Increased vascular permeability is a key feature of inflammatory conditions. In severe infections, leakage of plasma from the vasculature induces a life-threatening hypotension. Streptococcus pyogenes, a major human bacterial pathogen, causes a toxic shock syndrome (STSS) characterized by excessive plasma leakage and multi-organ failure. Here we find that M protein, released from the streptococcal surface, forms complexes with fibrinogen, which by binding to beta2 integrins of neutrophils, activate these cells. As a result, neutrophils release heparin binding protein, an inflammatory mediator inducing vascular leakage. In mice, injection of M protein or subcutaneous infection with S. pyogenes causes severe pulmonary damage characterized by leakage of plasma and blood cells. These lesions were prevented by treatment with a beta2 integrin antagonist. In addition, M protein/fibrinogen complexes were identified in tissue biopsies from a patient with necrotizing fasciitis and STSS, further underlining the pathogenic significance of such complexes in severe streptococcal infections.

Staphylococcus aureus is a prominent human pathogen. Here we report that intact S. aureus bacteria activate the contact system in human plasma in vitro, resulting in a massive release of the potent proinflammatory and vasoactive peptide bradykinin. In contrast, no such effect was recorded with Streptococcus pneumoniae. In the activation of the contact system, blood coagulation factor XII and plasma kallikrein play central roles, and a specific inhibitor of these serine proteinases inhibited the release of bradykinin by S. aureus in human plasma. Furthermore, fragments of the cofactor H-kininogen of the contact system efficiently blocked bradykinin release. The results suggest that activation of the contact system at the surface of S. aureus and the subsequent release of bradykinin could contribute to the hypovolemic hypotension seen in patients with severe S. aureus sepsis. The data also suggest that the contact system could be used as a target in the treatment of S. aureus infections.Gram-positive bacteria are currently as common as gramnegative bacteria in causing sepsis, and Staphylococcus aureus and Streptococcus pneumoniae are the most frequently isolated pathogens in gram-positive sepsis (5, 6, 16). These species can give rise to septic shock, a condition with a high mortality rate despite antibiotic treatment and improvements in intensive care. The pathogenesis of sepsis is not fully understood. However, there appears to be a common pathway by which both gram-negative and gram-positive bacteria induce the production of different inflammatory mediators, such as factors of the complement, coagulation, and contact systems, which act together with cytokines to form a complex inflammatory network (5, 9).The contact system consists of three enzymatic factors, factor XI (FXI), FXII, and plasma prekallikrein (PK), and the nonenzymatic cofactor H-kininogen (HK) (24). Activation of FXII is the initial step leading to the formation of kallikrein and activated FXI. As a result, bradykinin (BK), a nonapeptide, is released from HK. BK induces vasodilatation and increased microvascular permeability, effects that in part are mediated by the secondary release of other mediators (for instance, nitric oxide and platelet activating factor) via activation of BK receptors of the vascular endothelium. The contact system can also be activated directly by endotoxin and microbial proteinases (11,15,18).When injected into animals, BK reduces peripheral vascular resistance, leading to hypotension and elevated cardiac output (26), and several animal studies have shown that activation of the contact system correlates with irreversible hypotension during sepsis (25,26). Investigations of humans have revealed that factors of the contact system are consumed in plasma of patients with severe sepsis and, especially, that persistently low levels of FXII are a bad prognostic sign (14,21,27,28). A pathogenic role for the contact system is also suggested by observations that it can be activated by Streptococcus pyogenes, Salmonella, and Escher...

In PC12 cells, a well studied model for neuronal differentiation, an elevation in the intracellular cAMP level increases cell survival, stimulates neurite outgrowth, and causes activation of extracellular signalregulated protein kinase 1 and 2 (ERK1/2). Here we show that an increase in the intracellular cAMP concentration induces tyrosine phosphorylation of two receptor tyrosine kinases, i.e. the epidermal growth factor (EGF) receptor and the high affinity receptor for nerve growth factor (NGF), also termed Trk A . cAMP-induced tyrosine phosphorylation of the EGF receptor is rapid and correlates with ERK1/2 activation. It occurs also in Panc-1, but not in human mesangial cells. cAMP-induced tyrosine phosphorylation of the NGF receptor is slower and correlates with Akt activation. Inhibition of EGF receptor tyrosine phosphorylation, but not of the NGF receptor, reduces cAMP-induced neurite outgrowth. Expression of dominant-negative Akt does not abolish cAMP-induced survival in serum-free media, but increases cAMP-induced ERK1/2 activation and neurite outgrowth. Together, our results demonstrate that cAMP induces dual signaling in PC12 cells: transactivation of the EGF receptor triggering the ERK1/2 pathway and neurite outgrowth; and transactivation of the NGF receptor promoting Akt activation and thereby modulating ERK1/2 activation and neurite outgrowth.Neuronal development, differentiation, survival, and repair are subject to regulation by many different external signals under physiological and pathological conditions. For instance, the high affinity receptor for nerve growth factor (NGFR), 1 a receptor tyrosine kinase (RTK) also termed Trk A , is an important mediator of development, differentiation and survival of neurons (1, 2). The rat pheochromocytoma cell line PC12 is the best studied model of neuronal differentiation and survival. In these cells, nerve growth factor (NGF) causes survival upon serum-withdrawal and promotes neurite outgrowth. Activation of the epidermal growth factor receptor (EGFR), another RTK, can induce both proliferation and differentiation (3, 4); the latter response is strongly increased in EGFR-overexpressing cells (5). Activation of the extracellular signal-regulated kinases 1/2 (ERK1/2) pathway appears to play an important role in growth factor-mediated PC12 cell differentiation (5, 6). The mechanism of ERK1/2 activation by RTKs is well established and involves receptor autophosphorylation, recruitment of adaptor proteins such as Shc and Grb2 to the receptor, and activation of guanine nucleotide exchange factors acting on and thereby activating the small GTPase Ras. Active Ras recruits Raf kinases to the membrane, which leads to their activation and subsequent triggering of the ERK pathway (7).Studies on the pro-survival effect of NGF in PC12 cells show that activation of phosphatidylinositol 3-kinase (PI3K) is critical for its protective effect (8). Upon activation, PI3K phosphorylates membrane phosphoinositides at the D-3 position. These 3Ј-phosphorylated phospholipids act as s...

Endothelins are potent mitogens that stimulate extracellular signal-regulated kinases (ERK/MAP kinases) through their cognate G-protein-coupled receptors, ET A and ET B . To address the role of post-translational ET receptor modifications such as acylation on ERK activation and to identify relevant downstream effectors coupling the ET receptor to the ERK signaling cascades we have constructed a panel of palmitoylation-deficient ET receptor mutants with differential Ga protein binding capacity. Endothelin-1 stimulation of wild-type ET A or ET B induced a fivefold to sixfold increase in ERK in COS-7 and CHO cells whereas fulllength nonpalmitoylated ET A and ET B mutants failed to stimulate ERK. A truncated ET B lacking the C-terminal tail domain including putative phosphorylation and arrestin binding site(s) but retaining the critical palmitoylation site(s) was still able to fully stimulate ERK activation. Using mutated ET receptors with selective G-protein-coupling we found that endothelin-induced stimulation of Ga q , but not of

Post-translational modifications such as phosphorylation and palmitoylation play important roles for the function and regulation of receptors coupled to heterotrimeric guanyl nucleotide-binding proteins. Here we demonstrate that the human endothelin receptor A (ET A ) incorporates [ 3 H]palmitate. Mutation of a cluster of five cysteine residues present in the cytoplasmic tail of ET A into serine or alanine residues completely prevented palmitoylation of the receptor. The ligand binding affinity of the non-palmitoylated ET A mutants was essentially unchanged as compared to the palmitoylated wild type ET A suggesting that the replacement of the cysteine residues did not alter the overall structure of the receptor. Furthermore, the ligand-induced stimulation of adenylyl cyclase by the mutant ET A was unaffected by the mutation. In contrast, the mutated nonpalmitoylated receptors but not the wild type receptor failed to stimulate phosphatidylinositol hydrolysis by phospholipase C activation upon challenge by endothelin-1. Furthermore, the mutant receptors failed to stimulate the ligand-induced transient increase in the cytoplasmic calcium seen with the wild type ET A . Endothelin-1 induced mitogenic stimuli via the wild type receptors but not through the mutated receptors suggesting an important role for phospholipase C in this signal transduction pathway. The differential regulation of distinct signal transduction pathways by posttranslational modification suggests that palmitoylation of the ET A provides a novel mechanism of modulating ET A receptor activity.Post-translational modifications of receptors coupled to heterotrimeric guanyl nucleotide-binding proteins (G protein) modulate receptor function and activity (1-3). One such modification is the palmitoylation of conserved cysteine residues located in the cytoplasmic tail of many G protein-coupled receptors. It has been suggested that this acylation provides a membrane anchor that creates a fourth cytoplasmic receptor loop (4, 5). The functional role of palmitoylation has been studied for adrenergic receptors in some detail. Prevention of palmitoylation of a conserved cysteine residue, Cys 341 , of the ␤-adrenergic receptor causes functional uncoupling of the receptor from the adenylyl cyclase pathway (4). Furthermore, the nonpalmitoylated receptor shows an increased basal phosphorylation and a rapid desensitization in response to its ligand (6). Activation of the ␤ 2 -adrenergic receptor results in an enhanced palmitoylation thereby increasing the amount of functionally coupled receptor (7). Adrenergic receptor activation also increases palmitoylation of a receptor-associated stimulatory G protein (8). These findings clearly show the dynamic properties of protein/receptor palmitoylation. Unlike the ␤ 2 -adrenergic receptor a mutation of the corresponding palmitoylation site in the ␣ 2A -adrenergic receptor did not influence the signal transduction activity but decreased the ligand-promoted down-regulation of the receptor (9). On the other hand, purified nonpa...