Sphingosine-1-phosphate (S1P), formed by sphingosine kinases (SphKs), regulates cellular proliferation and migration by acting as an agonist at specific receptors or intracellularly. Since S1P's effects are probably dependent on subcellular localization of its formation and degradation, we have studied the influence of G protein-coupled receptors on the localization of SphK1. Activation of Gq-coupled receptors induced a profound, rapid (half-life 3-5 s) and long-lasting (> 2 h) translocation of SphK1 to the plasma membrane. This was mimicked by expression of constitutively active G protein alpha-subunits specifically of the Gq family. Classical Gq signalling pathways, or phosphorylation at Ser225, phospholipase D and Ca2+/calmodulin were not involved in M3 receptor-induced SphK1 translocation in HEK-293 cells. Translocation was associated with S1P receptor internalization, which was dependent on catalytic activity of SphK1 and S1P receptor binding and thus resulted from S1P receptor cross-activation. It is concluded that SphK1 is an important effector of Gq-coupled receptors, linking them via cross-activation of S1P receptors to G(i) and G12/13 signalling pathways.

Russell bodies (RBs) are intracellular inclusions filled with protein aggregates. In diverse lymphoid disorders these occur as immunoglobulin (Ig) deposits, accumulating in abnormal plasma or Mott cells. In heavy-chain deposition disease truncated antibody heavy-chains (HCs) are found, which bear a resemblance to diverse polypeptides produced in Ig light-chain (LC)-deficient (L(-/-)) mice. In L(-/-) animals, the known functions of LC, providing part of the antigen-binding site of an antibody and securing progression of B-cell development, may not be required. Here, we show a novel function of LC in preventing antibody aggregation. L(-/-) mice produce truncated HC naturally, constant region (C)gamma and Calpha lack C(H)1, and Cmicro is without C(H)1 or C(H)1 and C(H)2. Most plasma cells found in these mice are CD138(+) Mott cells, filled with RBs, formed by aggregation of HCs of different isotypes. The importance of LC in preventing HC aggregation is evident in knock-in mice, expressing Cmicro without C(H)1 and C(H)2, which only develop an abundance of RBs when LC is absent. These results reveal that preventing antibody aggregation is a major function of LC, important for understanding the physiology of heavy-chain deposition disease, and in general recognizing the mechanisms, which initiate protein conformational diseases.

Caloric restriction (CR) protects against aging and disease, but the mechanisms by which this affects mammalian life span are unclear. We show in mice that deletion of ribosomal S6 protein kinase 1 (S6K1), a component of the nutrient-responsive mTOR (mammalian target of rapamycin) signaling pathway, led to increased life span and resistance to age-related pathologies, such as bone, immune, and motor dysfunction and loss of insulin sensitivity. Deletion of S6K1 induced gene expression patterns similar to those seen in CR or with pharmacological activation of adenosine monophosphate (AMP)-activated protein kinase (AMPK), a conserved regulator of the metabolic response to CR. Our results demonstrate that S6K1 influences healthy mammalian life-span and suggest that therapeutic manipulation of S6K1 and AMPK might mimic CR and could provide broad protection against diseases of aging.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder caused by an increase in amyloid metabolism. The calcium hypothesis of AD explores how activation of the amyloidogenic pathway may function to remodel the neuronal Ca(2+) signaling pathways responsible for cognition. Hydrolysis of the beta-amyloid precursor protein (APP) yields two products that can influence Ca(2+) signaling. Firstly, the amyloids released to the outside form oligomers that enhance the entry of Ca(2+) that is pumped into the endoplasmic reticulum (ER). An increase in the luminal level of Ca(2+) within the ER enhances the sensitivity of the ryanodine receptors (RYRs) to increase the amount of Ca(2+) being released from the internal stores. Secondly, the APP intracellular domain may alter the expression of key signaling components such as the RYR. It is proposed that this remodeling of Ca(2+) signaling will result in the learning and memory deficits that occur early during the onset of AD. In particular, the Ca(2+) signaling remodeling may erase newly acquired memories by enhancing the mechanism of long-term depression that depends on activation of the Ca(2+)-dependent protein phosphatase calcineurin. The alteration in Ca(2+) signaling will also contribute to the neurodegeneration that characterizes the later stages of dementia.

We have hypothesized that variation in imprinted growth-promoting fetal genes may affect maternal glucose concentrations in pregnancy. To test this hypothesis we evaluated the effects of fetal disruption of murine H19(Delta13) on maternal glucose concentrations in pregnancy.

The study of small molecules in body fluids has become an important tool to monitor the state of biological organisms. Applications range from model studies using cell lines to applications where human body fluids are used to monitor disease states or drug responses. NMR spectroscopy has been an important tool for metabolomics although severe overlap of signals has limited the number of compounds, which can be unambiguously identified and quantified. Therefore, deconvolution of NMR spectra is one of the greatest challenges for NMR-based metabolomics. This has commonly been achieved by using multidimensional spectra that have the disadvantage of requiring significantly longer acquisition times. Recently, a number of methods have been described to record NMR spectra much faster. Here, we explore the use of Hadamard-encoded TOCSY spectra to simultaneously select multiple lines from crowded NMR spectra of blood serum samples to acquire pseudo-two-dimensional spectra in minutes which would otherwise require many hours. The potential of this approach is demonstrated for the detection of a signature for colorectal cancer from human blood samples.

The BCL-2 homology domain 3 (BH3)-only protein, B-cell lymphoma 2 interacting mediator of cell death (BIM) is a potent pro-apoptotic protein belonging to the B-cell lymphoma 2 protein family. In recent years, advances in basic biology have provided a clearer picture of how BIM kills cells and how BIM expression and activity are repressed by growth factor signalling pathways, especially the extracellular signal-regulated kinase 1/2 and protein kinase B pathways. In tumour cells these oncogene-regulated pathways are used to counter the effects of BIM, thereby promoting tumour cell survival. In parallel, a new generation of targeted therapeutics has been developed, which show remarkable specificity and efficacy in tumour cells that are addicted to particular oncogenes. It is now apparent that the expression and activation of BIM is a common response to these new therapeutics. Indeed, BIM has emerged from this marriage of basic and applied biology as an important mediator of tumour cell death in response to such drugs. The induction of BIM alone may not be sufficient for significant tumour cell death, as BIM is more likely to act in concert with other BH3-only proteins, or other death pathways, when new targeted therapeutics are used in combination with traditional chemotherapy agents. Here we discuss recent advances in understanding BIM regulation and review the role of BIM as a mediator of tumour cell death in response to novel oncogene-targeted therapeutics.

Pleckstrin homology (PH) domains are modules characterised by a conserved three-dimensional protein fold. Several PH domains bind phosphoinositides with high affinity and specificity whilst most others do not. ARAP3 is a dual GTPase activating protein for Arf6 and RhoA which was identified in a screen for phosphatidylinositol-(3,4,5)-trisphophate (PtdIns(3,4,5)P(3)) binding proteins. It is a regulator of cell shape and adhesion, and is itself regulated by PtdIns(3,4,5)P(3,) which acts to recruit ARAP3 to the plasma membrane and to catalytically activate it. We show here that ARAP3 binds to PtdIns(3,4,5)P(3) in an unusual, PH domain-dependent manner. None of the five PH domains are sufficient to bind PtdIns(3,4,5)P(3) in isolation. Instead, the minimal PtdIns(3,4,5)P(3) binding fragment comprises ARAP3's N-terminal tandem PH domains, and an N-terminal linker region. For substantial binding, the N-terminal sterile alpha motif (SAM) domain is also required. Site-directed mutagenesis of either of the two N-terminal PH domains within the fragment greatly reduces binding to PtdIns(3,4,5)P(3), however, in the context of the full-length protein, point mutations in the second PH domain have a lesser effect on binding, whilst deletion of any one of the five PH domains abolishes PtdIns(3,4,5)P(3) binding. We propose a mechanism by which basic residues from the N-terminal tandem PH domains, and from elsewhere in the protein synergise to mediate strong, specific PtdIns(3,4,5)P(3) binding.

Follicle-stimulating hormone (FSH) controls the proliferation and differentiation of Sertoli cells of the testis. FSH binds a G protein-coupled receptor (GPCR) to stimulate downstream effectors of the phosphoinositide-3 kinase (PI3K)-dependent pathway, without enhancing PI3K activity. To clarify this paradox, we explored the activity of phosphatase and tensin homolog deleted in chromosome 10 (PTEN), the PI3K major regulator, in primary cultures of rat Sertoli cells. We show that, within minutes, FSH increases PTEN neo-synthesis, requiring the proteasomal degradation of an unidentified intermediate, as well as PTEN enzymatic activity. Importantly, introducing an antisense cDNA of PTEN into differentiating Sertoli cells restores FSH-dependent cell proliferation. In conclusion, these results provide a new mechanism of PTEN regulation, which could serve to block entry into S phase of Sertoli cells, while they are proceeding through differentiation in prepubertal animals.

Glucocorticoids are potent immunosuppressive agents that block upstream signaling events required for T cell receptor (TCR) activation. However, the mechanism by which glucocorticoids inhibit downstream responses, such as inositol 1,4,5-trisphosphate (IP(3))-induced calcium signals, is not completely understood. Here we demonstrate that low concentrations of dexamethasone rapidly convert transient calcium elevations to oscillations after strong TCR stimulation. Dexamethasone converted the pattern of calcium signaling by inhibiting the Src family kinase Lck, which was shown to interact with and positively regulate Type I IP(3) receptor. In addition, low concentrations of dexamethasone were sufficient to inhibit calcium oscillations and interleukin-2 mRNA after weak TCR stimulation. Together, these findings indicate that by inhibiting Lck and subsequently down-regulating IP(3) receptors, glucocorticoids suppress immune responses by weakening the strength of the TCR signal.

Biological processes typically involve the interactions of a number of elements (genes, cells) acting on each others. Such processes are often modelled as networks whose nodes are the elements in question and edges pairwise relations between them (transcription, inhibition). But more often than not, elements actually work cooperatively or competitively to achieve a task. Or an element can act on the interaction between two others, as in the case of an enzyme controlling a reaction rate. We call "complex" these types of interaction and propose ways to identify them from time-series observations.

Polycomb group proteins have an essential role in the epigenetic maintenance of repressive chromatin states. The gene-silencing activity of the Polycomb repressive complex 2 (PRC2) depends on its ability to trimethylate lysine 27 of histone H3 (H3K27) by the catalytic SET domain of the EZH2 subunit, and at least two other subunits of the complex: SUZ12 and EED. Here we show that the carboxy-terminal domain of EED specifically binds to histone tails carrying trimethyl-lysine residues associated with repressive chromatin marks, and that this leads to the allosteric activation of the methyltransferase activity of PRC2. Mutations in EED that prevent it from recognizing repressive trimethyl-lysine marks abolish the activation of PRC2 in vitro and, in Drosophila, reduce global methylation and disrupt development. These findings suggest a model for the propagation of the H3K27me3 mark that accounts for the maintenance of repressive chromatin domains and for the transmission of a histone modification from mother to daughter cells.

The role of cellular phosphatidylinositol 5-phosphate (PtdIns5P), as a signalling molecule or as a substrate for the production of small, compartmentalized pools of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)], may be dependent on cell type and subcellular localization. PtdIns5P levels are primarily regulated by the PtdIns5P 4-kinases (type II PIP kinases or PIP4Ks), and we have investigated the expression and localization in the brain of the least-studied PIP4K isoform, PIP4Kgamma. In situ hybridization and immunohistochemistry, using antisense oligonucleotide probes and a PIP4Kgamma-specific antibody, revealed that this isoform has a restricted CNS expression profile. The use of antibodies to different cell markers showed that this expression is limited to neurons, particularly the cerebellar Purkinje cells, pyramidal cells of the hippocampus, large neuronal cell types in the cerebral cortex including pyramidal cells, and mitral cells in the olfactory bulb and is not expressed in cerebellar, hippocampal formation, or olfactory bulb granule cells. In neurons expressing this enzyme, PIP4Kgamma has a vesicular distribution and shows partial colocalization with markers of cellular compartments of the endomembrane trafficking pathway. The PIP4Kgamma isoform expression is established after day 7 of postnatal development. Overall, our data suggest that PIP4Kgamma may have a role in neuron function, specifically in the regulation of vesicular transport, in specific regions of the developed brain.

Previous work has shown that expression of Fos protein in neurons of the intermediate and medial mesopallium (IMM), a memory region in the forebrain of the domestic chick, increases in a learning-related manner after behavioural imprinting. We show here, using in situ hybridisation, that when chicks are trained for 15 min with an imprinting stimulus, expression of c-fos mRNA in the IMM rises to a maximum at or before the end of this training period. The results suggest that the learning-related increase in Fos protein production, which occurs in identifiable neuronal sub-populations in the IMM, reflects events that make an early contribution to learning and/or memory processing.

In sheep, the steroid control of gonadotropin-releasing hormone (GNRH) release is sexually differentiated such that estrogen can trigger a GNRH surge and attendant reproductive behaviors in the female, but not the male. Furthermore, female lambs that have been exposed to testosterone during a critical window of in utero development are also unable to generate a GNRH surge. This study tests the hypothesis that exposure of the ovine fetus to androgens alters the development of key steroid-receptive neuronal inputs to the GNRH neurons. In adulthood, this results in reduced activation of specific neurons by estrogen in the male and testosterone-treated female. To make this determination, groups of ewes, rams, and testosterone-exposed ewes were treated with estrogen, and the activation of neurons in the mediobasal hypothalamus and brain stem determined by immunocytochemistry. A lower percentage of neurons in the ventrolateral aspect of the ventromedial nucleus (vlVMN) and the caudal arcuate nucleus (cARC), but not the brainstem, was activated by a 6-h exposure to estrogen in the androgenized and male animals. In the vlVMN, some of these neurons contain somatostatin; however, the phenotype of activated neurons in the cARC remains unknown. These data suggest that specific neural populations in these brain regions are involved in the estrogen feedback control of GNRH release in the sheep, and that the defeminization of the surge-generating system by in utero androgen exposure results, in part, from a failure of estrogen to activate key neural phenotypes.

Recent developments in proteomics technology offer new opportunities for clinical applications in hospital or specialized laboratories including the identification of novel biomarkers, monitoring of disease, detecting adverse effects of drugs, and environmental hazards. Advanced spectrometry technologies and the development of new protein array formats have brought these analyses to a standard, which now has the potential to be used in clinical diagnostics. Besides standardization of methodologies and distribution of proteomic data into public databases, the nature of the human body fluid proteome with its high dynamic range in protein concentrations, its quantitation problems, and its extreme complexity present enormous challenges. Molecular cell biology (cytomics) with its link to proteomics is a new fast moving scientific field, which addresses functional cell analysis and bioinformatic approaches to search for novel cellular proteomic biomarkers or their release products into body fluids that provide better insight into the enormous biocomplexity of disease processes and are suitable for patient stratification, therapeutic monitoring, and prediction of prognosis. Experience from studies of in vitro diagnostics and especially in clinical chemistry showed that the majority of errors occurs in the preanalytical phase and the setup of the diagnostic strategy. This is also true for clinical proteomics where similar preanalytical variables such as inter- and intra-assay variability due to biological variations or proteolytical activities in the sample will most likely also influence the results of proteomics studies. However, before complex proteomic analysis can be introduced at a broader level into the clinic, standardization of the preanalytical phase including patient preparation, sample collection, sample preparation, sample storage, measurement, and data analysis is another issue which has to be improved. In this report, we discuss the recent advances and applications that fulfill the criteria for clinical proteomics with the focus on cellular proteomics (cytoproteomics) as related to preanalytical and analytical standardization and to quality control measures required for effective implementation of these technologies and analytes into routine laboratory testing to generate novel actionable health information. It will then be crucial to design and carry out clinical studies that can eventually identify novel clinical diagnostic strategies based on these techniques and validate their impact on clinical decision making.

V(D)J recombination in lymphocytes is the cutting and pasting together of antigen receptor genes in cis to generate the enormous variety of coding sequences required to produce diverse antigen receptor proteins. It is the key role of the adaptive immune response, which must potentially combat millions of different foreign antigens. Most antigen receptor loci have evolved to be extremely large and contain multiple individual V, D and J genes. The immunoglobulin heavy chain (Igh) and immunoglobulin kappa light chain (Igk) loci are the largest multigene loci in the mammalian genome and V(D)J recombination is one of the most complicated genetic processes in the nucleus. The challenge for the appropriate lymphocyte is one of macro-management-to make all of the antigen receptor genes in a particular locus available for recombination at the appropriate developmental time-point. Conversely, these large loci must be kept closed in lymphocytes in which they do not normally recombine, to guard against genomic instability generated by the DNA double strand breaks inherent to the V(D)J recombination process. To manage all of these demanding criteria, V(D)J recombination is regulated at numerous levels. It is restricted to lymphocytes since the Rag genes which control the DNA double-strand break step of recombination are only expressed in these cells. Within the lymphocyte lineage, immunoglobulin recombination is restricted to B-lymphocytes and TCR recombination to T-lymphocytes by regulation of locus accessibility, which occurs at multiple levels. Accessibility of recombination signal sequences (RSSs) flanking individual V, D and J genes at the nucleosomal level is the key micro-management mechanism, which is discussed in greater detail in other chapters. This chapter will explore how the antigen receptor loci are regulated as a whole, focussing on the Igh locus as a paradigm for the mechanisms involved. Numerous recent studies have begun to unravel the complex and complementary processes involved in this large-scale locus organisation. We will examine the structure of the Igh locus and the large-scale and higher-order chromatin remodelling processes associated with V(D)J recombination, at the level of the locus itself, its conformational changes and its dynamic localisation within the nucleus.

The mechanisms whereby G protein-coupled receptors (GPCR) activate signalling pathways involved in mRNA translation are ill-defined, in contrast to tyrosine kinase receptors (TKR). We compared a GPCR and a TKR, both endogenously expressed, for their ability to mediate phosphorylation of 70-kDa ribosomal S6 kinase p70S6K in primary rat Sertoli cells at two developmental stages. In proliferating cells stimulated with follicle-stimulating hormone (FSH), active p70S6K was phosphorylated on T389 and T421/S424, through cAMP-dependent kinase (PKA) and phosphatidyl-inositide-3 kinase (PI3K) antagonizing actions. In FSH-stimulated differentiating cells, active p70S6K was phosphorylated solely on T389, PKA and PI3K independently enhancing its activity. At both developmental stages, insulin-induced p70S6K regulation was consistent with reported data. Therefore, TKR and GPCR trigger distinct p70S6K active conformations. p70S6K developmental regulation was formalized in a dynamic mathematical model fitting the data, which led to experimentally inaccessible predictions on p70S6K phosphorylation rate.

The zinc-finger transcription factors Gata3 and ThPOK have both been implicated in positive selection of double positive (DP) thymocytes towards the CD4 lineage. As in the absence of Gata3, expression of ThPOK is lacking, Gata3 may directly regulate ThPOK expression. As ThPOK failed to promote CD4(+) lineage differentiation of Gata3-deficient cells, ThPOK cannot be the only Gata3 target gene essential for the induction of the CD4(+) lineage program. Therefore, it is conceivable that Gata3 is essential for selected DP T cells to reach the developmental stage at which ThPOK expression is induced. Here, we show that Gata3 overexpression does not affect ThPOK expression levels in DP or CD4(+) thymocytes, providing evidence that Gata3 does not directly regulate ThPOK. To identify additional target genes that clarify Gata3 function at the DP thymocyte stage, we performed gene expression profiling assays in wild-type mice and transgenice mice with enforced expression of Gata3, in the presence or absence of the MHC class II-restricted DO11.10 TCR. We found that Gata3 expression in DP cells undergoing positive selection was associated with downregulation of the V(D)J-recombination machinery genes Rag1, Rag2 and TdT. Moreover, Gata3 overexpression was associated with downregulation of many signaling molecules and the induction of modulators of TCR signaling, including Ctla-4 and thrombospondin 2. Together with our previous finding that Gata3 reduces expression of CD5, a negative regulator of TCR signaling, and upregulates TCR expression, these findings indicate that Gata3 in DP cells mainly functions to (i) terminate TCRalpha gene rearrangement, and (ii) regulate TCR signal intensity or duration in cells undergoing positive selection towards the CD4 lineage.

Long noncoding RNAs (lncRNAs) such as Xist, Air, and Kcnq1ot1 are required for epigenetic silencing of multiple genes in cis within large chromosomal domains, including distant genes located hundreds of kilobase pairs away. Recent evidence suggests that all three of these lncRNAs are functional and that they silence gene expression, in part, through an intimate interaction with chromatin. Here we provide an overview of lncRNA-dependent gene silencing, focusing on recent findings for the Air and Kcnq1ot1 lncRNAs. We review molecular evidence indicating that these lncRNAs interact with chromatin and correlate their presence with specific histone modifications associated with gene silencing. A general model for a lncRNA-dependent gene-silencing mechanism is presented based on the apparent ability of lncRNAs to recruit histone-modifying activities to chromatin. However, alternate mechanisms may be required to explain the silencing of some lncRNA-dependent genes. Finally, we discuss unanswered questions and future perspectives associated with these enigmatic lncRNA molecules.

We describe a method for identification of protein-protein interactions by combining two cell-free protein technologies, namely ribosome display and protein in situ immobilisation. The method requires only PCR fragments as the starting material, the target proteins being made through cell-free protein synthesis, either associated with their encoding mRNA as ribosome complexes or immobilised on a solid surface. The use of ribosome complexes allows identification of interacting protein partners from their attached coding mRNA. To demonstrate the procedures, we have employed the lymphocyte signalling proteins Vav1 and Grb2 and confirmed the interaction between Grb2 and the N-terminal SH3 domain of Vav1. The method has promise for library screening of pairwise protein interactions, down to the analytical level of individual domain or motif mapping.

Prolonged maintenance of trophoblast stem (TS) cells requires fibroblast growth factor (FGF) 4 and embryonic fibroblast feeder cells or feeder cell-conditioned medium. Previous studies have shown that TGF-beta and Activin are sufficient to replace embryonic fibroblast-conditioned medium. Nodal, a member of the TGF-beta superfamily, is also known to be important in vivo for the maintenance of TS cells in the developing placenta. Our current studies indicate that TS cells do not express the Nodal co-receptor, Cripto, and do not respond directly to active Nodal in culture. Conversely, Activin subunits and their receptors are expressed in the placenta and TS cell cultures, with Activin predominantly expressed by trophoblast giant cells (TGCs). Differentiation of TS cells in the presence of TGC-conditioned medium or exogenous Activin results in a reduction in the expression of TGC markers. In line with TGC-produced Activin representing the active component in TGC-conditioned medium, this differentiation-inhibiting effect can be reversed by the addition of follistatin. Additional experiments in which TS cells were differentiated in the presence or absence of exogenous Activin or TGF-beta show that Activin but not TGF-beta results in the maintenance of expression of TS cell markers, prolongs the expression of syncytiotrophoblast markers, and significantly delays the expression of spongiotrophoblast and TGC markers. These results suggest that Activin rather than TGF-beta (or Nodal) acts directly on TS cells influencing both TS cell maintenance and cell fate, depending on whether the cells are also exposed to FGF4.

The Vav family of proteins have the potential to act as both signalling adapters and GEFs for Rho GTPases. They have therefore been proposed as regulators of the cytoskeleton in various cell types. We have used macrophages from mice deficient in all three Vav isoforms to determine how their function affects cell morphology and migration. Macrophages lacking Vav proteins adopt an elongated morphology and have enhanced migratory persistence in culture. To investigate the pathways through which Vav proteins exert their effects we analysed the responses of macrophages to the chemoattractant CSF-1 and to adhesion. We found that morphological and signalling responses of macrophages to CSF-1 did not require Vav proteins. In contrast, adhesion-induced cell spreading, RhoA and Rac1 activation and cell signalling were all dependent on Vav proteins. We propose that Vav proteins affect macrophage morphology and motile behaviour by coupling adhesion receptors to Rac1 and RhoA activity and regulating adhesion signalling events such as paxillin and ERK1/2 phosphorylation by acting as adapters.

Although the presence of a BH4 domain distinguishes the antiapoptotic protein Bcl-2 from its proapoptotic relatives, little is known about its function. BH4 deletion converts Bcl-2 into a proapoptotic protein, whereas a TAT-BH4 fusion peptide inhibits apoptosis and improves survival in models of disease due to accelerated apoptosis. Thus, the BH4 domain has antiapoptotic activity independent of full-length Bcl-2. Here we report that the BH4 domain mediates interaction of Bcl-2 with the inositol 1,4,5-trisphosphate (IP3) receptor, an IP3-gated Ca(2+) channel on the endoplasmic reticulum (ER). BH4 peptide binds to the regulatory and coupling domain of the IP3 receptor and inhibits IP3-dependent channel opening, Ca(2+) release from the ER, and Ca(2+)-mediated apoptosis. A peptide inhibitor of Bcl-2-IP3 receptor interaction prevents these BH4-mediated effects. By inhibiting proapoptotic Ca(2+) signals at their point of origin, the Bcl-2 BH4 domain has the facility to block diverse pathways through which Ca(2+) induces apoptosis.