The activation of the Rac1 GTPase during cell signalling entails its translocation from the cytosol to membranes, release from sequestering Rho GDP dissociation inhibitors (RhoGDI), and GDP/GTP exchange. In addition to those steps, we show here that optimal Rac1 activation during cell signalling requires the engagement of a downstream, cytoskeletal-based feedback loop nucleated around the cytoskeletal protein coronin 1A and the Rac1 exchange factor ArhGEF7. These two proteins form a cytosolic complex that, upon Rac1-driven F-actin polymerization, translocates to juxtamembrane areas where it expands the pool of activated, membrane-bound Rac1. Such activity requires the formation of an F-actin/ArhGEF7-dependent physical complex of coronin 1A with Pak1 and RhoGDIa that, once assembled, promotes the Pak1-dependent dissociation of Rac1 from the Rac1/RhoGDIa complex and subsequent Rac1 activation. Genetic evidence demonstrates that this relay circuit is essential for generating sustained Rac1 activation levels during cell signalling.

Vav proteins are phosphorylation-dependent guanine nucleotide exchange factors (GEFs) that catalyze the activation of members of the Rho family of guanosine triphosphatases (GTPases). The current regulatory model holds that the nonphosphorylated, catalytically inactive state of these GEFs is maintained by intramolecular interactions among the amino-terminal domains and the central catalytic core, which block the binding of Vav proteins to GTPases. We showed that this autoinhibition is mechanistically more complex, also involving the bivalent association of the carboxyl-terminal Src homology 3 (SH3) region of Vav with its catalytic and pleckstrin homology (PH) domains. Such interactions occurred through proline-rich region-independent mechanisms. Full release from this double-locked state required synergistic weakening effects from multiple phosphorylated tyrosine residues, thus providing an optimized system to generate gradients of Vav GEF activity depending on upstream signaling inputs. This mechanism is shared by mammalian and Drosophila melanogaster Vav proteins, suggesting that it may be a common regulatory feature for this protein family.

In attempts to investigate the role of galectin-3 in innate immunity, we studied galectin-3-deficient (gal3 ؊/؊ ) mice with regard to their response to Toxoplasma gondii infection, which is characterized by inflammation in affected organs, Th-1-polarized immune response, and accumulation of cysts in the central nervous system. In wild-type (gal3 ؉/؉ ) mice, infected orally, galectin-3 was highly expressed in the leukocytes infiltrating the intestines, liver, lungs, and brain. Compared with gal3 ؉/؉ , infected gal3 ؊/؊ mice developed reduced inflammatory response in all of these organs but the lungs. Brain of gal3 ؊/؊ mice displayed a significantly reduced number of infiltrating monocytes/macrophages and CD8 ؉ cells and a higher parasite burden. Furthermore, gal3 ؊/؊ mice mounted a higher Th1-polarized response and had comparable survival rates on peroral T. gondii infection, even though they were more susceptible to intraperitoneal infection. Interestingly, splenic cells and purified CD11c ؉ dendritic cells from gal3 ؊/؊ mice produced higher amounts of interleukin-12 than cells from gal3 ؉/؉ mice, possibly explaining the higher Th1 response verified in the gal3 ؊/؊ mice. We conclude that galectin-3 exerts an important role in innate immunity, including not only a proinflammatory effect but also a regulatory role on dendritic cells, capable of interfering in the adaptive immune response.