Activated heterotrimeric G protein αi subunits inhibit Rap-dependent cell adhesion and promote cell migration.

Our recent work uncovered novel roles for activated Gαi signaling in the regulation of neutrophil polarity and adhesion. GαiGTP-mediated enhancement of neutrophil polarization was dependent on inhibition of cAMP/PKA signaling, whereas reversal of Gßγ-stimulated adhesion was cAMP/PKA independent. To uncover the mechanism for Gαi regulation of adhesion, we analyzed the effects of constitutively active Gαi1(Q204L) expression on adhesion driven by constitutively active Rap1a(G12V) or its downstream effector Radil in neutrophil-like HL-60 cells, or in HT-1080 fibrosarcoma cells. In HT-1080 cells, Rap1a(G12V) or Radil cause an increase in cell spreading and adhesion to fibronectin, which are both reversed by Gαi1(Q204L) but not WT Gαi1 In contrast, Gαi1(Q204L) did not reverse Rap1-GTP-interacting adaptor molecule (RIAM)-dependent increases in cell adhesion. This indicates that adhesion regulation by Gαi-GTP occurs downstream of Rap1a and Radil, but is upstream of components such as integrins and talin that are regulated by both Radil and RIAM. HL-60 neutrophil-like cells expressing Rap1a(G12V) or Radil have an elongated phenotype because of enhanced uropod adhesion as they attempt to migrate on fibronectin. This elongated phenotype driven by Rap1a(G12V) or Radil is reversed by Gαi1(Q204L), but not by WT Gαi1 expression, suggesting that Gαi-GTP also regulates adhesion in immune cells at the level of, or downstream of, Radil. These data identify a novel role of Gαi-GTP in regulation of cell adhesion and migration. Cell migration involves cycles of adhesion and de-adhesion, and we propose that the dynamic spatiotemporal balance between Gßγ-promoted adhesion and Gαi-GTP reversal of adhesion is important for this process.

Inhibition of G Protein ßγ Subunit Signaling Abrogates Nephritis in Lupus-Prone Mice.

OBJECTIVE: Despite considerable advances in the understanding of systemic lupus erythematosus (SLE), there is still an urgent need for new and more targeted treatment approaches. We previously demonstrated that small-molecule blockade of G protein ßγ subunit (Gßγ) signaling inhibits acute inflammation through inhibition of chemokine receptor signal transduction. We undertook this study to determine whether inhibition of Gßγ signaling ameliorates disease in a mouse model of SLE. METHODS: Lupus-prone (NZB × NZW)F1 female mice were prophylactically or therapeutically treated with the small-molecule Gßγ inhibitor gallein. Tissue samples were analyzed by flow cytometry and immunohistochemistry. The development and extent of nephritis were assessed by monitoring proteinuria and by immunohistochemical analysis. Serum immunoglobulin levels were measured by enzyme-linked immunosorbent assay, and total IgG and anti-double-stranded DNA (anti-dsDNA) antibody-secreting cells were measured by enzyme-linked immunospot assay. RESULTS: Gallein inhibited accumulation of T cells and germinal center (GC) B cells in the spleen. Both prophylactic and therapeutic treatment reduced GC size, decreased antibody-secreting cell production in the spleen, and markedly decreased accumulation of autoreactive anti-dsDNA antibody-secreting cells in kidneys. Gallein also reduced immune complex deposition in kidneys. Finally, gallein treatment dramatically inhibited kidney inflammation, prevented glomerular damage, and decreased proteinuria. Mechanistically, gallein inhibited immune cell migration and signaling in response to chemokines in vitro, which suggests that its mechanisms of action in vivo are inhibition of migration of immune cells to sites of inflammation and inhibition of immune cell maturation. CONCLUSION: Overall, these data demonstrate the potential use of gallein or novel inhibitors of Gßγ signaling in SLE treatment.

Dynamic regulation of neutrophil polarity and migration by the heterotrimeric G protein subunits Gαi-GTP and Gßγ.

Activation of the Gi family of heterotrimeric guanine nucleotide-binding proteins (G proteins) releases ßγ subunits, which are the major transducers of chemotactic G protein-coupled receptor (GPCR)-dependent cell migration. The small molecule 12155 binds directly to Gßγ and activates Gßγ signaling without activating the Gαi subunit in the Gi heterotrimer. We used 12155 to examine the relative roles of Gαi and Gßγ activation in the migration of neutrophils on surfaces coated with the integrin ligand intercellular adhesion molecule-1 (ICAM-1). We found that 12155 suppressed basal migration by inhibiting the polarization of neutrophils and increasing their adhesion to ICAM-1-coated surfaces. GPCR-independent activation of endogenous Gαi and Gßγ with the mastoparan analog Mas7 resulted in normal migration. Furthermore, 12155-treated cells expressing a constitutively active form of Gαi1 became polarized and migrated. The extent and duration of signaling by the second messenger cyclic adenosine monophosphate (cAMP) were enhanced by 12155. Inhibiting the activity of cAMP-dependent protein kinase (PKA) restored the polarity of 12155-treated cells but did not decrease their adhesion to ICAM-1 and failed to restore migration. Together, these data provide evidence for a direct role of activated Gαi in promoting cell polarization through a cAMP-dependent mechanism and in inhibiting adhesion through a cAMP-independent mechanism.