Guanine nucleotide exchange factors of the cytohesin family and their roles in signal transduction

Summary:  Members of the cytohesin protein family, a group of guanine nucleotide exchange factors for adenosine diphosphate ribosylation factor (ARF) guanosine triphosphatases, have recently emerged as important regulators of signal transduction in vertebrate and invertebrate biology. These proteins share a modular domain structure, comprising carboxy-terminal membrane recruitment elements, a Sec7 homology effector domain, and an amino-terminal coiled-coil domain that serve as a platform for their integration into larger signaling complexes. Although these proteins have a highly similar overall build, their individual biological functions appear to be at least partly specific. Cytohesin-1 had been identified as a regulator of β2 integrin inside-out regulation in immune cells and was subsequently shown to be involved in mitogen-associated protein kinase signaling in tumor cell proliferation as well as in T-helper cell activation and differentiation. Cytohesin-3, which had been discovered to be strongly associated with T-cell anergy, was very recently described as an essential component of insulin signal transduction in Drosophila and in human and murine liver cells. Future work will aim to dissect the mechanistic details of the modes of action of the cytohesins as well as to define the precise roles of these versatile proteins in vertebrates at the genetic level.     

Modulation of agonist binding by guanine nucleotides in CHO cells expressing muscarinic m1 – m5 receptors

In membranes prepared from CHO-m2 cells, inhibition of [³H]-N-methylscopolamine ([³H]NMS) binding by several muscarinic agonists resulted in competition curves with Hill slopes significantly different from unity. Addition of 5-guanylylimidodiphosphate (Gpp(NH)p) led to an increase in the IC₅₀ value of the agonists with significant steepening of the inhibition curves. The shift in potency induced by Gpp(NH)p differed among the agonists with a rank order of oxotremorine-M = carbachol > oxotremorine > McN-A-343 = pilocarpine. In CHO-m4 membranes, Gpp(NH)p was less efficacious than in CHO-m2 membranes whereas no effect of the guanine nucleotide was found in membranes prepared from CHO-m1, -m3, and-m5 cells. No major differences in the effect of Gpp(NH)p among agonists were found in CHO-m4 cells. Atropine binding was not affected by the guanine nucleotide. Together, these results indicate that coupling of G-proteins to muscarinic receptors linked to inhibition of cyclic adenosine monophosphate (cAMP) (m2 and m4) but not of those linked to phosphoinositol turnover (m1, m3 and m5) can be perturbed by Gpp(NH)p. The differential effects observed with Gpp(NH)p between agonist binding to m2 and m4 receptors appear to be receptor-specific and may reflect differences in the G proteins activated by these receptors in CHO cells.     

Cross GTPase-activating protein (CrossGAP)/Vilse links the Roundabout receptor to Rac to regulate midline repulsion

The regulators of the Rho-family GTPases, GTPase-activating proteins (GAPs) and guanine exchange factors (GEFs), play important roles in axon guidance. By means of a functional genomic study of the Rho-family GEFs and GAPs in Drosophila, we have identified a Rho-family GAP, CrossGAP (CrGAP), which is involved in Roundabout (Robo) receptor-mediated repulsive axon guidance. CrGAP physically associates with the Robo receptor. Too much or too little CrGAP activity leads to defects in Robo-mediated repulsion at the midline choice point. The CrGAP gain-of-function phenotype mimics the loss-of-function phenotypes of both Robo and Rac. Dosage-sensitive genetic interactions among CrGAP, Robo, and Rac support a model in which CrGAP transduces signals downstream of Robo receptor to regulate Rac-dependent cytoskeletal changes.     

High expression of guanine nucleotide-binding protein-like-3-like is associated with poor prognosis in esophageal cancer

Guanine nucleotide-binding protein-like-3-like (GNL3L) is required for processing ribosomal pre-rRNA and cell proliferation and is upregulated in many types of cancer. This study is aimed to investigate the clinical significance of GNL3L in esophageal cancer. The mRNA and protein expression levels of GNL3L were determined by using quantitative real-time polymerase chain reaction and immunohistochemistry, respectively. GNL3L was localized in both cytoplasm and nucleus. The expression levels of GNL3L in esophageal cancer tissues were significantly higher than those in adjacent nonmalignant tissues. High GNL3L expression was associated with pathologic type and poor differentiation. Patients with high GNL3L expression had shorter overall survival (OS) than those with low GNL3L expression. Multivariate Cox regression analysis revealed that GNL3L expression was an independently predictive factor for the OS of patient with esophageal cancer. The Gene Expression Profiling Interactive Analysis (GEPIA) databases also showed that GNL3L was upregulated in esophageal cancer, which was closely associated with an unfavorable prognosis of patients with esophageal cancer. Taken together, our findings suggest that GNL3L is upregulated in esophageal cancer, which is linked to the progression of the disease. As a result, GNL3L could be used as a biomarker for esophageal cancer.     

Deletion of WASp and N-WASp in B cells cripples the germinal center response and results in production of IgM autoantibodies

Humoral immunodeficiency caused by mutations in the Wiskott-Aldrich syndrome protein (WASp) is associated with failure to respond to common pathogens and high frequency of autoimmunity. Here we addressed the question how deficiency in WASp and the homologous protein N-WASp skews the immune response towards autoreactivity. Mice devoid of WASp or both WASp and N-WASp in B cells formed germinal center to increased load of apoptotic cells as a source of autoantigens. However, the germinal centers showed abolished polarity and B cells retained longer and proliferated less in the germinal centers. While WASp-deficient mice had high titers of autoreactive IgG, B cells devoid of both WASp and N-WASp produced mainly IgM autoantibodies with broad reactivity to autoantigens. Moreover, B cells lacking both WASp and N-WASp induced somatic hypermutation at reduced frequency. Despite this, IgG1-expressing B cells devoid of WASp and N-WASp acquired a specific high affinity mutation, implying an increased BCR signaling threshold for selection in germinal centers. Our data provides evidence for that N-WASp expression alone drives WASp-deficient B cells towards autoimmunity.     

Systemic immune responses to an inactivated,whole H9N2 avian influenza virus vaccine using class B CpG oligonucleotides in chickens

Commercial vaccines against avian influenza viruses (AIV) in chickens consist mainly of inactivated AIV, requiring parenteral administration and co-delivery of an adjuvant. Limitations in T helper 1 or T helper 2 biased responses generated by these vaccines emphasize the need for alternative, more efficacious adjuvants. The Toll-like receptor (TLR) 21 ligand, CpG oligodeoxynucleotides (ODN), has been established as immunomodulatory in chickens. Therefore, the objective of this study was to investigate the adjuvant potential of high (20 μg) and low (2 μg) doses of CpG ODN 2007 (CpG 2007) and CpG ODN 1826 (CpG 1826) when administered to chickens with a formalin-inactivated H9N2 AIV. Antibody responses in sera were evaluated in 90 specific pathogen free (SPF) chickens after intramuscular administration of vaccine formulations at 7 and 21 days post-hatch. Antibody responses were assessed based on haemagglutination inhibition (HI) and virus neutralization (VN) assays; virus-specific IgM and IgY antibody responses were evaluated by ELISA. The results suggest that the vaccine formulation containing low dose CpG 2007 was significantly more effective at generating neutralizing (both HI and VN) responses than formulations with high or low doses of CpG 1826 or high dose CpG 2007. Neutralizing responses elicited by low dose CpG 2007 significantly exceeded those generated by a squalene-based adjuvanted vaccine formulation during peak responses. A significantly higher IgM response was elicited by the formulation containing low dose CpG 2007 compared to high and low doses of 1826. Although the low dose of CpG 2007 elicited a higher IgY response than CpG 1826, the difference was not statistically significant. In conclusion, 2 μg of CpG 2007 is potentially promising as a vaccine adjuvant when delivered intramuscularly with inactivated H9N2 virus to chickens. Future studies may be directed at determining the mucosal antibody responses to the same vaccine formulations.     

Rho guanine nucleotide exchange factor is an NFL mRNA destabilizing factor that forms cytoplasmic inclusions in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is an adult-onset progressive disorder of unknown etiology characterized by the selective degeneration of motor neurons. Recent evidence supports the hypothesis that alterations in RNA metabolism in motor neurons can explain the development of protein inclusions, including neurofilamentous aggregates, observed in this pathology. In mice, p190RhoGEF, a guanine nucleotide exchange factor, is involved in neurofilament protein aggregation in an RNA-triggered transgenic model of motor neuron disease. Here, we observed that rho guanine nucleotide exchange factor (RGNEF), the human homologue of p190RhoGEF, binds low molecular weight neurofilament mRNA and affects its stability via 3′ untranslated region destabilization. We observed that the overexpression of RGNEF in a stable cell line significantly decreased the level of low molecular weight neurofilament protein. Furthermore, we observed RGNEF cytoplasmic inclusions in ALS spinal motor neurons that colocalized with ubiquitin, p62/sequestosome-1, and TAR (trans-active regulatory) DNA-binding protein 43 (TDP-43). Our results provide further evidence that RNA metabolism pathways are integral to ALS pathology. This is also the first described link between ALS and an RNA binding protein with aggregate formation that is also a central cell signaling pathway molecule.