The TNFR, TNF-R1, is localized to lipid raft and nonraft regions of the plasma membrane. Ligand binding sets in motion signaling cascades that promote the activation of p42mapk/erk2 and NF-κB. However, the role of receptor localization in the activation of downstream signaling events is poorly understood. In this study, we investigated the dynamics of TNF-R1 localization to lipid rafts and the consequences of raft localization on the activation of p42mapk/erk2 and NF-κB in primary cultures of mouse macrophages. Using sucrose density gradient ultracentrifugation and a sensitive ELISA to detect TNF-R1, we show that TNF-R1 is rapidly and transiently recruited to lipid rafts in response to TNF-α. Disruption of lipid rafts by cholesterol depletion prevented the TNF-α-dependent recruitment of TNF-R1 to lipid rafts and inhibited the activation of p42mapk/erk2, while the activation of NF-κB was unaffected. In addition, phosphorylated p42mapk/erk2, but not receptor interacting protein, I-κB kinase-γ, or I-κBα was detected in raft-containing fractions following TNF-α stimulation. These findings suggest that TNF-R1 is localized to both lipid raft and nonraft regions of the plasma membrane and that each compartment is capable of initiating different signaling responses. We propose that segregation of TNF-R1 to raft and nonraft regions of the plasma membrane contributes to the diversity of signaling responses initiated by TNF-R1.

Chem. 276, 17213-17220). We now examine the role of Class IA PI 3-kinases isoforms in degranulation itself, using a single-cell degranulation assay that measures the binding of fluorescently tagged annexin V to phosphatidylserine in the outer leaflet of the plasma membrane of degranulated mast cells. Consistent with previous data, antibodies against p110␦ and p110␤ blocked Fc⑀R1-mediated degranulation in response to Fc⑀RI ligation. However, antigen-stimulated degranulation was also inhibited by antibodies against p110␣, despite the fact that these antibodies have no effect on antigeninduced calcium flux. These data suggest that p110␣ mediates a calcium-independent signal during degranulation. In contrast, only p110␤ was required for enhancement of antigen-stimulated degranulation by adenosine, which augments mast cell-mediated airway inflammation in asthma. Finally, we examined carbachol-stimulated degranulation in RBL2H3 cells stably expressing the M1 muscarinic receptor (RBL-2H3-M1 cells). Surprisingly, carbachol-stimulated degranulation was blocked by antibody-mediated inhibition of the Class III PI 3-kinase hVPS34 or by titration of its product with FYVE domains. Antibodies against Class IA PI 3-kinases had no effect. These data demonstrate: (a) a calcium-independent role for p110␣ in antigen-stimulated degranulation; (b) a requirement for p110␤ in adenosine receptor signaling; and (c) a requirement for hVPS34 during M1 muscarinic receptor signaling. Elucidation of the intersections between these distinct pathways will lead to new insights into mast cell degranulation.Mast cells are important cellular mediators of allergic responses in humans (1). Moreover, increased levels of mast cells and mast cell-derived inflammatory mediators are found in brochoalveolar lavage fluid from asthmatics, suggesting a role for mast cells in the etiology of clinical asthma (2-4). Crosslinking of cell surface Fc⑀RI receptors leads to the release of pre-formed mediators present in mast cell granules, as well as the induction of cytokines and bioactive lipids (5). Release of these inflammatory molecules in the lung is likely to contribute to inflammation and vasoconstriction during asthma. Antigenmediated degranulation is enhanced by co-stimulation of mast cells with adenosine, which is an important contributor to airway inflammation in asthma (6).The initial signaling events during antigen-stimulated degranulation have been well studied. Fc⑀RI cross-linking leads to recruitment and activation of lyn and syk tyrosine kinases, with subsequent phosphorylation of tyrosine residues in the Fc⑀RI ␥-chain (5, 7). This leads to the recruitment, phosphorylation and activation of phospholipase C␥, and generation of inositol trisphosphate and diacylglycerol from the hydrolysis of plasma membrane phosphatidylinositol (4,5)-bisphosphate. Inositol trisphosphate-mediated release of intracellular calcium stores and activation of classical and novel isoforms of protein kinase C (8) are required for the opening of plasma membrane calcium channels...

Phosphoinositide 3-kinases (PI 3-kinases) are important regulators of endocytic trafficking. Previous studies have shown that mutant human platelet-derived growth factor-␤ receptors (PDGFR), which contain Phe in place of Tyr at the two p85/p110 PI 3-kinase binding sites (PDGFR-F/F), are defective for both p85 binding and ligand-stimulated degradation. This suggested that p85/p110 regulates PDGFR trafficking. However, more recent work has identified hVPS34, and not p85/p110, as the major PI 3-kinase regulating the movement of receptors through the endosomal system. To reconcile this discrepancy, we hypothesized that YXXM motifs in the PDGFR might play a second role as Tyr-based lysosomal sorting motifs (YXX⌽). To test this, we replaced both YXXM motifs with a motif from LAMP-1, YQTI. This mutant PDGFR (PDGFR-YQTI) still underwent PDGF-stimulated autophosphorylation but did not bind p85. In CHO cells, both wild-type and YQTI receptors showed PDGF-stimulated turnover, whereas F/F receptors did not. In addition, uptake and degradation of cell surfacelabeled YXXM and YQTI receptors was fast relative to F/F receptors. We also constructed chimeras containing extracellular and membrane-spanning domains from CD25 (Tac) and cytoplasmic tails containing the YQTI motif, two YXXM motifs, or two mutant FXXM motifs. The YXXM and YQTI chimeras mediated lysosomal delivery of fluorescein isothiocyanate-labeled anti-CD25 antibodies, whereas the F/F chimera was defective. Thus, YQTI motifs can target PDGFR for degradation in the absence of p85/p110 binding, and the p85/p110 binding motifs from PDGFR are sufficient to target Tac chimeras to the lysosome. These data suggest that the YXXM motifs in the PDGFR serve two distinct functions: PI 3-kinase recruitment and lysosomal targeting.PI 3-kinases 1 are important regulators of endocytic trafficking (1, 2). The production of PI(3)P in endosomal and late endosomal/multivesicular body membranes leads to the recruitment of multiple FYVE and Phox (PX) homology domaincontaining proteins (3, 4). These include endosomal tethering proteins (5), proteins involved in the regulation of ubiquitinated receptors (6), RGS (regulators of G-protein signaling) proteins (7), lipid kinases (8 -10), and protein kinases (11). The Class III PI 3-kinase, VPS34, is largely responsible for the production of endosomal PIP 3 in yeast, mammalian cells, and the nematode Caenorhabditis elegans (12-14), although contributions from Class II PI 3-kinases are possible in metazoans.Class I PI 3-kinases have also been implicated in the regulation of vesicular trafficking. Inhibition of the Class IA enzyme p85/p110␣ blocks insulin-stimulated translocation of Glut4 (15). p85/p110␤ has been detected in clathrin-coated vesicles (16), and the p85/p110 product, PI(3,4,5)P 3 , increases the affinity of AP2 adaptins for tyrosine-based endocytic sorting motifs (17). The Class IB PI 3-kinase, PI3K␥, interacts directly with ␤-arrestin kinase I, leading to an increase in ␤-adrenergic receptor endocytosis (18).The role of Class IA PI ...

No abstract