Modulation of the cAMP response by Gαi and Gßγ: a computational study of G protein signaling in immune cells.

Cyclic AMP is important for the resolution of inflammation, as it promotes anti-inflammatory signaling in several immune cell lines. In this paper, we present an immune cell specific model of the cAMP signaling cascade, paying close attention to the specific isoforms of adenylyl cyclase (AC) and phosphodiesterase that control cAMP production and degradation, respectively, in these cells. The model describes the role that G protein subunits, including Gαs, Gαi, and Gßγ, have in regulating cAMP production. Previously, Gαi activation has been shown to increase the level of cAMP in certain immune cell types. This increase in cAMP is thought to be mediated by ßγ subunits which are released upon Gα activation and can directly stimulate specific isoforms of AC. We conduct numerical experiments in order to explore the mechanisms through which Gαi activation can increase cAMP production. An important conclusion of our analysis is that the relative abundance of different G protein subunits is an essential determinant of the cAMP profile in immune cells. In particular, our model predicts that limited availability of ßγ subunits may both (i) enable immune cells to link inflammatory Gαi signaling to anti-inflammatory cAMP production thereby creating a balanced immune response to stimulation with low concentrations of PGE2, and (ii) prohibit robust anti-inflammatory cAMP signaling in response to stimulation with high concentrations of PGE2.

An anti-sulfatide antibody O4 immunoprecipitates sulfatide rafts including Fyn, Lyn and the G protein α subunit in rat primary immature oligodendrocytes.

The association of sulfatide with specific proteins in oligodendrocytes was examined by co-immunoprecipitation with an anti-sulfatide antibody. Protein kinase activity was detected in precipitates with a monoclonal antibody to sulfatide (O4) from the rat primary immature oligodendrocytes. We conducted in vitro kinase assay of tyrosine phosphorylated proteins of 80, 59, 56, 53 and 40 kDa by gel electrophoresis. Of these proteins, the proteins of 59 kDa and 53/56 kDa were identified as the Src family tyrosine kinases Fyn and Lyn on the basis of their sequential immunoprecipitation with anti-Fyn and anti-Lyn antibodies, respectively. The 40 kDa protein was identified as the α subunit of the heterotrimeric G protein. These observations suggest that O4 immunoprecipitates sulfatide rafts including Fyn, Lyn and the α subunit of the heterotrimeric G protein.

Heterotrimeric G-protein signaling is critical to pathogenic processes in Entamoeba histolytica.

Heterotrimeric G-protein signaling pathways are vital components of physiology, and many are amenable to pharmacologic manipulation. Here, we identify functional heterotrimeric G-protein subunits in Entamoeba histolytica, the causative agent of amoebic colitis. The E. histolytica Gα subunit EhGα1 exhibits conventional nucleotide cycling properties and is seen to interact with EhGßγ dimers and a candidate effector, EhRGS-RhoGEF, in typical, nucleotide-state-selective fashions. In contrast, a crystal structure of EhGα1 highlights unique features and classification outside of conventional mammalian Gα subfamilies. E. histolytica trophozoites overexpressing wildtype EhGα1 in an inducible manner exhibit an enhanced ability to kill host cells that may be wholly or partially due to enhanced host cell attachment. EhGα1-overexpressing trophozoites also display enhanced transmigration across a Matrigel barrier, an effect that may result from altered baseline migration. Inducible expression of a dominant negative EhGα1 variant engenders the converse phenotypes. Transcriptomic studies reveal that modulation of pathogenesis-related trophozoite behaviors by perturbed heterotrimeric G-protein expression includes transcriptional regulation of virulence factors and altered trafficking of cysteine proteases. Collectively, our studies suggest that E. histolytica possesses a divergent heterotrimeric G-protein signaling axis that modulates key aspects of cellular processes related to the pathogenesis of this infectious organism.

C5a receptor-dependent cell activation by physiological concentrations of desarginated C5a: insights from a novel label-free cellular assay.

The complement anaphylatoxins C3a, C5a, and desarginated C5a (C5a(desArg)) play critical roles in the induction of inflammation and the modulation of innate and acquired immune responses after binding to their G protein-coupled receptors, C3a receptor and C5a receptor (C5aR). The role of C5a(desArg) in inducing cell activation has been often neglected, because the affinity of C5a(desArg) for C5aR has been reported to be much lower than that of C5a. We have used a novel label-free cellular assay to reassess the potential of C5a(desArg) to induce activation of transfected and primary immune cells. Our results indicate that physiological levels of C5a(desArg) induce significant levels of cell activation that are even higher than those achieved by stimulating cells with analogous concentrations of C5a. Such activation was strictly dependent on C5aR, because it was completely abrogated by PMX-53, a C5aR antagonist. Pharmacological inhibition of specific G proteins located downstream of C5aR indicated differential involvement of G(α) proteins upon C5aR engagement by C5a or C5a(desArg). Further, mass spectrometric characterization of plasma-derived C5a and C5a(desArg) provided important insight into the posttranslational modification pattern of these anaphylatoxins, which includes glycosylation at Asn(64) and partial cysteinylation at Cys(27). Although the context-specific physiological contribution of C5a(desArg) has to be further explored, our data suggest that C5a(desArg) acts as a key molecule in the triggering of local inflammation as well as the maintenance of blood surveillance and homeostatic status.

Immunolocalization of G protein α subunits in the olfactory system of Polypterus senegalus (Cladistia, Actinopterygii).

In vertebrates, the receptor neurons of the olfactory/vomeronasal systems express different receptor gene families and related G-protein types (in particular the G protein alpha subunit). There are no data in the literature about the molecular features of the olfactory/vomeronasal systems of Cladistia thus, in this work, the presence and distribution of different types of G protein alpha subunits were investigated in the olfactory organs of the bichir Polypterus senegalus, using immunohistochemistry. Gαo-like immunoreactivity was detected in the microvillous receptor neurons, with the cell body in the basal zone of the sensory epithelium, and in the crypt neurons. Gαo-like ir glomeruli were mainly localized in the anterior part of the olfactory bulb. Gαolf-like immunoreactivity in the sensory epithelium was detected in the ciliated receptor neurons, while the immunoreactive glomeruli in the olfactory bulb were mainly localized in the ventral-posterior part. No Gαq nor Gαi3 immunoreactivity was detected. These data are partially in agreement with studies that show the distribution of G protein alpha subunits in teleosts, allowing to hypothesize a common organization of the olfactory/vomeronasal systems in the group of Actinopterigians.

Differential regulation of lipopolysaccharide and Gram-positive bacteria induced cytokine and chemokine production in macrophages by Galpha(i) proteins.

Heterotrimeric G(i) proteins play a role in signalling activated by lipopolysaccharide (LPS), Staphylococcus aureus (SA) and group B streptococci (GBS), leading to production of inflammatory mediators. We hypothesized that genetic deletion of G(i) proteins would alter cytokine and chemokine production induced by LPS, SA and GBS stimulation. LPS-induced, heat-killed SA-induced and heat-killed GBS-induced cytokine and chemokine production in peritoneal macrophages from wild-type (WT), Galpha(i2) (-/-) or Galpha(i1/3) (-/-) mice were investigated. LPS induced production of tumour necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), IL-10 and interferon-gamma-inducible protein-10 (IP-10); SA induced TNF-alpha, and IL-1beta production; and GBS induced TNF-alpha, IL-6, IL-1beta, macrophage inflammatory protein-1alpha (MIP-1alpha) and keratinocyte chemoattract (KC) production were all decreased (P < 0.05) in Galpha(i2) (-/-) or Galpha(i1/3) (-/-) mice compared with WT mice. In contrast to the role of G(i) proteins as a positive regulator of mediators, LPS-induced production of MIP-1alpha and granulocyte-macrophage colony-stimulating factor (GM-CSF) were increased in macrophages from Galpha(i1/3) (-/-) mice, and SA-induced MIP-1alpha production was increased in both groups of Galpha(i) protein-depleted mice. LPS-induced production of KC and IL-1beta, SA-induced production of GM-CSF, KC and IP-10, and GBS-induced production of IL-10, GM-CSF and IP-10 were unchanged in macrophages from Galpha(i2) (-/-) or Galpha(i1/3) (-/-) mice compared with WT mice. These data suggest that G(i2) and G(i1/3) proteins are both involved and differentially regulate murine inflammatory cytokine and chemokine production in response to both LPS and Gram-positive microbial stimuli.

G-protein coupled activation of potassium channels by endogenous neuropeptides in snail neurons.

Members of the mytilus inhibitory peptide (MIP) family play an important role in the modulation of many physiological processes in molluscs. The signal transduction pathways affected by the MIP effect have not, however, been elucidated. Application of guanosine 5'-[gamma-thio]triphosphate tetralithium salt (GTPgammaS), guanosine 5'-[beta-thio]diphosphate trilithium salt (GDPbetaS), the G-protein inhibitor suramin and pertussis toxin (PTX) demonstrated the involvement of the PTX-insensitive G-protein in the signal transduction pathway mediating MIP effects. Both G-protein alpha(i) and betagamma subunits were identified in D-neurons of Helix pomatia by immunoblotting. Their role in signal transduction was shown in electrophysiological experiments, which supported the notion that, in addition to the Galpha subunit, the betagamma dimer also participates in the neuropeptide-induced activation of K-channels in snail neurons. Finally, neuropeptide-activated responses were inhibited by the activation of adenylyl cyclase and by blockers of the phospholipase pathway. We suggest that bifurcation of the signal transduction takes place at the level of G-protein subunits. The alpha subunit may have a direct effect on adenylyl cyclase, while the betagamma subunit may have a direct effect on phospholipase enzymes.

Identification and quantitation of G-protein alpha-subunits.

The demonstration that many intracellular signaling processes are mediated by a family of closely related guanine nucleotide binding proteins (G-proteins) has led to the development of specific techniques that can be used to identify which of these polypeptide(s) is involved on receptor activation by ligand. In addition, these methods can be used to probe the specificity of the interaction and to yield information about the stoichiometries involved.