subfamily RGS proteins. However, their expression patterns and functional roles in hematopoietic stem and progenitor cells (HSC) are poorly characterized. Here, we showed that human CD34+ HSC derived from cord blood (CB, n = 12) expressed RGS1 , RGS2 , RGS3 , RGS5 , RGS13 , RGS16 and RGS18 at mRNA level, whereas RGS4 , RGS8 and RGS21 were undetectable. Exposure of CB CD34+ cells to the critical homing-related chemokine stromal cell-derived factor-1 (SDF-1) significantly increased RGS1 , RGS2 , RGS13 and RGS16 , and decreased RGS3 and RGS18 expressions (P ≤ 0.040, n = 5). Expressions of RGS1 , RGS13 and RGS16 were significantly higher in bone marrow (BM, n = 14) CD34+ cells when compared to mobilized peripheral blood (MPB, n = 14) CD34+ cells (P ≤ 0.002), while RGS3 and RGS18 expressions were lower in BM CD34+ cells (P ≤ 0.036), suggesting a SDF-1 and niche-dependent regulation of RGS expressions. Lentivirus-mediated overexpression of RGS1 , RGS13 or RGS16 significantly inhibited migration of CD34+ cells to a SDF-1 gradient (P ≤ 0.039, n = 4-5) butdid not alter SDF-1-induced actin polymerization (n = 3). In addition, phosphorylation of signaling molecules downstream of the SDF-1/CXCR4 axis, including Akt, ERK and STAT3 was significantly suppressed in RGS1 - and RGS16 -overexpressing CD34+ cells. In the NOD/SCID mouse xenotransplantation model, overexpression of RGS1 , RGS13 or RGS16 significantly reduced BM engraftment of CD34+ cells by 98% (P = 0.023, n = 7), 89% (P = 0.014, n = 8) and 87% (P = 0.015, n = 8) respectively. Transcriptome profiling (n = 4) of CD34+ cells identified 312 genes with differential expressions upon overexpression of RGS1 , RGS13 or RGS16 , with 32 genes commonly regulated. Gene function enrichment analysis revealed significant downregulation of genes involved in the migration (PROS1 , CCL1 , THBS1 , F2RL2 ), complement activation (C3AR1 , C5AR1 , C5AR2 ), and proteolysis (MMP14 , TIMP3 ) pathways, which were validated by qPCR (n = 8). Taken together, we provided the first evidence that R4 RGS members are expressed and regulated by the SDF-1 in human CD34+ HSC. We also presented data showing that RGS1 , RGS13 or RGS16 negatively regulated in vitro SDF-1-mediated migration and signaling as well as in vivo engraftment of CD34+ cells, suggesting that these RGS members might serve as a feedback mechanism to regulate the SDF-1/CXCR4 axis. Strategies to inhibit specific R4 RGS proteins could potentially enhance efficiency of HSC homing and engraftment, which are particularly important in the setting of CB transplantation. DisclosuresNo relevant conflicts of interest to declare.