SUMMARY The precise role of caveolae, the characteristic plasma membrane invaginations present in many cells, still remains debated. The high density of caveolae in cells experiencing mechanical stress led us to investigate their role in membrane-mediated mechanical response. Acute mechanical stress induced by cell osmotic swelling or by uniaxial stretching results in the immediate disappearance of caveolae, which is associated with a reduced caveolin/Cavin1 interaction, and an increase of free caveolins at the plasma membrane. Tether pulling force measurements in live cells and in plasma membrane spheres demonstrate that caveola flattening and disassembly is the primary actin and ATP-independent cell response which buffers membrane tension surges during mechanical stress. Conversely, stress release leads to complete caveola reassembly in an actin and ATP-dependent process. The absence of a functional caveola reservoir in myotubes from muscular dystrophic patients enhanced membrane fragility under mechanical stress. Our findings support a new role for caveolae as a physiological membrane reservoir that allows cells to quickly accommodate sudden and acute mechanical stresses.

The P2X7 receptor (P2X7R) is an ATP-gated cationic channel expressed by hematopoietic, epithelial, and neuronal cells. Prolonged ATP exposure leads to the formation of a nonselective pore, which can result in cell death. We show that P2X7R is associated with detergent-resistant membranes (DRMs) in both transfected human embryonic kidney (HEK) cells and primary macrophages independently from ATP binding. The DRM association requires the posttranslational modification of P2X7R by palmitic acid. Treatment of cells with the palmitic acid analog 2-bromopalmitate as well as mutations of cysteine to alanine residues abolished P2X7R palmitoylation. Substitution of the 17 intracellular cysteines of P2X7R revealed that 4 regions of the carboxyl terminus domain are involved in palmitoylation. Palmitoylation-defective P2X7R mutants showed a dramatic decrease in cell surface expression because of their retention in the endoplasmic reticulum and proteolytic degradation. Taken together, our data demonstrate that P2X7R palmitoylation plays a critical role in its association with the lipid microdomains of the plasma membrane and in the regulation of its half-life.

Understanding how membrane nanoscale organization controls transmembrane receptors signaling activity remains a challenge. We studied interferon-γ receptor (IFN-γR) signaling in fibroblasts from homozygous patients with a T168N mutation in IFNGR2. By adding a neo-N-glycan on IFN-γR2 subunit, this mutation blocks IFN-γ activity by unknown mechanisms. We show that the lateral diffusion of IFN-γR2 is confined by sphingolipid/cholesterol nanodomains. In contrast, the IFN-γR2 T168N mutant diffusion is confined by distinct actin nanodomains where conformational changes required for Janus-activated tyrosine kinase/signal transducer and activator of transcription (JAK/STAT) activation by IFN-γ could not occur. Removing IFN-γR2 T168N-bound galectins restored lateral diffusion in lipid nanodomains and JAK/STAT signaling in patient cells, whereas adding galectins impaired these processes in control cells. These experiments prove the critical role of dynamic receptor interactions with actin and lipid nanodomains and reveal a new function for receptor glycosylation and galectins. Our study establishes the physiological relevance of membrane nanodomains in the control of transmembrane receptor signaling in vivo. VIDEO ABSTRACT.

The amyloid precursor protein (APP) is cleaved by ␤-and ␥-secretases to generate the ␤-amyloid (A␤) peptides, which are present in large amounts in the amyloid plaques of Alzheimer disease (AD) patient brains. Non-amyloidogenic processing of APP by ␣-secretases leads to proteolytic cleavage within the A␤ peptide sequence and shedding of the soluble APP ectodomain (sAPP␣), which has been reported to be endowed with neuroprotective properties. In this work, we have shown that activation of the purinergic receptor P2X7 (P2X7R) stimulates sAPP␣ release from mouse neuroblastoma cells expressing human APP, from human neuroblastoma cells and from mouse primary astrocytes or neural progenitor cells. sAPP␣ shedding is inhibited by P2X7R antagonists or knockdown of P2X7R with specific small interfering RNA (siRNA) and is not observed in neural cells from P2X7R-deficient mice. P2X7R-dependent APP-cleavage is independent of extracellular calcium and strongly inhibited by hydroxamate-based metalloprotease inhibitors, TAPI-2 and GM6001. However, knockdown of a disintegrin and metalloproteinase-9 (ADAM9), ADAM10 and ADAM17 by specific siRNA, known to have ␣-secretase activity, does not block the P2X7R-dependent non-amyloidogenic pathway. Using several specific pharmacological inhibitors, we demonstrate that the mitogen-activated protein kinase modules Erk1/2 and JNK are involved in P2X7R-dependent ␣-secretase activity. Our study suggests that P2X7R, which is expressed in hippocampal neurons and glial cells, is a potential therapeutic target in AD.The amyloid precursor protein (APP) 3 is a transmembrane protein expressed by neurons, astrocytes, microglia, and neural progenitor cells (NPCs) in the central nervous system (CNS). APP can be cleaved by three different secretases called: ␣, ␤, and ␥, depending on their cleavage sites on the APP (1, 2). The amyloid-␤ (A␤) peptides generated by ␤-and ␥-secretases are found in senile plaques of patients with Alzheimer Disease (AD). The ␣-secretase cleaves APP within the A␤ peptide sequence, precluding the formation of neurotoxic A␤ peptides. The soluble fragment of APP, sAPP␣ produced after cleavage by the ␣-secretase, has been shown to have neurotrophic and neuroprotective properties (3-5). Several enzymes capable of mediating non-amyloidogenic ␣-processing of APP have been identified: ADAM9, -10, and -17 (6). However, several proteases may cooperate in the physiological ␣-cleavage of APP in CNS. Various reports have shown that ADAM activation is induced by G protein-coupled receptor (GPCR) agonists. Ca 2ϩ increase, ROS production, and phosphorylation of ADAMs by protein kinases C (PKC) or mitogen-activated protein kinases (MAP kinases), are involved in ADAM activation following stimulation of GPCR (7,8). However, receptors other than GPCR are able to activate PKC and MAP kinases and/or to induce Ca 2ϩ increase and ROS production. Indeed, the purinergic receptor P2X7 (P2X7R) is endowed with such properties (9).P2X7R is a non-selective ATP-gated cation channel, expressed by various ce...

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J. Neurochem. (2009) 109, 846–857. Abstract Neural progenitor cells (NPCs) are capable of self‐renewal and differentiation into neurons, astrocytes and oligodendrocytes, and have been used to treat several animal models of CNS disorders. In the present study, we show that the P2X7 purinergic receptor (P2X7R) is present on NPCs. In NPCs, P2X7R activation by the agonists extracellular ATP or benzoyl ATP triggers opening of a non‐selective cationic channel. Prolonged activation of P2X7R with these nucleotides leads to caspase independent death of NPCs. P2X7R ligation induces NPC lysis/necrosis demonstrated by cell membrane disruption accompanied with loss of mitochondrial membrane potential. In most cells that express P2X7R, sustained stimulation with ATP leads to the formation of a non‐selective pore allowing the entry of solutes up to 900 Da, which are reportedly involved in P2X7R‐mediated cell lysis. Surprisingly, activation of P2X7R in NPCs causes cell death in the absence of pore formation. Our data support the notion that high levels of extracellular ATP in inflammatory CNS lesions may delay the successful graft of NPCs used to replace cells and repair CNS damage.

Cytokines belonging to the common gamma chain (γ) family depend on the shared γ receptor subunit for signaling. We report the existence of a fast, cytokine-induced pathway cross-talk acting at the receptor level, resulting from a limiting amount of γ on the surface of T cells. We found that this limited abundance of γ reduced interleukin-4 (IL-4) and IL-21 responses after IL-7 preexposure but not vice versa. Computational modeling combined with quantitative experimental assays indicated that the asymmetric cross-talk resulted from the ability of the "private" IL-7 receptor subunits (IL-7Rα) to bind to many of the γ molecules even before stimulation with cytokine. Upon exposure of T cells to IL-7, the high affinity of the IL-7Rα:IL-7 complex for γ further reduced the amount of free γ in a manner dependent on the concentration of IL-7. Measurements of bioluminescence resonance energy transfer (BRET) between IL-4Rα and γ were reduced when IL-7Rα was overexpressed. Furthermore, in a system expressing IL-7Rα, IL-4Rα, and γ, BRET between IL-4Rα and γ increased after IL-4 binding and decreased when cells were preexposed to IL-7, supporting the assumption that IL-7Rα and the IL-7Rα:IL-7 complex limit the accessibility of γ for other cytokine receptor complexes. We propose that in complex inflammatory environments, such asymmetric cross-talk establishes a hierarchy of cytokine responsiveness.

Type I interferons (IFNs) bind IFNAR receptors and activate Jak kinases and Stat transcription factors to stimulate the transcription of genes downstream from IFN-stimulated response elements. In this study, we analyze the role of protein palmitoylation, a reversible post-translational lipid modification, in the functional properties of IFNAR. We report that pharmacological inhibition of protein palmitoylation results in severe defects of IFN receptor endocytosis and signaling. We generated mutants of the IFNAR1 subunit of the type I IFN receptor, in which each or both of the two cysteines present in the cytoplasmic domain are replaced by alanines. We show that cysteine 463 of IFNAR1, the more proximal of the two cytoplasmic cysteines, is palmitoylated. A thorough microscopic and biochemical analysis of the palmitoylation-deficient IFNAR1 mutant revealed that IFNAR1 palmitoylation is not required for receptor endocytosis, intracellular distribution, or stability at the cell surface. However, the lack of IFNAR1 palmitoylation affects selectively the activation of Stat2, which results in a lack of efficient Stat1 activation and nuclear translocation and IFN-␣-activated gene transcription. Thus, receptor palmitoylation is a previously undescribed mechanism of regulating signaling activity by type I IFNs in the Jak/Stat pathway.Type I interferons (IFN 7 ␣/␤) are potent cellular mediators essential for several key cell functions, including immunomodulatory, antiviral, and antiproliferative activities. These pleiotropic effects occur through the transcriptional regulation of many IFN-stimulated genes (ISGs) (1). IFN signal transduction relies mainly on the activation of the Janus tyrosine kinase (Jak)/signal-transducing activators of transcription (Stat) pathways, although several other signaling cascades have also been associated with IFN-regulated transcription (2, 3). In general, the binding of type I IFNs to the cell surface receptor IFNAR1 and IFNAR2 subunits induces tyrosine phosphorylation in trans of the IFNAR-associated Jak kinases (Tyk2 with IFNAR1 and Jak1 with IFNAR2), which in turn leads to IFNAR tyrosine phosphorylation. Several members of the Stat family can be activated by type I IFNs, and Stat1 and Stat2 are the main downstream effectors of the type I IFN transcriptional response. Upon IFN-␣ stimulation, cytosolic Stat2 is recruited to the activated IFNAR complex where it becomes tyrosine-phosphorylated by the receptor-associated Jak kinases. Stat2 activation is a key event in IFN-␣ signaling because it is required for the indirect recruitment, through binding to Stat2, of Stat1 to IFNAR1 and its activation. There is some debate as to whether cytosolic Stat2 is preferentially recruited to IFNAR1 or to IFNAR2 (4 -9). Whereas the SH2 domain of Stat2 binds to a region surrounding the phosphorylated tyrosine 466 of IFNAR1, Stat2 can bind to IFNAR2 whether it is tyrosine-phosphorylated or not. This series of sequential tyrosine phosphorylations precedes the translocation of the IFN-stimulated gene factor 3...