T cell receptor/CD3 negative variants are unresponsive to stimulation through the Ly-6 encoded molecule, TAP

TAP is a phosphatidylinositol-anchored Ly-6-encoded, glycoprotein that is involved in the activation of murine T-inducer cells. Anti-TAP antibodies can stimulate T cells directly and also synergize with Ag stimulation. To determine the relationship between TAP and TCR-mediated activation, we have derived TCR/CD3 loss variants of functional T hybrid clones. T-T hybrid variants that have lost TCR expression are not responsive to anti-TAP stimulation. Isolation and analysis of revertant clones, obtained from one of the TCR/CD3-negative mutant cell lines, demonstrate a concordant re-expression of the TCR/CD3 complex and responsiveness to anti-TAP stimulation. A similar loss of responsiveness to TAP stimulation is observed after antibody-induced modulation of the TCR/CD3 complex from the surface of T cell hybrids. In contrast, both the TCR/CD3 loss variants and TCR/CD3-modulated T-T hybrids remain fully responsive to stimulation with a calcium ionophore and phorbol esters. This structure-function correlation has been observed repeatedly in independent isolations of variants, and with TCR/CD3 modulated cells, from two different T-T hybridomas. Given the apparent functional interrelationship between TAP and the TCR/CD3 complex, we have also analyzed if these molecules were significantly associated on the T cell surface. Antibody-induced modulation of the TCR/CD3 complex does not affect the cell surface expression of TAP molecules. Moreover, the expression of these two sets of proteins is also independent as evidenced by the selective loss of either set of these proteins in the TCR/CD3 expression mutants.     

sgp-60, a signal-transducing glycoprotein concerned with T cell activation through the T cell receptor/CD3 complex

T cell activation depends not only on the expression of a TCR, but also on that of accessory molecules that function in cell-cell adhesion and/or signal transduction. The subject of this report is the biochemical and functional characterization of what appears to be a novel murine lymphocyte cell surface antigen, provisionally termed sgp-60. Extensive, higher-order cross-linking of this glycoprotein with an anti-sgp-60 mAb and a second-step antibody reagent results in the activation of resting CD4+ T cells in the presence of a second signal. Monovalent or bivalent engagement of sgp-60 by the anti-sgp-60 antibody results in profound and direct inhibition of anti-CD3- or Con A-driven T cell activation, whereas alternative T cell activation via the phosphatidylinositol-linked proteins Thy-1 and TAP/Ly-6A is not affected. These findings raise the possibility that the sgp-60 molecule may be specifically involved in signal transduction through the TCR/CD3 complex and thus point to an important physiologic role for this protein in CD4+ T cells.     

Internalization of phosphatidylinositol-anchored lymphocyte proteins. I. Documentation and potential significance for T cell stimulation

Several proteins that are anchored to the surface of T lymphocytes via a phosphatidylinositol (PI) moiety can initiate cell stimulation upon cross-linking. Inasmuch as these proteins do not traverse the plasma membrane, it is not clear how they are capable of signaling across the membrane. Herein we report two distinct sets of experiments that examine the consequence of cross-linking PI-anchored molecules on murine T cells. We first analyzed the fate of antibody cross-linked TAP (Ly-6A.2) and Thy-1 molecules on T-T hybrids. Using an assay to measure receptor-mediated endocytosis, an intracellular accumulation of 125I labeled anti-TAP and anti-Thy-1 mAb was documented that was specific and Ag dependent. The internalization of these molecules was confirmed by cytotoxicity assays using antibody-toxin conjugates, and electron microscopic studies. Although the PI-anchored proteins lack a cytoplasmic domain that is necessary for the internalization of many receptors, they nevertheless can be induced to enter the cell upon cross-linking. The rate of entry of cross-linked TAP and Thy-1 into cells was shown to be 10 and 2% per hour, respectively, which is considerably less than that observed for the transferrin receptor or TCR/CD3 complex. To assess whether the internalization of TAP and Thy-1 might be of importance in their ability to stimulate T cells, we attempted to cross-link these molecules under conditions where the mAb or its cross-linked complex can not enter the cell. We observed that anti-TAP and anti-Thy-1 mAb conjugated to a cell impermeant matrix fail to stimulate T cells. This loss of stimulatory activity was observed with multiple T-T hybridomas and mAb over a wide titration of antibody concentration and was independent of the mAb isotype. Results from experiments with anti-Ig cross-linking of the mAb-PI anchored protein complex suggested that the loss of T cell stimulation upon mAb immobilization is not simply due to an alteration in the degree of antibody cross-linking. These findings were generalized to three distinct PI-anchored proteins: TAP, Thy-1, and Ly6C on normal T cells. When the same cells were stimulated through the TCR/CD3 complex, only immobilized mAb are stimulatory. These results demonstrate a marked difference in the cross-linking requirements for stimulating T cells through PI-anchored molecules in contrast to the transmembrane TCR complex. Furthermore, these findings raise the possibility that molecular internalization of Ab-PI-anchored complexes may be necessary in signaling through these molecules.     

T cell-activating protein on murine lymphocytes

A functional T cell surface molecule, T cell-activating protein (TAP), has been identified on murine lymphocytes. TAP is a protein with an apparent molecular mass of 10-12 kilodaltons (kDa) nonreduced, 16-17 kDa reduced. Cross-linking of TAP can result in profound activation of T lymphocytes to produce lymphokines and to enter the cell cycle. Furthermore, antibody binding to TAP can modulate antigen-driven T cell stimulation. Current data suggest that TAP is physically distinct from the T cell receptor complex. On unstimulated lymphocytes, TAP is expressed on T cells and defines heterogeneity within the major T cell subsets. Its profile of expression is rapidly altered on cell activation. Ontologically, it is first detected in the thymus, where it is found on both the most immature and the most mature cell subsets, and it is functional on both. Together, these TAP+ cells constitute a small fraction of thymocytes. TAP expression, however, defines the immunocompetent compartment of the thymus, and thus could be involved in functional maturation. Finally, the gene controlling TAP expression has been mapped, and is tightly linked to a locus of hematopoietic antigens (Ly-6). TAP is molecularly distinct from these antigens. Furthermore, all of these proteins show a markedly distinct developmental regulation in their cell surface expression.     

Stimulation of T cells via the TAP molecule, a member in a family of activating proteins encoded in the Ly-6 locus

T-cell activating protein, TAP, is a Ly-6 encoded 12,000 dalton glycoprotein involved in the activation of murine T cells. TAP is distinct from other known surface activating structures, such as the T cell receptor/T3 complex and Thy-1. This study investigates the mechanism of activation via the TAP molecule. Soluble alpha TAP monoclonal antibody (MAb) is sufficient for T cell activation. This, however, requires cross-linking because F(ab) monovalent antibody is not stimulatory unless cross-linked by a second antibody. Immediately after cross-linking, there is a rapid influx of calcium, which is comparable with concanavalin A or T cell receptor triggered responses. Subsequently, interleukin 2 (IL 2) is produced, and IL 2 receptors (IL 2R) are expressed. TAP-stimulated T cell proliferation is driven by this autocrine pathway because it is inhibited by alpha IL 2R MAb. Thus TAP-mediated activation appears to share a common final pathway with other mitogenic stimuli. A nonactivating alpha TAP MAb was observed to stimulate T cells upon additional cross-linking. Given this observation, we asked whether other Ly-6 linked proteins might share similar activating potential. We show that at least three distinct Ly-6 linked molecules are capable of stimulating T hybrid clones and/or heterogeneous T lymphocytes. Thus it appears that the Ly-6 locus encodes a family of activating cell surface molecules.     

Functional analysis of T-cell mutants defective in the biosynthesis of glycosylphosphatidylinositol anchor. Relative importance of glycosylphosphatidylinositol anchor versus N-linked glycosylation in T-cell activation.

The glycosylphosphatidylinositol (GPI) anchor, potentially capable of generating a number of second messengers, such as diacylglycerol, phosphatidic acid, and inositol phosphate glycan, has been postulated to be involved in signal transduction in various cell types, including T-cells. We have identified a panel of T-cell hybridoma mutants that are defective at various steps of GPI anchor biosynthesis. Since they were derived from a functional T-T hybridoma, we were able to determine the precise role of the GPI anchor in T-cell activation. Two mutants were chosen for this analysis. The first mutant is defective at the first step of GPI anchor biosynthesis, i.e. in the transfer of N-acetylglucosamine to a phosphatidylinositol acceptor. Thus, it cannot form any GPI precursors or GPI-like compounds. Interestingly, this mutant can be activated by antigen, superantigen, and concanavalin A in a manner comparable to the wild-type hybridoma. These data strongly suggest that the GPI anchor, its precursor, or its potential cleavage product, inositol phosphate glycan, is not required for the early phase of T-cell activation. The second mutant is able to synthesize the first two GPI precursors, but is not able to add mannose residues to them due to a deficiency in dolichol-phosphate-mannose (Dol-P-Man) biosynthesis. Unexpectedly, all of the Dol-P-Man mutants are defective in activation by antigen, suprantigen, and concanavalin A despite normal T-cell receptor expression. Here, we show that the activation defect was due to a pleiotropic glycosylation abnormality because Dol-P-Man is required for both GPI anchor and N-linked oligosaccharide biosynthesis. When the yeast Dol-P-Man synthase gene was stably transfected into the mutants, full expression of surface GPI-anchored proteins was restored. However, N-linked glycosylation was either partially or completely corrected in different transfectants. Reconstitution of activation defects correlates well with the status of N-linked glycosylation, but not with the expression of GPI-anchored proteins. These results thus reveal an unexpected role of N-linked glycosylation in T-cell activation.     

The expression, function, and ontogeny of a novel T cell-activating protein, TAP, in the thymus

The TAP molecule is an allelic 12,000 m.w. membrane protein that participates in T cell activation. This report analyzes the expression, function, and ontogeny of this molecule in the thymus. TAP is expressed on a small subset (10 to 20%) of thymocytes which is distinct from its expression on a majority (70%) of peripheral T lymphocytes. In the adult thymus, the majority of the TAP-bearing thymocytes are cortisone-resistant, Thy-1+, TL-, J11D-, and PNA-, which localizes TAP expression to medullary thymocytes. Cortical thymocytes do not bear this determinant. Parallel functional studies demonstrated that TAP+ thymocytes are required for Con A and MLR responsiveness. Anti-TAP MAb plus PMA specifically induces proliferation of mature thymocytes comparable in magnitude to the Con A response. These results demonstrate that TAP expression defines the immunocompetent thymocyte compartment and, further, that this molecule is functional on these cells. The ontogeny of TAP expression was also analyzed. TAP is expressed early in fetal thymic development at a time when most T cell markers (except Thy-1 and the iL2-R) are absent. The small sub-population of adult L3T4- and Lyt-2- thymocytes, which resemble early fetal thymocytes, also express TAP. These early thymocytes are capable of being activated through the TAP molecule. The implications of these findings for T cell development and, in particular, the relationship of TAP to T cell receptor expression and acquisition of immunocompetence are discussed.     

Impaired autoreactive T cell-induced T cell-T cell interaction in aged mice

Self-Ia-reactive (autoreactive) L3T4+ T cell clones have been shown earlier to stimulate the proliferation of syngeneic naive L3T4+ T cells and initiate a T cell-T cell (T-T) interaction leading to the generation of immunoregulatory circuits. Since aging has been shown to be associated with a decline of the immune responsiveness, age-related alterations in the T-T interaction was investigated in the present study. Using several I-Ed-specific autoreactive T cell clones isolated from 2- to 3-month-old (young) DBA/2 mice as stimulators, it was observed that L3T4+ T cells from 22- to 24-month-old (aged) DBA/2 mice, failed to demonstrate a significant response to the autoreactive T cells. In contrast, L3T4+ T cells from young mice responded strongly to the autoreactive T cell clones. The deficient T-T cell interaction in aged mice correlated with an impaired syngeneic mixed lymphocyte reaction in these mice, thereby suggesting that aging induces a defect both in the autoreactive T cells and in T cells which react with the autoreactive T cells. When exogenous recombinant interleukin 2 (rIL-2), recombinant interleukin 4 (rIL-4), or a combination of these was added to the interaction, it was observed that rIL-4 but not rIL-2 enhanced the T-T interaction in young mice. However, rIL-4 or a combination of rIL-2 and rIL-4 failed to correct the defective T-T interaction in aged mice. Since the T cell network is believed to play an important role in the maintenance of normal immune system homeostasis, the present study suggests that age-related alterations in T and B cell functions and increased susceptibility to autoimmune diseases with age may result from a defect in the T cell network regulation.     

Discovery of a novel Toxoplasma gondii conoid-associated protein important for parasite resistance to reactive nitrogen intermediates

Toxoplasma gondii modifies its host cell to suppress its ability to become activated in response to IFN-γ and TNF-α and to develop intracellular antimicrobial effectors, including NO. Mechanisms used by T. gondii to modulate activation of its infected host cell likely underlie its ability to hijack monocytes and dendritic cells during infection to disseminate to the brain and CNS where it converts to bradyzoites contained in tissue cysts to establish persistent infection. To identify T. gondii genes important for resistance to the effects of host cell activation, we developed an in vitro murine macrophage infection and activation model to identify parasite insertional mutants that have a fitness defect in infected macrophages following activation but normal invasion and replication in naive macrophages. We identified 14 independent T. gondii insertional mutants out of >8000 screened that share a defect in their ability to survive macrophage activation due to macrophage production of reactive nitrogen intermediates (RNIs). These mutants have been designated counter-immune mutants. We successfully used one of these mutants to identify a T. gondii cytoplasmic and conoid-associated protein important for parasite resistance to macrophage RNIs. Deletion of the entire gene or just the region encoding the protein in wild-type parasites recapitulated the RNI-resistance defect in the counter-immune mutant, confirming the role of the protein in resistance to macrophage RNIs.     

Genetic evidence for the involvement of the lck tyrosine kinase in signal transduction through the T cell antigen receptor.

Signaling through the T cell antigen receptor (TCR) results both in rapid increases in tyrosine phosphorylation on a number of proteins and in the activation of the phosphatidylinositol pathway. It is not clear how stimulation of the TCR leads to these signaling events. Mutants of the Jurkat T cell line have been previously isolated that fail to show increases in calcium following receptor stimulation. Analysis of one of these mutants, JCaM1, which is defective in the induction of tyrosine phosphorylation, revealed a defect in the expression of functional lck tyrosine kinase. The lack of lck activity was caused in part by a splicing defect. Expression of the lck cDNA in JCaM1 restores the ability of the cell to respond to TCR stimulation. These results indicate that lck is required for normal signal transduction through the TCR.     

Role of ICAM-1 in antigen presentation demonstrated by ICAM-1 defective mutants

We report a methodology for selecting APC with mutations that have impaired their ability to present Ag to T cells. A20 B lymphoblastoid cells were mutagenized and then repeatedly cocultured with murine T-T hybridomas in the presence of specific Ag. During these cocultures, the T-T hybridomas kill the competent APC, allowing the outgrowth of inactive variants. Two variants, A20.M1 and A20.M2, were isolated and studied in detail. These variants are impaired in their ability to present multiple Ag to T cells. This defect is also observed for the presentation of processing independent peptides by fixed APC indicating that a lesion exists in a post-Ag processing step. The level of expression of MHC molecules is unaffected and the functional defect in the APC is not localized to a particular MHC molecule. In contrast, these mutants were found to have a selective decrease in the expression of the murine homolog of ICAM-1, and the residual ability of these cells to present Ag was not blocked by anti-ICAM-1 mAb. Conversely, Ag presentation by the wild-type A20 is inhibited by anti-ICAM-1 mAb. Similarly, anti-LFA-1 mAb inhibited the response of T cells to Ag presented by the wild-type A20 to a much greater degree than by the mutant cells, indicating that LFA-1 is involved in interaction of T cells with the former, but not latter, APC. In the apparent absence of a contribution of LFA-1 to the T cell-APC interaction, either as a result of mAb blocking or the disruption of the APC membrane, the mutant and wild-type APC have a similar level of Ag-presenting activity. Reconstitution of ICAM-1 expression in these mutants by transfection with murine ICAM-1 cDNA fully restores their ability to present Ag. Together these results demonstrate that a murine ICAM-1 homolog is expressed on A20 B cells, where it functions as a major cell interaction molecule. The degree of functional impairment in these mutant APC gives insight into the contribution of cell interaction molecules to efficient Ag presentation and T cell-B cell interaction. Finally, these results also demonstrate the feasibility of selecting APC with mutations affecting Ag presentation.     

Different expression levels of the TAP peptide transporter lead to recognition of different antigenic peptides by tumor-specific CTL

Decreased antigenicity of cancer cells is a major problem in tumor immunology. This is often acquired by an expression defect in the TAP. However, it has been reported that certain murine Ags appear on the target cell surface upon impairment of TAP expression. In this study, we identified a human CTL epitope belonging to this Ag category. This epitope is derived from preprocalcitonin (ppCT) signal peptide and is generated within the endoplasmic reticulum by signal peptidase and signal peptide peptidase. Lung cancer cells bearing this antigenic peptide displayed low levels of TAP, but restoration of their expression by IFN-γ treatment or TAP1 and TAP2 gene transfer abrogated ppCT Ag presentation. In contrast, TAP upregulation in the same tumor cells increased their recognition by proteasome/TAP-dependent peptide-specific CTLs. Thus, to our knowledge, ppCT(16-25) is the first human tumor epitope whose surface expression requires loss or downregulation of TAP. Lung tumors frequently display low levels of TAP molecules and might thus be ignored by the immune system. Our results suggest that emerging signal peptidase-generated peptides represent alternative T cell targets, which permit CTLs to destroy TAP-impaired tumors and thus overcome tumor escape from CD8(+) T cell immunity.     

The CD2 ligand LFA-3 activates T cells but depends on the expression and function of the antigen receptor

The T cell Ag receptor (CD3/Ti) and the sheep E receptor (CD2) expressed on the surface of human T cells are both capable of initiating intracellular signals necessary for T cell activation. CD3/Ti interacts with Ag to initiate cellular immune responses. Although the exact function of CD2 is unknown, lymphocyte function-associated Ag 3 (LFA-3), a 55- to 70-kDa receptor expressed on a broad spectrum of hemopoietic and nonhemopoietic cells, has recently been shown to be its natural ligand. We show here that although purified multimeric LFA-3 is not capable of initiating transmembrane signaling events on its own, the combination of LFA-3 and the anti-CD2 mAb CD2.1 induces intracellular calcium increases, phosphatidylinositol second messenger generation and lymphokine secretion in the T cell leukemic line Jurkat. In order to study the signaling requirements of CD2, we compared the ability of CD2 mAb and LFA-3 to initiate activation signals in Jurkat and in three Jurkat-derived mutants. A CD3-CD2+ mutant failed to increase calcium or exhibit phosphatidylinositol hydrolysis to either the combination of agonist CD2 mAb 9-1 and 9.6 or LFA-3 and CD2.1. Reconstitution of the Ag receptor by transfection of the Ti-beta-chain restored the expression of the CD3/Ti complex and the ability to respond to either combination of CD2 ligands. However, no response to CD2 ligands was detected in a CD3+CD2+ mutant selected for signaling defects to CD3/Ti ligands. Complementation of the CD3/Ti signaling defect by cell fusion also restored competency to respond to CD2 agonists. These results demonstrate that LFA-3 under appropriate conditions can activate T cells via the CD2 complex and that this activation requires not only the cell surface expression of the CD3/Ti complex but also a functional Ag receptor pathway.     

MHC class II-restricted interaction between thymocytes plays an essential role in the production of innate CD8+ T cells

We have recently shown that MHC class II-dependent thymocyte-thymocyte (T-T) interaction successfully generates CD4(+) T cells (T-T CD4(+) T cells), and that T-T CD4(+) T cells expressing promyelocytic leukemia zinc finger protein (PLZF) show an innate property both in mice and humans. In this article, we report that the thymic T-T interaction is essential for the conversion of CD8(+) T cells into innate phenotype in the physiological condition. CD8(+) T cells developed in the presence of PLZF(+) CD4(+) T cells showed marked upregulation of eomesodermin (Eomes), activation/memory phenotype, and rapid production of IFN-γ on ex vivo stimulation. Their development was highly dependent on the PLZF expression in T-T CD4(+) T cells and the IL-4 secreted by PLZF(+) T-T CD4(+) T cells. The same events may take place in humans, as a substantial number of Eomes expressing innate CD8(+) T cells were found in human fetal thymi and spleens. It suggests that PLZF(+) T-T CD4(+) T cells in combination with Eomes(+) CD8(+) T cells might actively participate in the innate immune response against various pathogens, particularly in human perinatal period.     

NK cells stimulate proliferation of T and NK cells through 2B4/CD48 interactions

Few studies have addressed the consequences of physical interactions between NK and T cells, as well as physical interactions among NK cells themselves. We show in this study that NK cells can enhance T cell activation and proliferation in response to CD3 cross-linking and specific Ag through interactions between 2B4 (CD244) on NK cells and CD48 on T cells. Furthermore, 2B4/CD48 interactions between NK cells also enhanced proliferation of NK cells in response to IL-2. Overall, these results suggest that NK cells augment the proliferation of neighboring T and NK cells through direct cell-cell contact. These results provide new insights into NK cell-mediated control of innate and adaptive immunity and demonstrate that receptor/ligand-specific cross talk between lymphocytes may occur in settings other than T-B cell or T-T cell interactions.     

A novel IgA receptor expressed on a murine B cell lymphoma.

The specificity and properties of a novel IgA receptor expressed on the surface of a tissue culture-adapted B cell lymphoma, T560, that originated in murine gut-associated lymphoid tissue, have been explored. Like the IgA receptors of murine T and splenic B cells studied by others, the T560 IgA receptor is trypsin sensitive and neuraminidase resistant and is up-regulated on T560 cells by exposing them overnight to high concentrations of polymeric IgA. Unlike them, the T560 IgA receptor is inhibited by low concentrations of IgM and high concentrations of IgG2a and IgG2b, binds at pH 4.0 but not at pH 8.0, is down-regulated by activation of protein kinase C and is sensitive to phosphatidylinositol-specific phospholipase C, indicating that it is glycosyl phosphatidylinositol-linked to the cell membrane. It is not a cell-bound form of galactosyl transferase, does not appear to bind to Ig through carbohydrate residues and does not react specifically with antibody to secretory component. It may be a completely new, cross-reactive receptor, perhaps related in some way to the polymeric Ig receptor or to the receptor for IgA expressed on the apical surface of Peyer's patch M cells, which is known to cross-react with IgG. Alternatively, it may be homologous to the highly IgA-specific Fc alpha R of T cells but, perhaps because of its glycosyl phosphatidylinositol linker, may have an ability to move and interact with other Ig receptors on the cell surface such that Ig bound to them are cross-inhibitory.     

Susceptibility to cell death is a dominant phenotype: triggering of activation-driven T-cell death independent of the T-cell antigen receptor complex.

The failure of Thy-1 and Ly-6 to trigger interleukin-2 production in the absence of surface T-cell antigen receptor complex (TCR) expression has been interpreted to suggest that functional signalling via these phosphatidylinositol-linked alternative activation molecules is dependent on the TCR. We find, in contrast, that stimulation of T cells via Thy-1 or Ly-6 in the absence of TCR expression does trigger a biological response, the cell suicide process of activation-driven cell death. Activation-driven cell death is a process of physiological cell death that likely represents the mechanism of negative selection of T cells. The absence of the TCR further reveals that signalling leading to activation-driven cell death and to lymphokine production are distinct and dissociable. In turn, the ability of alternative activation molecules to function in the absence of the TCR raises another issue: why immature T cells, thymomas, and hybrids fail to undergo activation-driven cell death in response to stimulation via Thy-1 and Ly-6. One possibility is that these activation molecules on immature T cells are defective. Alternatively, susceptibility to activation-driven cell death may be developmentally regulated by TCR-independent factors. We have explored these possibilities with somatic cell hybrids between mature and immature T cells, in which Thy-1 and Ly-6 are contributed exclusively by the immature partner. The hybrid cells exhibit sensitivity to activation-driven cell death triggered via Thy-1 and Ly-6. Thus, the Thy-1 and Ly-6 molecules of the immature T cells can function in a permissive environment. Moreover, with regard to susceptibility to Thy-1 and Ly-6 molecules of the immature T cells can function in a permissive environment. Moreover, with regard to susceptibility to Thy-1 and Ly-6 triggering, the mature phenotype of sensitivity to cell death is genetically dominant.     

Modulation of Ca2+ signals by phosphatidylinositol-linked novel D1 dopamine receptor in hippocampal neurons

Recent evidence indicates the existence of a putative novel phosphatidylinositol-linked D1 dopamine receptor in brain that mediates phosphatidylinositol hydrolysis via activation of phospholipase Cbeta. The present work was designed to characterize the Ca(2+) signals regulated by this phosphatidylinositol-linked D(1) dopamine receptor in primary cultures of hippocampal neurons. The results indicated that stimulation of phosphatidylinositol-linked D1 dopamine receptor by its newly identified selective agonist SKF83959 induced a long-lasting increase in basal [Ca(2+)](i) in a time- and dose-dependent manner. Stimulation was observable at 0.1 microm and reached the maximal effect at 30 microm. The [Ca(2+)](i) increase induced by 1 microm SKF83959 reached a plateau in 5 +/- 2.13 min, an average 96 +/- 5.6% increase over control. The sustained elevation of [Ca(2+)](i) was due to both intracellular calcium release and calcium influx. The initial component of Ca(2+) increase through release from intracellular stores was necessary for triggering the late component of Ca(2+) rise through influx. We further demonstrated that activation of phospholipase Cbeta/inositol triphosphate was responsible for SKF83959-induced Ca(2+) release from intracellular stores. Moreover, inhibition of voltage-operated calcium channel or NMDA receptor-gated calcium channel strongly attenuated SKF83959-induced Ca(2+) influx, indicating that both voltage-operated calcium channel and NMDA receptor contribute to phosphatidylinositol-linked D(1) receptor regulation of [Ca(2+)](i).     

Deregulation of the Egfr/Ras signaling pathway induces age-related brain degeneration in the Drosophila mutant vap

Ras signaling has been shown to play an important role in promoting cell survival in many different tissues. Here we show that upregulation of Ras activity in adult Drosophila neurons induces neuronal cell death, as evident from the phenotype of vacuolar peduncle (vap) mutants defective in the Drosophila RasGAP gene, which encodes a Ras GTPase-activating protein. These mutants show age-related brain degeneration that is dependent on activation of the EGF receptor signaling pathway in adult neurons, leading to autophagic cell death (cell death type 2). These results provide the first evidence for a requirement of Egf receptor activity in differentiated adult Drosophila neurons and show that a delicate balance of Ras activity is essential for the survival of adult neurons.     

HIV-1 Nef perturbs the function, structure, and signaling of the Golgi through the Src kinase Hck

The interaction between HIV-1 Nef and the Src kinase Hck in macrophages has been shown to accelerate the progression to AIDS. We previously showed that Nef disturbed the N-glycosylation/trafficking of Fms, a cytokine receptor essential for maintaining macrophages in an anti-inflammatory state, in an Hck-dependent manner. Here, we show the underlying molecular mechanism of this effect. Using various Hck isoforms and their mutants and Golgi-targeting Hck mutants, we confirmed that Hck activation at the Golgi causes the Nef-induced Fms N-glycosylation defect. Importantly, we found that both the co-expression of Nef and Hck and the expression of a Golgi-targeted active Hck mutant caused alterations in the distribution of GM130, a Golgi protein that was shown to be required for efficient protein glycosylation. Moreover, the activation of Hck at the Golgi caused strong serine phosphorylation of the GM130-interacting Golgi structural protein GRASP65, which is known to induce Golgi cisternal unstacking. Using pharmacological inhibitors, we also found that the activation of Hck at the Golgi followed by the activation of the MAP kinase ERK-GRASP65 cascade is involved in the Fms N-glycosylation defect. These results suggest that Nef perturbs the structure and signaling of the Golgi by activating Hck at the Golgi, and thereby, induces the N-glycosylation/trafficking defect of Fms, which is in line with the idea that Src family kinases are crucial Golgi regulators.