Molecular mechanism of regulation of RhoA GTPase by phosphorylation of RhoGDI.

Rho-specific guanine nucleotide dissociation inhibitors (RhoGDIs) play a crucial role in the regulation of Rho family GTPases. They act as negative regulators that prevent the activation of Rho GTPases by forming complexes with the inactive GDP-bound state of GTPase. Release of Rho GTPase from the RhoGDI-bound complex is necessary for Rho GTPase activation. Biochemical studies provide evidence of a "phosphorylation code," where phosphorylation of some specific residues of RhoGDI selectively releases its GTPase partner (RhoA, Rac1, Cdc42, etc.). This work attempts to understand the molecular mechanism behind this specific phosphorylation-induced reduction in binding affinity. Using several microseconds long atomistic molecular dynamics simulations of the wild-type and phosphorylated states of the RhoA-RhoGDI complex, we propose a molecular-interaction-based mechanistic model for the dissociation of the complex. Phosphorylation induces major structural changes, particularly in the positively charged polybasic region (PBR) of RhoA and the negatively charged N-terminal region of RhoGDI that contribute most to the binding affinity. Molecular mechanics Poisson-Boltzmann surface area binding energy calculations show a significant weakening of interaction on phosphorylation at the RhoA-specific site of RhoGDI. In contrast, phosphorylation at a Rac1-specific site does not affect the overall binding affinity significantly, which confirms the presence of a phosphorylation code. RhoA-specific phosphorylation leads to a reduction in the number of contacts between the PBR of RhoA and the N-terminal region of RhoGDI, which manifests a reduction of the binding affinity. Using hydrogen bond occupancy analysis and energetic perturbation network, we propose a mechanistic model for the allosteric response, i.e., long-range signal propagation from the site of phosphorylation to the PBR and buried geranylgeranyl group in the form of rearrangement and rewiring of hydrogen bonds and salt bridges. Our results highlight the crucial role of specific electrostatic interactions in manifestation of the phosphorylation code.

Identification of an Aurora-A/PinsLINKER/Dlg spindle orientation pathway using induced cell polarity in S2 cells.

Asymmetric cell division is intensely studied because it can generate cellular diversity as well as maintain stem cell populations. Asymmetric cell division requires mitotic spindle alignment with intrinsic or extrinsic polarity cues, but mechanistic detail of this process is lacking. Here, we develop a method to construct cortical polarity in a normally unpolarized cell line and use this method to characterize Partner of Inscuteable (Pins; LGN/AGS3 in mammals) -dependent spindle orientation. We identify a previously unrecognized evolutionarily conserved Pins domain (Pins(LINKER)) that requires Aurora-A phosphorylation to recruit Discs large (Dlg; PSD-95/hDlg in mammals) and promote partial spindle orientation. The well-characterized Pins(TPR) domain has no function alone, but placing the Pins(TPR) in cis to the Pins(LINKER) gives dynein-dependent precise spindle orientation. This "induced cortical polarity" assay is suitable for rapid identification of the proteins, domains, and amino acids regulating spindle orientation or cell polarity.

The atypical Rab GTPase associated with Parkinson's disease, Rab29, is localized to membranes.

Several genetic studies have shown that the small GTPase Rab29 is involved in the pathogenesis of Parkinson's Disease (PD). It has also been shown that overexpression of Rab29 increases the activity of leucine-rich repeat kinase 2, a protein kinase often mutated in familial PD, although the mechanism underlying this activation remains unclear. Here, we employed biochemical analyses to characterize the localization of Rab29 and found that, unlike general Rab proteins, Rab29 is predominantly fractionated into the membrane fraction by ultracentrifugation. We also found that Rab29 is resistant to extraction from membranes by GDP-dissociation inhibitors (GDIs) in vitro. Furthermore, Rab29 failed to interact with GDIs, and its membrane localization was not affected by the knockout of GDIs in cells. We show that the knockout of Rab geranylgeranyltransferase decreased the hydrophobicity of Rab29, suggesting that Rab29 is geranylgeranylated at its carboxyl terminus as is with typical Rab proteins. Notably, we demonstrated that membrane-bound Rab29 retains some hydrophilicity, indicating that mechanisms other than geranylgeranylation might also be involved in the membrane localization of Rab29. Taken together, these findings uncover the atypical nature of Rab29 among Rab proteins, which will provide important clues for understanding how Rab29 is involved in the molecular pathomechanism of PD.

Implementation of pharmacogenetic testing in medication reviews in a hospital setting.

AIM: To investigate whether it is feasible to perform pharmacogenetic testing and implement the test results as part of medication reviews during hospitalization of multimorbid patients. METHODS: Patients with ≥2 chronic conditions and ≥5 regular drugs with at least one potential gene-drug interaction (GDI) were included from one geriatric and one cardiology ward for pharmacogenetic testing. After inclusion by the study pharmacist, blood samples were collected and shipped to the laboratory for analysis. For patients still hospitalized at the time when the pharmacogenetic test results were available, the information was used in medication reviews. Recommendations from the pharmacist on actionable GDIs were communicated to the hospital physicians, who subsequently decided on potential immediate changes or forwarded suggestions in referrals to general practitioners. RESULTS: The pharmacogenetic test results were available for medication review in 18 of the 46 patients (39.1%), where median length of hospital stay was 4.7 days (1.6-18.3). The pharmacist recommended medication changes for 21 of 49 detected GDIs (42.9%). The hospital physicians accepted 19 (90.5%) of the recommendations. The most commonly detected GDIs involved metoprolol (CYP2D6 genotype), clopidogrel (CYP2C19 genotype) and atorvastatin (CYP3A4/5 and SLCOB1B1 genotype). CONCLUSIONS: The study shows that implementation of pharmacogenetic testing for medication review of hospitalized patients has the potential to improve drug treatment before being transferred to primary care. However, the logistics workflow needs to be further optimized, as test results were available during hospitalization for less than half of the patients included in the study.

The 'invisible hand': regulation of RHO GTPases by RHOGDIs.

The 'invisible hand' is a term originally coined by Adam Smith in The Theory of Moral Sentiments to describe the forces of self-interest, competition and supply and demand that regulate the resources in society. This metaphor continues to be used by economists to describe the self-regulating nature of a market economy. The same metaphor can be used to describe the RHO-specific guanine nucleotide dissociation inhibitor (RHOGDI) family, which operates in the background, as an invisible hand, using similar forces to regulate the RHO GTPase cycle.

The effects of the first versus second glaucoma drainage implant surgery in patients with primary open-angle glaucoma.

BACKGROUND: To investigate the outcome of non-valved glaucoma drainage implant surgery (GDIS) in primary open-angle glaucoma (POAG) patients divided in the first GDI group (patients who underwent the first GDIS) and the second GDI group (patients who underwent the second GDIS because of the failed first GDIS). METHODS: Intraocular pressure (IOP), visual acuity (VA), visual field defect (VFD), medication score (MS), survival rate of GDIS, complications, and patient background was retrospectively analyzed. Two success criteria were set: Criteria (1) IOP reduction ≥ 20% and 5 < IOP ≤ 21, Criteria (2) IOP reduction ≥ 20% and 5 < IOP ≤ 14. RESULTS: There were 136 eyes of 109 patients in the first GDI group and 32 eyes of 27 patients in the second GDI group. In the first GDI group and II, mean preoperative IOP was 26.7 ± 6.7 mmHg and 23.7 ± 3.5 mmHg, respectively (P = 0.09). No statistically significant difference in postoperative IOP reduction was found between the two groups (P = 0.39). At 5-years postoperative, the Criteria 1 (Criteria 2) survival rate in the first GDI group and the second GDI group was 60.4% (31.7%) and 61.2% (25.6%), respectively (Criteria 1: hazard ratio [HR]: 0.64, 95% confidence interval [CI]: 0.30-1.35 [P = 0.24]; Criteria 2: HR: 0.81, 95% CI: 0.46-1.44, P = 0.48). No significant difference in VA, VFD change, MS, or complications was observed. Young patient age was the only significant factor for failure in the first GDI group (odds ratio: 0.95, 95% confidence interval: 0.91-1.00, P = 0.03). CONCLUSION: The second GDIS may be as effective as the first GDIS for IOP reduction in POAG patients, however, there is a high risk of failure in young-age patients and the surgery may be ineffective in eyes requiring Criteria 2.

AGS3-dependent trans-Golgi network membrane trafficking is essential for compaction in mouse embryos.

Activator of G-protein signaling 3 (AGS3, also known as GPSM1) regulates the trans-Golgi network. The AGS3 GoLoco motif binds to Gαi and thereby regulates the transport of proteins to the plasma membrane. Compaction of early embryos is based on the accumulation of E-cadherin (Cdh1) at cell-contacted membranes. However, how AGS3 regulates the transport of Cdh1 to the plasma membrane remains undetermined. To investigate this, AGS3 was knocked out using the Cas9-sgRNA system. Both trans-Golgi network protein 46 (TGN46, also known as TGOLN2) and transmembrane p24-trafficking protein 7 (TMED7) were tracked in early mouse embryos by tagging these proteins with a fluorescent protein label. We observed that the majority of the AGS3-edited embryos were developmentally arrested and were fragmented after the four-cell stage, exhibiting decreased accumulation of Cdh1 at the membrane. The trans-Golgi network and TMED7-positive vesicles were also dispersed and were not polarized near the membrane. Additionally, increased Gαi1 (encoded by GNAI1) expression could rescue AGS3-overexpressed embryos. In conclusion, AGS3 reinforces the dynamics of the trans-Golgi network and the transport of TMED7-positive cargo containing Cdh1 to the cell-contact surface during early mouse embryo development.

Epithelial tricellular junctions act as interphase cell shape sensors to orient mitosis.

The orientation of cell division along the long axis of the interphase cell--the century-old Hertwig's rule--has profound roles in tissue proliferation, morphogenesis, architecture and mechanics. In epithelial tissues, the shape of the interphase cell is influenced by cell adhesion, mechanical stress, neighbour topology, and planar polarity pathways. At mitosis, epithelial cells usually adopt a rounded shape to ensure faithful chromosome segregation and to promote morphogenesis. The mechanisms underlying interphase cell shape sensing in tissues are therefore unknown. Here we show that in Drosophila epithelia, tricellular junctions (TCJs) localize force generators, pulling on astral microtubules and orienting cell division via the Dynein-associated protein Mud independently of the classical Pins/Gαi pathway. Moreover, as cells round up during mitosis, TCJs serve as spatial landmarks, encoding information about interphase cell shape anisotropy to orient division in the rounded mitotic cell. Finally, experimental and simulation data show that shape and mechanical strain sensing by the TCJs emerge from a general geometric property of TCJ distributions in epithelial tissues. Thus, in addition to their function as epithelial barrier structures, TCJs serve as polarity cues promoting geometry and mechanical sensing in epithelial tissues.

A Complete Survey of RhoGDI Targets Reveals Novel Interactions with Atypical Small GTPases.

There are three RhoGDIs in mammalian cells, which were initially defined as negative regulators of Rho family small GTPases. However, it is now accepted that RhoGDIs not only maintain small GTPases in their inactive GDP-bound form but also act as chaperones for small GTPases, targeting them to specific intracellular membranes and protecting them from degradation. Studies to date with RhoGDIs have usually focused on the interactions between the "typical" or "classical" small GTPases, such as the Rho, Rac, and Cdc42 subfamily members, and either the widely expressed RhoGDI-1 or the hematopoietic-specific RhoGDI-2. Less is known about the third member of the family, RhoGDI-3 and its interacting partners. RhoGDI-3 has a unique N-terminal extension and is found to localize in both the cytoplasm and the Golgi. RhoGDI-3 has been shown to target RhoB and RhoG to endomembranes. In order to facilitate a more thorough understanding of RhoGDI function, we undertook a systematic study to determine all possible Rho family small GTPases that interact with the RhoGDIs. RhoGDI-1 and RhoGDI-2 were found to have relatively restricted activity, mainly binding members of the Rho and Rac subfamilies. RhoGDI-3 displayed wider specificity, interacting with the members of Rho, Rac, and Cdc42 subfamilies but also forming complexes with "atypical" small Rho GTPases such as Wrch2/RhoV, Rnd2, Miro2, and RhoH. Levels of RhoA, RhoB, RhoC, Rac1, RhoH, and Wrch2/RhoV bound to GTP were found to decrease following coexpression with RhoGDI-3, confirming its role as a negative regulator of these small Rho GTPases.

GDI2 is a target of paclitaxel that affects tumorigenesis of prostate cancer via the p75NTR signaling pathway.

BACKGROUND: Prostate cancer (PCa) refers to malignant tumors derived from prostate epithelial cells, whose morbidity and mortality rates have been increasing every year. Although new drugs for treating prostate cancer continue to emerge, the unclear mechanism underlying drug targets limits this therapy, thereby constraining identification of effective therapeutic targets. Although GDP dissociation inhibitor 2(GDI2) is highly expressed and closely associated with occurrence and development of many tumors, its role in prostate cancer remains unclear. In this study, we investigated the role of GDI2 and elucidated its underlying mechanism of action in prostate cancer. Moreover, we screened chemotherapeutic drugs that affect GDI2 expression with a view of identifying novel targets for diagnosis and treatment of prostate cancer. METHODS: We performed sequence analyses and functional assays to precisely elucidate the GDI2 role in prostate cancer. Moreover, we induced tumorigenesis in nude mice to verify the role of GDI2 in vivo. Finally, we used the CCK8 assay to ascertain the most suitable IC50 across the three drugs and performed quantitative real time polymerase chain reaction (qRT-PCR) and Western Blot to analyze the effects of drugs on expression of GDI2, p75NTR, and p-NFκB. RESULTS: GDI2 was up-regulated in prostate cancer cells and tissues. Knocking down GDI2 suppressed cell proliferation but promoted cell apoptosis. Interestingly, knocking down GDI2 activated the p75NTR signaling pathway, indicating, for the first time, that p75NTR is negatively correlated with GDI2 expression. CONCLUSION: Taken together, these results indicate that GDI2 is a therapeutic target of paclitaxel. Knocking down of GDI2 inhibits cell proliferation and promotes cell apoptosis via the p75NTR signaling pathway in prostate cancer. Notably, paclitaxel inhibits GDI2 expression, implying that GDI2 may be a promising therapeutic target in prostate cancer.

CARD9 facilitates microbe-elicited production of reactive oxygen species by regulating the LyGDI-Rac1 complex.

In response to invading microorganisms, macrophages engage in phagocytosis and rapidly release reactive oxygen species (ROS), which serve an important microbicidal function. However, how phagocytosis induces ROS production remains largely unknown. CARD9, a caspase-recruitment domain (CARD)-containing protein, is important for resistance to fungal and bacterial infection. The mechanism of CARD9-mediated bacterial clearance is still mostly unknown. Here we show that CARD9 is required for killing intracellular bacteria in macrophages. CARD9 associated with the GDP-dissociation inhibitor LyGDI in phagosomes after bacterial and fungal infection and binding of CARD9 suppressed LyGDI-mediated inhibition of the GTPase Rac1, thereby leading to ROS production and bacterial killing in macrophages. Thus, our studies identify a key pathway that leads to microbe-elicited ROS production.

Adipose tissue depot-specific intracellular and extracellular cues contributing to insulin resistance in obese individuals.

Adipose tissue dysregulation in obesity strongly influences systemic metabolic homeostasis and is often linked to insulin resistance (IR). However, the molecular mechanisms underlying adipose tissue dysfunction in obesity are not fully understood. Herein, a proteomic analysis of subcutaneous (SC) and omental (OM) fat from lean subjects and obese individuals with different degrees of insulin sensitivity was performed to identify adipose tissue biomarkers related to obesity-associated metabolic disease. Our results suggest that dysregulation of both adipose tissue extracellular matrix (ECM) organization and intracellular trafficking processes may be associated with IR in obesity. Thus, abnormal accumulation of the small leucine-rich proteoglycan, lumican, as observed in SC fat of IR obese individuals, modifies collagen I organization, impairs adipogenesis and activates stress processes [endoplasmic reticulum and oxidative stress] in adipocytes. In OM fat, IR is associated with increased levels of the negative regulator of the Rab family of small GTPases, GDI2, which alters lipid storage in adipocytes by inhibiting insulin-stimulated binding of the Rab protein, Rab18, to lipid droplets. Together, these results indicate that lumican and GDI2 might play depot-dependent, pathogenic roles in obesity-associated IR. Our findings provide novel insights into the differential maladaptive responses of SC and OM adipose tissue linking obesity to IR.

eIF2B promotes eIF5 dissociation from eIF2*GDP to facilitate guanine nucleotide exchange for translation initiation.

Protein synthesis factor eIF2 delivers initiator tRNA to the ribosome. Two proteins regulate its G-protein cycle: eIF5 has both GTPase-accelerating protein (GAP) and GDP dissociation inhibitor (GDI) functions, and eIF2B is the guanine nucleotide exchange factor (GEF). In this study, we used protein-protein interaction and nucleotide exchange assays to monitor the kinetics of eIF2 release from the eIF2•GDP/eIF5 GDI complex and determine the effect of eIF2B on this release. We demonstrate that eIF2B has a second activity as a GDI displacement factor (GDF) that can recruit eIF2 from the eIF2•GDP/eIF5 GDI complex prior to GEF action. We found that GDF function is dependent on the eIF2Bε and eIF2Bγ subunits and identified a novel eIF2-eIF2Bγ interaction. Furthermore, GDF and GEF activities are shown to be independent. First, eIF2B GDF is insensitive to eIF2α phosphorylation, unlike GEF. Second, we found that eIF2Bγ mutations known to disrupt GCN4 translational control significantly impair GDF activity but not GEF function. Our data therefore define an additional step in the protein synthesis initiation pathway that is important for its proper control. We propose a new model to place eIF2B GDF function in the context of efficient eIF2 recycling and its regulation by eIF2 phosphorylation.

Electrostatic Forces Mediate the Specificity of RHO GTPase-GDI Interactions.

Three decades of research have documented the spatiotemporal dynamics of RHO family GTPase membrane extraction regulated by guanine nucleotide dissociation inhibitors (GDIs), but the interplay of the kinetic mechanism and structural specificity of these interactions is as yet unresolved. To address this, we reconstituted the GDI-controlled spatial segregation of geranylgeranylated RHO protein RAC1 in vitro. Various biochemical and biophysical measurements provided unprecedented mechanistic details for GDI function with respect to RHO protein dynamics. We determined that membrane extraction of RHO GTPases by GDI occurs via a 3-step mechanism: (1) GDI non-specifically associates with the switch regions of the RHO GTPases; (2) an electrostatic switch determines the interaction specificity between the C-terminal polybasic region of RHO GTPases and two distinct negatively-charged clusters of GDI1; (3) a non-specific displacement of geranylgeranyl moiety from the membrane sequesters it into a hydrophobic cleft, effectively shielding it from the aqueous milieu. This study substantially extends the model for the mechanism of GDI-regulated RHO GTPase extraction from the membrane, and could have implications for clinical studies and drug development.

Regulation of small GTPases by GEFs, GAPs, and GDIs.

Small GTPases use GDP/GTP alternation to actuate a variety of functional switches that are pivotal for cell dynamics. The GTPase switch is turned on by GEFs, which stimulate dissociation of the tightly bound GDP, and turned off by GAPs, which accelerate the intrinsically sluggish hydrolysis of GTP. For Ras, Rho, and Rab GTPases, this switch incorporates a membrane/cytosol alternation regulated by GDIs and GDI-like proteins. The structures and core mechanisms of representative members of small GTPase regulators from most families have now been elucidated, illuminating their general traits combined with scores of unique features. Recent studies reveal that small GTPase regulators have themselves unexpectedly sophisticated regulatory mechanisms, by which they process cellular signals and build up specific cell responses. These mechanisms include multilayered autoinhibition with stepwise release, feedback loops mediated by the activated GTPase, feed-forward signaling flow between regulators and effectors, and a phosphorylation code for RhoGDIs. The flipside of these highly integrated functions is that they make small GTPase regulators susceptible to biochemical abnormalities that are directly correlated with diseases, notably a striking number of missense mutations in congenital diseases, and susceptible to bacterial mimics of GEFs, GAPs, and GDIs that take command of small GTPases in infections. This review presents an overview of the current knowledge of these many facets of small GTPase regulation.

Role of G-proteins and phosphorylation in the distribution of AGS3 to cell puncta.

Activator of G-protein signaling 3 (AGS3, also known as GPSM1) exhibits broad functional diversity and oscillates among different subcellular compartments in a regulated manner. AGS3 consists of a tetratricopeptide repeat (TPR) domain and a G-protein regulatory (GPR) domain. Here, we tested the hypothesis that phosphorylation of the AGS3 GPR domain regulates its subcellular distribution and functionality. In contrast to the cortical and/or diffuse non-homogeneous distribution of wild-type (WT) AGS3, an AGS3 construct lacking all 24 potential phosphorylation sites in the GPR domain localized to cytosolic puncta. This change in localization was revealed to be dependent upon phosphorylation of a single threonine amino acid (T602). The punctate distribution of AGS3-T602A was rescued by co-expression of Gαi and Gαo but not Gαs or Gαq Following treatment with alkaline phosphatase, both AGS3-T602A and WT AGS3 exhibited a gel shift in SDS-PAGE as compared to untreated WT AGS3, consistent with a loss of protein phosphorylation. The punctate distribution of AGS3-T602A was lost in an AGS3-A602T conversion mutant, but was still present upon T602 mutation to glutamate or aspartate. These results implicate dynamic phosphorylation as a discrete mechanism to regulate the subcellular distribution of AGS3 and associated functionality.

Rab GDP-dissociation inhibitor gdiA is an essential gene required for cell wall chitin deposition in Aspergillus niger.

The cell wall is a distinctive feature of filamentous fungi, providing them with structural integrity and protection from both biotic and abiotic factors. Unlike plant cell walls, fungi rely on structurally strong hydrophobic chitin core for mechanical strength together with alpha- and beta-glucans, galactomannans and glycoproteins. Cell wall stress conditions are known to alter the cell wall through the signaling cascade of the cell wall integrity (CWI) pathway and can result in increased cell wall chitin deposition. A previously isolated set of Aspergillus niger cell wall mutants was screened for increased cell wall chitin deposition. UV-mutant RD15.8#16 was found to contain approximately 60% more cell wall chitin than the wild type. In addition to the chitin phenotype, RD15.8#16 exhibits a compact colony morphology and increased sensitivity towards SDS. RD15.8#16 was subjected to classical genetic approach for identification of the underlying causative mutation, using co-segregation analysis and SNP genotyping. Genome sequencing of RD15.8#16 revealed eight SNPs in open reading frames (ORF) which were individually checked for co-segregation with the associated phenotypes, and showed the potential relevance of two genes located on chromosome IV. In situ re-creation of these ORF-located SNPs in a wild type background, using CRISPR/Cas9 genome editing, showed the importance Rab GTPase dissociation inhibitor A (gdiA) for the phenotypes of RD15.8#16. An alteration in the 5' donor splice site of gdiA reduced pre-mRNA splicing efficiency, causing aberrant cell wall assembly and increased chitin levels, whereas gene disruption attempts showed that a full gene deletion of gdiA is lethal.

Genome-wide meta-analysis associates GPSM1 with type 2 diabetes, a plausible gene involved in skeletal muscle function.

Genome-wide association studies (GWASs) have identified many genetic variations associated with type 2 diabetes mellitus (T2DM) in Asians, but understanding the functional genetic variants that influence traits is often a complex process. In this study, fine mapping and other analytical strategies were performed to investigate the effects of G protein signaling modulator 1 (GPSM1) on insulin resistance in skeletal muscle. A total of 128 single-nucleotide polymorphisms (SNPs) within GPSM1 were analysed in 21,897 T2DM cases and 32,710 healthy controls from seven GWASs. The SNP rs28539249 in intron 9 of GPSM1 showed a nominally significant association with T2DM in Asians (OR = 1.07, 95% CI = 1.04-1.10, P < 10-4). The GPSM1 mRNA was increased in skeletal muscle and correlated with T2DM traits across obese mice model. An eQTL for the cis-acting regulation of GPSM1 expression in human skeletal muscle was identified for rs28539249, and the increased GPSM1 expression related with T2DM traits within GEO datasets. Another independent Asian cohort showed that rs28539249 is associated with the skeletal muscle expression of CACFD1, GTF3C5, SARDH, and FAM163B genes, which are functionally enriched for endoplasmic reticulum stress (ERS) and unfolded protein response (UPR) pathways. Moreover, rs28539249 locus was predicted to disrupt regulatory regions in human skeletal muscle with enriched epigenetic marks and binding affinity for CTCF. Supershift EMSA assays followed luciferase assays demonstrated the CTCF specifically binding to rs28539249-C allele leading to decreased transcriptional activity. Thus, the post-GWAS annotation confirmed the Asian-specific association of genetic variant in GPSM1 with T2DM, suggesting a role for the variant in the regulation in skeletal muscle.

Specific inhibition of GPCR-independent G protein signaling by a rationally engineered protein.

Activation of heterotrimeric G proteins by cytoplasmic nonreceptor proteins is an alternative to the classical mechanism via G protein-coupled receptors (GPCRs). A subset of nonreceptor G protein activators is characterized by a conserved sequence named the Gα-binding and activating (GBA) motif, which confers guanine nucleotide exchange factor (GEF) activity in vitro and promotes G protein-dependent signaling in cells. GBA proteins have important roles in physiology and disease but remain greatly understudied. This is due, in part, to the lack of efficient tools that specifically disrupt GBA motif function in the context of the large multifunctional proteins in which they are embedded. This hindrance to the study of alternative mechanisms of G protein activation contrasts with the wealth of convenient chemical and genetic tools to manipulate GPCR-dependent activation. Here, we describe the rational design and implementation of a genetically encoded protein that specifically inhibits GBA motifs: GBA inhibitor (GBAi). GBAi was engineered by introducing modifications in Gαi that preclude coupling to every known major binding partner [GPCRs, Gßγ, effectors, guanine nucleotide dissociation inhibitors (GDIs), GTPase-activating proteins (GAPs), or the chaperone/GEF Ric-8A], while favoring high-affinity binding to all known GBA motifs. We demonstrate that GBAi does not interfere with canonical GPCR-G protein signaling but blocks GBA-dependent signaling in cancer cells. Furthermore, by implementing GBAi in vivo, we show that GBA-dependent signaling modulates phenotypes during Xenopus laevis embryonic development. In summary, GBAi is a selective, efficient, and convenient tool to dissect the biological processes controlled by a GPCR-independent mechanism of G protein activation mediated by cytoplasmic factors.

AGS3 and Gαi3 Are Concomitantly Upregulated as Part of the Spindle Orientation Complex during Differentiation of Human Neural Progenitor Cells.

Adult neurogenesis is modulated by many Gi-coupled receptors but the precise mechanism remains elusive. A key step for maintaining the population of neural stem cells in the adult is asymmetric cell division (ACD), a process which entails the formation of two evolutionarily conserved protein complexes that establish the cell polarity and spindle orientation. Since ACD is extremely difficult to monitor in stratified tissues such as the vertebrate brain, we employed human neural progenitor cell lines to examine the regulation of the polarity and spindle orientation complexes during neuronal differentiation. Several components of the spindle orientation complex, but not those of the polarity complex, were upregulated upon differentiation of ENStem-A and ReNcell VM neural progenitor cells. Increased expression of nuclear mitotic apparatus (NuMA), Gαi subunit, and activators of G protein signaling (AGS3 and LGN) coincided with the appearance of a neuronal marker (ß-III tubulin) and the concomitant loss of neural progenitor cell markers (nestin and Sox-2). Co-immunoprecipitation assays demonstrated that both Gαi3 and NuMA were associated with AGS3 in differentiated ENStem-A cells. Interestingly, AGS3 appeared to preferentially interact with Gαi3 in ENStem-A cells, and this specificity for Gαi3 was recapitulated in co-immunoprecipitation experiments using HEK293 cells transiently overexpressing GST-tagged AGS3 and different Gαi subunits. Moreover, the binding of Gαi3 to AGS3 was suppressed by GTPγS and pertussis toxin. Disruption of AGS3/Gαi3 interaction by pertussis toxin indicates that AGS3 may recognize the same site on the Gα subunit as G protein-coupled receptors. Regulatory mechanisms controlling the formation of spindle orientation complex may provide novel means to manipulate ACD which in turn may have an impact on neurogenesis.