Astrocytes are involved in normal and pathological brain functions, where they become activated and undergo reactive gliosis. Astrocytes have been shown to respond to extracellular nucleotides via the activation of P2 receptors, either G protein-coupled P2Y receptors or P2X receptors that are ligand-gated ion channels. In this study, we have examined the manner in which activation of the P2X(7) nucleotide receptor, an extracellular ATP-gated ion channel expressed in astrocytes, can lead to the phosphorylation of ERK1/2. Results showed that the P2X(7) receptor agonist 2',3'-O-(4-benzoyl)benzoyl-ATP induced ERK1/2 phosphorylation in human astrocytoma cells overexpressing the recombinant rat P2X(7) receptor (rP2X(7)-R), a response that was inhibited by the P2X(7) receptor antagonist, oxidized ATP. Other results suggest that rP2X(7)-R-mediated ERK1/2 phosphorylation was linked to the phosphorylation of the proline-rich/Ca(2+)-activated tyrosine kinase Pyk2, c-Src, phosphatidylinositol 3'-kinase, and protein kinase Cdelta activities and was dependent on the presence of extracellular Ca(2+). These results support the hypothesis that the P2X(7) receptor and its signaling pathways play a role in astrocyte-mediated inflammation and neurodegenerative disease.

Molecular determinants of P2Y 2 receptor desensitization and sequestration have been investigated. Wildtype P2Y 2 receptors and a series of five C-terminal truncation mutants of the receptor were epitope-tagged and stably expressed in 1321N1 cells. These constructs were used to assess the importance of the intracellular C terminus on 1) UTP-stimulated increases in intracellular calcium concentration, 2) homologous desensitization of the receptor, and 3) agonist-induced decreases in cellsurface density (receptor sequestration) of epitopetagged receptors using fluorescence-activated cell sorting. The potency and efficacy of UTP were similar for the wild-type and all mutant P2Y 2 receptors. Truncation of 18 or more amino acids from the C terminus increased by ϳ30-fold the concentration of UTP necessary to desensitize the receptor. Both the rate and magnitude of UTP-induced receptor sequestration were decreased with progressively larger truncations of the C terminus. Furthermore, the recovery from sequestration was slower for the most extensively truncated receptor. Complete desensitization was obtained with >50% of the original receptor complement remaining on the cell surface. Protein kinase C activation, which desensitizes the P2Y 2 receptor, had no effect on sequestration, consistent with the ideas that desensitization and sequestration are discrete events and that agonist occupancy is required for receptor sequestration.Responses to extracellular nucleotides are mediated by P2 receptors that belong to two receptor superfamilies: P2Y G protein-coupled receptors (GPCRs) 1 and P2X ligand-gated ion channels. Multiple P2Y subtypes have been classified pharmacologically and molecularly and are predominantly linked to activation of phospholipase C and increased levels of inositol 1,4,5-trisphosphate and diacylglycerol, leading to elevations in the intracellular free calcium concentration ([Ca 2ϩ ] i ) and the activation of protein kinase C (PKC) (1-4). The P2Y 2 nucleotide receptor subtype (formerly the P 2U receptor) is distinguished pharmacologically from the other known mammalian P2Y receptor subtypes by the equal potency and efficacy of the naturally occurring agonists ATP and UTP.Activation of P2Y 2 receptors present in airway epithelium increases Cl Ϫ secretion through Ca 2ϩ -dependent and outwardly rectifying Cl Ϫ channels (5). It has been shown that P2Y 2 receptor activation by UTP in airway epithelia of cystic fibrosis patients can increase Cl Ϫ secretion, thereby effectively bypassing the defective cAMP-dependent Cl Ϫ transport (6). Like other members of the GPCR superfamily, P2Y 2 receptors undergo agonist-induced desensitization (7), but little is known about the mechanisms involved in desensitization of the P2Y 2 receptor. It seems likely that a fuller understanding of desensitization and the signaling pathways that affect the P2Y 2 receptor may lead to improved therapies targeted to this receptor.Desensitization of GPCRs is a complex process involving phosphorylation of the receptors by multiple pro...

Bethesda, Md., and was propagated in modified Hayflick medium (26) supplemented with 10% (vol/vol) heat-inactivated horse serum (JRH Biosciences, Lenexa, Kans.). M. fennentans Incognitus was isolated and propagated in SP4 medium as described previously (8). Unless specified otherwise, organisms were harvested from logarithmic-phase cultures by centrifugation, washed in phosphate-buffered saline (PBS; 2.7 mM KCl, 1.5 mM KH2PO4, 137 mM NaCl, 8 mM Na2HPO4; pH 7.2), and stored at-70°C prior to use. Other human mycoplasmas were obtained from J. Tully, Frederick Cancer Research and Development Center, Frederick, Md., as frozen broth cultures. These were thawed, and organisms were harvested by centrifugation at 19,000 x g for 10 min. Cell pellets were washed by centrifugation and suspended in PBS. Human mycoplasma species included M.

The wall-less mycoplasmas have revealed unusual microbial strategies for adaptive variation of antigenic membrane proteins exposed during their surface colonization of host cells. In particular, high-frequency mutations affecting the expression of selected surface lipoproteins have been increasingly documented for this group of organisms. A novel manifestation of mutational phase variation is shown here to occur in Mycoplasma fermentans, a chronic human infectious agent and possible AIDS-associated pathogen. A putative ABC type transport operon encoding four gene products is identified. The 3 distal gene encoding P78, a known surface-exposed antigen and the proposed substrate-binding lipoprotein of the transporter, is subject to localized hypermutation in a short homopolymeric tract of adenine residues located in the N-terminal coding region of the mature product. High-frequency, reversible insertion/deletion frameshift mutations lead to selective phase variation in P78 expression, whereas the putative nucleotide-binding protein, P63, encoded by the most 5 gene of the operon, is continually expressed. Mutation-based phase variation in specific surfaceexposed microbial transporter components may provide an adaptive advantage for immune evasion, while continued expression of other elements of the same transporter may preserve essential metabolic functions and confer alternative substrate specificity. These features could be critical in mycoplasmas, where limitations in both transcriptional regulators and transport systems may prevail. This study also documents that P63 contains an uncharacteristic hydrophobic sequence between predicted nucleotide binding motifs and displays an amphiphilic character in detergent fractionation. Both features are consistent with an evolutionary adaptation favoring integral association of this putative energy-transducing component with the single mycoplasma membrane.With an ability to establish persistent or chronic infections in its mammalian host (30, 54), Mycoplasma fermentans typifies many of the nearly 100 species in the genus Mycoplasma (44). This agent has been implicated as a human pathogen associated with fulminant respiratory distress syndrome (31, 33) and pathologic lesions in immunocompromised individuals with AIDS (7, 32). Like all mycoplasmas, this species is evolutionarily related to the low-GϩC-content branch of gram-positive eubacteria (61) and contains a small genome (20, 23). Mycoplasmas offer interesting and useful models as minimal prokaryotic pathogens. Notably, these organisms endure an extracellular existence exposed to host defenses, yet they possess only a single limiting plasma membrane without the additional protective outer membrane system or cell wall matrix characteristic of other eubacteria. Consequently, critical systems for transport and other metabolic pathways associated with "periplasmic" functions in both gram-negative and gram-positive eubacteria (4, 36) are on the membrane surface of mycoplasmas. How such components are concealed or otherwise ev...

Mycoplasma fermentans, a wall-less prokaryote, is currently under investigation as a potential human pathogen. Recently, several surface lipoproteins have been shown to vary in expression between M. fermentans strains. Using specific antibodies to these lipoproteins, we investigated the extent and nature of antigenic variation within this species. Immunoscreening of type strain PG18 agar-grown colonies revealed marked heterogeneity in expression of distinct surface lipoproteins. Subsequent isolation and propagation of clonal isolates established isogenic lineages which displayed high-frequency (10-2 to 10-5 per generation) antigenic phase variation.[35S]cysteine-labeled protein profiles and Western immunoblots of phase-variant clones showed that several distinct integral membrane proteins undergo noncoordinate variation in expression. In addition to differential expression of epitope-bearing lipoproteins, differential accessibility of epitopes to antibodies was also documented as a mechanism generating surface phenotypic variation. Examination of one strain-variant antigen showed high-frequency phase variation to underlie previously observed antigenic differences between strains of this species. Thus, M. fermentans has a complex system capable of creating rapid changes in surface mosaics. This may profoundly affect mycoplasma-host interactions and may limit the methods by which populations of M. fermentans may be studied in vivo.