Nitric Oxide Produced in Response to Engagement of β2 Integrins on Human Neutrophils Activates the Monomeric GTPases Rap1 and Rap2 and Promotes Adhesion

Abstract

We found that engagement of ␤2 integrins on human neutrophils increased the levels of GTP-bound Rap1 and Rap2. Also, the activation of Rap1 was blocked by PP1, SU6656, LY294002, GF109203X, or BAPTA-AM, which indicates that the downstream signaling events in Rap1 activation involve Src tyrosine kinases, phosphoinositide 3-kinase, protein kinase C, and release of calcium. Surprisingly, the ␤2 integrin-induced activation of Rap2 was not regulated by any of the signaling pathways mentioned above. However, we identified nitric oxide as the signaling molecule involved in ␤2 integrin-induced activation of Rap1 and Rap2. This was illustrated by the fact that engagement of ␤2 integrins increased the production of nitrite, a stable end-product of nitric oxide. Furthermore, pretreatment of neutrophils with N -monomethyl-L-arginine, or 1400W, which are inhibitors of inducible nitric-oxide synthase, blocked ␤2 integrin-induced activation of Rap1 and Rap2. Similarly, R p -8pCPT-cGMPS, an inhibitor of cGMP-dependent serine/threonine kinases, also blunted the ␤2 integrin-induced activation of Rap GTPases. Also nitric oxide production and its downstream activation of cGMP-dependent serine/threonine kinases were essential for proper neutrophil adhesion by ␤2 integrins. Thus, we made the novel findings that ␤2 integrin engagement on human neutrophils triggers production of nitric oxide and its downstream signaling is essential for activation of Rap GTPases and neutrophil adhesion.

Integrin α9β1 mediates enhanced cell migration through nitric oxide synthase activity regulated by Src tyrosine kinase

“…It also appears that the RGD sequence itself is not essential for activation as integrin α9β1 does not use RGD as a binding motif (Yokasaki and Sheppard, 2000). Furthermore, specific β-subunits do not appear to be essential because β2 integrins also induce NO production in neutrophils (Jenei et al, 2006). Instead, our results suggest that the α-subunit, and specifically its cytoplasmic domain, is the key determinant for integrin-induced iNOS activation.…”

“…As components of the protein signaling pathways that transduce integrin and NO effects are shared, such as Src and Rac-1 noted above, we wished to determine whether there are interactions between integrins and iNOS and whether this interaction might modulate cell migration. Previous studies have reported that, when activated, β2 integrins expressed on neutrophils can stimulate NO production (Jenei et al, 2006). However, whether this effect occurs by means of β1 integrins and the nature by which NO might subsequently modulate integrin-associated cell migration are incompletely understood.…”

Characterization of Interactions of Adapter Protein RAPL/Nore1B with RAP GTPases and Their Role in T Cell Migration

“…Previous studies of Rap2 proteins were limited and often focused on comparison with Rap1 (36,(41)(42)(43)(44), whereas Nore1B/RAPL protein was mainly considered as an effector of Rap1 in lymphocytes (13,14). Although Rap1 and Rap2 proteins are closely related (40), the functional link between Rap2 and RAPL has not been explored.…”

p190RhoGAP and Rap-dependent RhoGAP (ARAP3) inactivate RhoA in response to nerve growth factor leading to neurite outgrowth from PC12 cells

“…Transfection of DN-Rap1 (Rap1N17) significantly inhibited neurite outgrowth, whereas WT-and CA-Rap1 (Rap1 V12) did not affect it in response to NGF ( Figure 3B). A putative inhibitor of Rap1, 8CPT-cGMP (Jenei et al, 2006), significantly The interaction of Rap1 and RhoA in response to NGF was examined. Serum-starved PC12 cells were treated with 100 ng/ml NGF for various times as indicated and lysed.…”