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A genetic selection in Saccharomyces cerevisiae for mutants that stimulate the mating pathway uncovered a mutant that had a hyperactive pheromone response pathway and also had hyperpolarized growth. Cloning and segregation analysis demonstrated that BUD14 was the affected gene. Disruption of BUD14 in wild-type cells caused mild stimulation of pheromone response pathway reporters, an increase in sensitivity to mating factor, and a hyperelongated shmoo morphology. The bud14 mutant also had hyperfilamentous growth. Consistent with a role in the control of cell polarity, a Bud14p-green fluorescent protein fusion was localized to sites of polarized growth in the cell. Bud14p shared morphogenetic functions with the Ste20p and Bni1p proteins as well as with the type 1 phosphatase Glc7p. The genetic interactions between BUD14 and GLC7 suggested a role for Glc7p in filamentous growth, and Glc7p was found to have a positive function in filamentous growth in yeast.

In haploid strains of Saccharomyces cerevisiae, glucose depletion causes invasive growth, a foraging response that requires a change in budding pattern from axial to unipolar-distal. To begin to address how glucose influences budding pattern in the haploid cell, we examined the roles of bud-site-selection proteins in invasive growth. We found that proteins required for bipolar budding in diploid cells were required for haploid invasive growth. In particular, the Bud8p protein, which marks and directs bud emergence to the distal pole of diploid cells, was localized to the distal pole of haploid cells. In response to glucose limitation, Bud8p was required for the localization of the incipient bud site marker Bud2p to the distal pole. Three of the four known proteins required for axial budding, Bud3p, Bud4p, and Axl2p, were expressed and localized appropriately in glucose-limiting conditions. However, a fourth axial budding determinant, Axl1p, was absent in filamentous cells, and its abundance was controlled by glucose availability and the protein kinase Snf1p. In the bud8 mutant in glucose-limiting conditions, apical growth and bud site selection were uncoupled processes. Finally, we report that diploid cells starved for glucose also initiate the filamentous growth response.

Calcium is a universal intracellular signal that is responsible for controlling a plethora of cellular processes. Understanding how such a simple ion can regulate so many diverse cellular processes is a key goal of calcium- and cell-biologists. One molecule that is sensitive to changes in intracellular calcium levels is Ras. This small GTPase operates as a binary molecular switch, and regulates cell proliferation and differentiation. Here, we focus on examining the link between calcium and Ras signalling and, in particular, we speculate as to how the complexity of calcium signalling could regulate Ras activity.

The membrane phospholipid phosphatidylinositol is the precursor of a family of lipid second-messengers, known as phosphoinositides, which differ in the phosphorylation status of their inositol group. A major advance in understanding phosphoinositide signalling has been the identification of a number of highly conserved modular protein domains whose function appears to be to bind various phosphoinositides. Such 'cut and paste' modules are found in a diverse array of multidomain proteins and recruit their host protein to specific regions in cells via interactions with phosphoinositides. Here, with particular reference to proteins involved in membrane traffic pathways, we discuss recent advances in our understanding of phosphoinositide-binding domains.

Ca(2+) is a universal second messenger that is critical for cell growth and is intimately associated with many Ras-dependent cellular processes such as proliferation and differentiation. Ras is a small GTP binding protein that operates as a molecular switch regulating the control of gene expression, cell growth, and differentiation through a pathway from receptors to mitogen-activated protein kinases (MAPKs). A role for intracellular Ca(2+) in the activation of Ras has been previously demonstrated, e.g., via the nonreceptor tyrosine kinase PYK2 and by Ca(2+)/calmodulin-dependent guanine nucleotide exchange factors (GEFs) such as Ras-GRF; however, there is no Ca(2+)-dependent mechanism for direct inactivation. An important advance toward greater understanding of the complex coordination within the Ras-signaling network is the spatio-temporal analysis of signaling events in vivo. Here, we describe the identification of CAPRI (Ca(2+)-promoted Ras inactivator), a Ca(2+)-dependent Ras GTPase-activating protein (GAP) that switches off the Ras-MAPK pathway following a stimulus that elevates intracellular Ca(2+). Analysis of the spatio-temporal dynamics of CAPRI indicates that Ca(2+) regulates the GAP by a fast C2 domain-dependent translocation mechanism.

Mammalian casein kinases I (CKI) belong to a family of serine/threonine protein kinases involved in diverse cellular processes including cell cycle progression, membrane trafficking, circadian rhythms, and Wnt signaling. Here we show that CKIalpha co-purifies with centaurin-alpha(1) in brain and that they interact in vitro and form a complex in cells. In addition, we show that the association is direct and occurs through the kinase domain of CKI within a loop comprising residues 217-233. These residues are well conserved in all members of the CKI family, and we show that centaurin-alpha(1) associates in vitro with all mammalian CKI isoforms. To date, CKIalpha represents the first protein partner identified for centaurin-alpha(1). However, our data suggest that centaurin-alpha(1) is not a substrate for CKIalpha and has no effect on CKIalpha activity. Centaurin-alpha(1) has been identified as a phosphatidylinositol 3,4,5-trisphosphate-binding protein. Centaurin-alpha(1) contains a cysteine-rich domain that is shared by members of a newly identified family of ADP-ribosylation factor guanosine trisphosphatase-activating proteins. These proteins are involved in membrane trafficking and actin cytoskeleton rearrangement, thus supporting a role for CKIalpha in these biological events.

The debate over whether activated Ras can regulate phosphoinositide-specific phospholipase C (PLC) has been contentious and at times heated. The argument may be resolved by the recent identification of a novel Ras-regulated PLC, but some unexpected properties of this protein are sure to stimulate further controversy.

Activation of the collagen receptor glycoprotein VI (GPVI) by a collagen-related peptide (CRP) induces stimulation of platelets and megakaryocytes through the phosphatidylinositol (PI) 3-kinase-dependent pathway leading to activation of Bruton tyrosine kinase (Btk) and phospholipase Cgamma2 (PLCgamma2). Here, we present evidence that both proteins undergo PI 3-kinase-dependent translocation to the plasma membrane on CRP stimulation that is markedly inhibited by wortmannin and LY294002. Translocation of PLCgamma2 but not Btk is also seen in megakaryocytes from X-linked immunodeficiency mice, which have a mutation that reduces the affinity of the pleckstrin homology (PH) domain of Btk for PI 3,4,5-trisphosphate (PI 3,4,5-P3). Activation of PC12 cells by epidermal growth factor (EGF) results in increased PI 3-kinase activity and high PI 3,4,5-P3 levels that trigger translocation of the green fluorescent protein (GFP)-labeled PH of Btk, but not the GFP-labeled PH and tandem Src homology 2 (SH2) domains of PLCgamma2. In contrast to the results with CRP, the G protein-coupled receptor agonist thrombin stimulates PI 3-kinase-independent translocation of Btk but not PLCgamma2. In conclusion, these results demonstrate that in mouse megakaryocytes, CRP leads to PI 3-kinase-dependent translocation of PLCgamma2 and Btk that are independent of one another, whereas thrombin only induces translocation of Btk through a pathway that is independent of PI 3-kinase activity.

Ins(1,4,5)P(3) 3-kinase (IP3K) phosphorylates the Ca(2+)-mobilizing second messenger Ins(1,4,5)P(3) to yield the putative second messenger Ins(1,3,4,5)P(4). A HeLa cell line was established expressing the rat B isoform of IP3K under the control of an inducible promoter. The IP3KB-transfected cell line possessed 23-fold greater IP3K activity than untransfected cells after induction of IP3KB expression, but only 0.23-fold greater activity when IP3KB expression was not induced. Elevating IP3KB expression significantly reduced levels of Ins(1,4,5)P(3) and increased levels of Ins(1,3,4,5)P(4) after stimulation of cells with histamine, but had no effect on basal levels. Histamine- and ATP-evoked cytosolic Ca(2+) responses were dramatically reduced upon elevation of IP3KB expression. On stimulation with a supramaximal dose of histamine, 67% of cells induced to express IP3KB gave no detectable elevation in cytosolic Ca(2+), compared with 3% of uninduced cells. The quantity of Ca(2+) within thapsigargin-sensitive and -insensitive stores was unaffected by elevation of IP3KB expression, as was capacitative Ca(2+) entry. These data suggest that IP3KB may play a significant role in the regulation of Ins(1,4,5)P(3) levels, and consequently in Ca(2+) responses following stimulation of cells with Ins(1,4,5)P(3)-elevating agonists.

Haploid yeast invades solid agar in response to nutrient limitation. To decipher the cues that underlie invasion, we have developed a single cell invasive growth assay. Using this assay, as well as the traditional plate-washing assay, we show that invasive growth occurs in response to glucose depletion. In the absence of glucose (or other fermentable sugar), individual cells adopted a nonaxial budding pattern and elongated morphology within the first cell divisions, and invasion into the agar was observed in microcolonies containing as few as 10 cells. In support of this observation, we found that glucose suppressed the hyperinvasive growth morphology of STE11-4, pbs2, hsl7, and RAS2V19 mutations. In addition, removal of glucose from YPD medium caused constitutive invasion in wild-type cells. We tested glucose control proteins for a role in invasion and found that Snf1, a protein required for derepression of glucose-repressed genes, was required for invasive growth. The transcription factor Sip4, which interacts with Snf1 and is induced during the diauxic shift, had an inhibitory role on invasive growth, suggesting that multiple mechanisms are required for glucose depletion-dependent invasion.

Pleckstrin homology domains are modular domains that direct membrane targeting of their host proteins by binding to polyphosphoinositides; recent results have increased our appreciation of how some of these domains actually bind 3-phosphoinositides, and along the way thrown up some unexpected observations.

The group I family of pleckstrin homology (PH) domains are characterized by their inherent ability to specifically bind phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) and its corresponding inositol head-group inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P(4)). In vivo this interaction results in the regulated plasma membrane recruitment of cytosolic group I PH domain-containing proteins following agonist-stimulated PtdIns(3,4,5)P(3) production. Among group I PH domain-containing proteins, the Ras GTPase-activating protein GAP1(IP4BP) is unique in being constitutively associated with the plasma membrane. Here we show that, although the GAP1(IP4BP) PH domain interacts with PtdIns(3,4, 5)P(3), it also binds, with a comparable affinity, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) (K(d) values of 0.5 +/- 0.2 and 0.8 +/- 0.5 microm, respectively). Intriguingly, whereas this binding site overlaps with that for Ins(1,3,4,5)P(4), consistent with the constitutive plasma membrane association of GAP1(IP4BP) resulting from its PH domain-binding PtdIns(4,5)P(2), we show that in vivo depletion of PtdIns(4,5)P(2), but not PtdIns(3,4,5)P(3), results in dissociation of GAP1(IP4BP) from this membrane. Thus, the Ins(1,3,4,5)P(4)-binding PH domain from GAP1(IP4BP) defines a novel class of group I PH domains that constitutively targets the protein to the plasma membrane and may allow GAP1(IP4BP) to be regulated in vivo by Ins(1,3,4,5)P(4) rather than PtdIns(3,4,5)P(3).

GAP1(IP4BP) is a Ras GTPase-activating protein (GAP) that in vitro is regulated by the cytosolic second messenger inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P(4)]. We have studied Ins(1,3,4,5)P(4) binding to GAP1(IP4BP), and shown that the inositol phosphate specificity and binding affinity are similar to Ins(1,3,4,5)P(4) binding to Bruton's tyrosine kinase (Btk), evidence which suggests a similar mechanism for Ins(1,3,4,5)P(4) binding. The crystal structure of the Btk pleckstrin homology (PH) domain in complex with Ins(1,3,4,5)P(4) has shown that the binding site is located in a partially buried pocket between the beta 1/beta 2- and beta 3/beta 4-loops. Many of the residues involved in the binding are conserved in GAP1(IP4BP). Therefore we generated a model of the PH domain of GAP1(IP4BP) in complex with Ins(1,3,4,5)P(4) based on the Btk-Ins(1,3,4,5)P(4) complex crystal structure. This model had the typical PH domain fold, with the proposed binding site modelling well on the Btk structure. The model has been verified by site-directed mutagenesis of various residues in and around the proposed binding site. These mutations have markedly reduced affinity for Ins(1,3,4,5)P(4), indicating a specific and tight fit for the substrate. The model can also be used to explain the specificity of inositol phosphate binding.

In haploid Saccharomyces cerevisiae, mating occurs by activation of the pheromone response pathway. A genetic selection for mutants that activate this pathway uncovered a class of mutants defective in cell wall integrity. Partial loss-of-function alleles of PGI1, PMI40, PSA1, DPM1, ALG1, MNN10, SPT14, and OCH1, genes required for mannose utilization and protein glycosylation, activated a pheromone-response-pathway-dependent reporter (FUS1) in cells lacking a basal signal (ste4). Pathway activation was suppressed by the addition of mannose to hexose isomerase mutants pgi1-101 and pmi40-101, which bypassed the requirement for mannose biosynthesis in these mutants. Pathway activation was also suppressed in dpm1-101 mutants by plasmids that contained RER2 or PSA1, which produce the substrates for Dpm1. Activation of FUS1 transcription in the mannose utilization/protein glycosylation mutants required some but not all proteins from three different signaling pathways: the pheromone response, invasive growth, and HOG pathways. We specifically suggest that a Sho1 --> Ste20/Ste50 --> Ste11 --> Ste7 --> Kss1 --> Ste12 pathway is responsible for activation of FUS1 transcription in these mutants. Because loss of pheromone response pathway components leads to a synthetic growth defect in mannose utilization/protein glycosylation mutants, we suggest that the Sho1 --> Ste12 pathway contributes to maintenance of cell wall integrity in vegetative cells.

ADP-ribosylation factor (ARF) 6 regulates plasma membrane trafficking and cortical actin formation by cycling between inactive GDP and active GTP-bound conformations. Here we show that agonist stimulation of phosphatidylinositide 3-kinase (PI 3-kinase) activates a pathway that leads to ARF6 activation. We also describe experiments that propose a central role in this pathway for the PI 3-kinase-dependent plasma membrane recruitment of the cytohesin-1 family of PtdIns(3,4,5)P(3)-binding ARF-exchange factors.

The activation of phosphatidylinositol 3-kinase (PI 3-kinase) and production of PtdIns(3,4,5)P(3) is crucial in the actions of numerous extracellular stimuli, including insulin-stimulated glucose uptake. Platelet-derived growth factor (PDGF) also stimulates PI 3-kinase, but only weakly promotes glucose uptake when compared with insulin. Insulin and PDGF have thus been proposed to have differential effects on the subcellular targeting of PI 3-kinase. However, owing to a lack of suitable methodologies, the subcellular localization of the PtdIns(3,4,5)P(3) generated has not been examined. The pleckstrin-homology (PH) domains of the nucleotide exchange factors, ADP-ribosylation factor nucleotide-binding-site opener (ARNO) and general receptor for 3-phosphoinositides (GRP1), which have a high affinity and specificity for PtdIns(3,4,5)P(3), were fused to green fluorescent protein and used to examine the subcellular localization of PtdIns(3,4,5)P(3) generation in living 3T3-L1 adipocytes. PtdIns(3,4,5)P(3) was produced almost exclusively in the plasma membrane in response to both agonists, although the response to insulin was greater in magnitude and occurred in considerably more cells. The results suggest that the greater ability of insulin to stimulate glucose uptake may be the result of its ability to generate significantly more plasma-membrane PtdIns(3, 4,5)P(3) than PDGF. ARNO and GRP1 are nucleotide exchange factors for the small GTP-binding protein ADP-ribosylation factor 6 (ARF6). The inability of a constitutively active GTPase-deficient mutant of ARF6 (ARF6-Q67L; Gln(67)-->Leu) to cause glucose transporter GLUT4 translocation suggests that activation of this pathway is not sufficient to cause GLUT4 translocation.

We report here the molecular cloning, expression, and characterisation of a human homologue of rat centaurin-alpha, which we have termed centaurin-alpha(1). The cDNA contains a single open reading frame, which encodes a 373-amino-acid protein with a calculated molecular weight of 43,429 Daltons. Centaurin-alpha(1) shows high identity at the amino acid level with the other centaurin-alpha homologues, p42(IP4) and PIP(3)BP. Northern analysis revealed that centaurin-alpha(1) expresses as a single 2.5-kb transcript, mainly in the brain. Recombinant centaurin-alpha(1) binds the inositol head group of PtdIns(3,4,5)P(3) and Ins(1,3,4, 5)P(4), with high affinity (K(d) 139.7 +/- 10.5 nM) and inositol phosphate specificity, consistent with it functioning as a putative PtdIns(3,4,5)P(3) receptor. In keeping with this conclusion, we have shown that GFP-tagged centaurin-alpha(1) recruits to the plasma membrane in a PI 3-kinase-dependent manner and the recruitment is inhibited by the PI 3-kinase inhibitor wortmannin. These results suggest that centaurin-alpha(1) can function as an in vivo PtdIns(3, 4,5)P(3) receptor., (Copyright 1999 Academic Press.)

In this review we have described data supporting the conclusion that PtdIns(3,4,5)P3 may regulate the activation state of ARF6 through an ability to recruit the ARF exchange factors ARNO, GRP1 and cytohesin-1 to the plasma membrane. The downstream consequences of such a PtdIns(3,4,5)P3-dependent activation of ARF6 are presently unclear. However, given the role of ARF6 in fusion events at the plasma membrane, we have proposed that PtdIns(3,4,5)P3 may regulate vesicle trafficking at this membrane through its ability to activate ARF6. This is an attractive possibility given the number of PtdIns(3,4,5)P3-dependent pathways which involve some aspect of vesicle trafficking at the plasma membrane, for instance glucose transport, membrane ruffling and cell movement.

Centaurin-alpha is a 46 kDa in vitro binding protein for the lipid second messenger PtdIns(3,4,5)P3. In this report we have addressed whether centaurin-alpha1, a human homologue of centaurin-alpha, binds PtdIns(3,4,5)P3 in vivo and furthermore, identified a potential physiological function for centaurin-alpha1. Using confocal microscopy of live PC12 cells, transiently transfected with a chimera of green fluorescent protein (GFP) fused to the N-terminus of centaurin-alpha1 (GFP-centaurin-alpha1), we demonstrated the rapid plasma membrane recruitment of cytosolic GFP-centaurin-alpha1 following stimulation with either nerve growth factor or epidermal growth factor. This recruitment was dependent on the centaurin-alpha1 pleckstrin homology domains and was blocked by the PtdIns(4,5)P2 3-kinase (PI 3-kinase) inhibitors wortmannin (100 nM) and LY294002 (50 microM), and also by co-expression with a dominant negative p85. Functionally, we demonstrated that centaurin-alpha1 could complement a yeast strain deficient in the ADP-ribosylation factor (ARF) GTPase-activating protein Gcs1; a complementation that was blocked by mutagenesis of conserved cysteine residues within the ARF GTPase-activating protein analogous domain of centaurin-alpha1. Taken together, our data demonstrated that centaurin-alpha1 could potentially function as an ARF GTPase-activating protein that, on agonist stimulation, was recruited to the plasma membrane possibly through an ability to interact with PtdIns(3,4,5)P3.

ADP-ribosylation factors (ARFs) are small GTP-binding proteins that function as regulators of eukaryotic vesicle trafficking. Cytohesin-1 is a member of a family of ARF guanine nucleotide-exchange factors that contain a C-terminal pleckstrin homology (PH) domain which has been proposed to bind the lipid second messenger phosphatidylinositol 3,4,5-trisphosphate (PIP3). Here we demonstrate that in vitro, recombinant cytohesin-1 binds, via its PH domain, the inositol head group of PIP3, inositol 1,3,4, 5-tetrakisphosphate (IP4), with an affinity greater than 200-fold higher than the inositol head group of either phosphatidylinositol 4, 5-bisphosphate or phosphatidylinositol 3,4-bisphosphate. Moreover, addition of glycerol or diacetylglycerol to the 1-phosphate of IP4 does not alter the ability to interact with cytohesin-1, data which is entirely consistent with cytohesin-1 functioning as a putative PIP3 receptor. To address whether cytohesin-1 binds PIP3 in vivo, we have expressed a chimera of green fluorescent protein (GFP) fused to the N terminus of cytohesin-1 in PC12 cells. Using laser scanning confocal microscopy we demonstrate that either EGF- or NGF-stimulation of transiently transfected PC12 cells results in a rapid translocation of GFP-cytohesin-1 from the cytosol to the plasma membrane. This translocation is dependent on the cytohesin-1 PH domain and occurs with a time course that parallels the rate of plasma membrane PIP3 production. Furthermore, the translocation requires the ability of either agonist to activate PI 3-kinase, since it is inhibited by wortmannin (100 nM), LY294002 (50 microM) and by coexpression with a dominant negative p85. This data therefore suggests that in vivo cytohesin-1 can interact with PIP3 via its PH domain.

GAP1(m) is a member of the GAP1 family of Ras GTPase-activating proteins (GAPs) [1]. In vitro, it has been shown to bind inositol 1, 3,4,5-tetrakisphosphate (IP4), the water-soluble inositol head group of the lipid second messenger phosphatidylinositol 3,4, 5-trisphosphate (PIP3) [2] [3]. This has led to the suggestion that GAP1(m) might function as a PIP3 receptor in vivo [4]. Here, using rat pheochromocytoma PC12 cells transiently transfected with a plasmid expressing a chimera of green fluorescent protein fused to GAP1(m) (GFP-GAP1(m)), we show that epidermal growth factor (EGF) induces a rapid (less than 60 seconds) recruitment of GFP-GAP1(m) from the cytosol to the plasma membrane. This recruitment required a functional GAP1(m) pleckstrin homology (PH) domain, because a specific point mutation (R629C) in the PH domain that inhibits IP4 binding in vitro [5] totally blocked EGF-induced GAP1(m) translocation. Furthermore, the membrane translocation was dependent on PI 3-kinase, and the time course of translocation paralleled the rate by which EGF stimulates the generation of plasma membrane PIP3 [6]. Significantly, the PIP3-induced recruitment of GAP1(m) did not appear to result in any detectable enhancement in its basal Ras GAP activity. From these results, we conclude that GAP1(m) binds PIP3 in vivo, and it is recruited to the plasma membrane, but does not appear to be activated, following agonist stimulation of PI 3-kinase.

GAP1(IP4BP) and GAP1(m) belong to the GAP1 family of Ras GTPase-activating proteins that are candidate InsP4 receptors. Here we show they are ubiquitously expressed in human tissues and are likely to have tissue-specific splice variants. Analysis by subcellular fractionation of RBL-2H3 rat basophilic leukemia cells confirms that endogenous GAP1(IP4BP) is primarily localised to the plasma membrane, whereas GAP1(m) appears localised to the cytoplasm (cytosol and internal membranes) but not the plasma membrane. Subcellular fractionation did not indicate a specific co-localisation between membrane-bound GAP1(m) and several Ca2+ store markers, consistent with the lack of co-localisation between GAP1(m) and SERCA1 upon co-expression in COS-7 cells. This difference suggests that GAP1(m) does not reside at a site where it could regulate the ability of InsP4 to release intracellular Ca2+. As GAP1(m) is primarily localised to the cytosol of unstimulated cells it may be spatially regulated in order to interact with Ras at the plasma membrane., (Copyright 1999 Academic Press.)

ADP-ribosylation factors (ARFs) are small GTP-binding proteins that are regulators of vesicle trafficking in eukaryotic cells. GRP1 is a member of a family of ARF guanine-nucleotide-exchange factors that binds in vitro the lipid second messenger phosphatidylinositol 3,4, 5-trisphosphate [PtdIns(3,4,5)P3]. In order to study the effects of PtdIns(3,4,5)P3 on the function of GRP1, we have cloned the human homologue of GRP1, encoding for a protein which is 98.8% identical to mouse brain GRP1. Human GRP1 binds, via its pleckstrin homology (PH) domain, the inositol head group of PtdIns(3,4,5)P3, inositol 1, 3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4], with high affinity (Kd 32. 2+/-5.2 nM) and inositol phosphate specificity [Kd values for Ins(1, 3,4,5,6)P5, InsP6, Ins(1,3,4)P3 and Ins(1,4,5)P3: 283+/-32, >10000, >10000 and >10000 nM, respectively). Furthermore, GRP1 can accommodate addition of glycerol or diacetylglycerol to the 1-phosphate of Ins(1,3,4,5)P4, data that are consistent with its proposed role as a putative PtdIns(3,4,5)P3 receptor. To address whether GRP1 binds PtdIns(3,4,5)P3 in vivo, we have expressed a chimaera of green fluorescent protein (GFP) fused to the N-terminus of GRP1 in PC12 cells and, using confocal microscopy, examined its resultant localization in live cells. Stimulation with either nerve growth factor or epidermal growth factor (both at 100 ng/ml) results in a rapid, PH-domain dependent, translocation of GFP-GRP1 from the cytosol to the plasma membrane, which occurs with a time course that parallels the production of PtdIns(3,4,5)P3. This translocation is dependent on the activation of phosphatidylinositol 3-kinase, since it is inhibited by wortmannin (100 nM), LY294002 (50 microM) and by the co-expression with dominant negative p85. Taken together these data strongly suggest that GRP1 interacts in vivo with plasma membrane-located PtdIns(3,4,5)P3 and hence constitutes a true PtdIns(3,4,5)P3 receptor.

Previously we have purified and cloned a high affinity isomerically specific inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4)-binding protein which, because it is clearly a member of the GAP1 family of Ras GTPase-activating proteins (GAP), we have termed GAP1(IP4BP). Here we show that expressed full-length GAP1(IP4BP) binds Ins(1,3,4, 5)P4 with an affinity and specificity similar to that of the originally purified protein, a binding activity which is dependent on a functional PH/Btk domain. Furthermore, we highlight a fundamental distinction between GAP1(IP4BP) and its homologue GAP1(m), namely that both proteins function as Ras GAPs but only GAP1(IP4BP) displays Rap GAP activity., (Copyright 1998 Academic Press.)

The Rhodobacter capsulatus NtrC protein is a bacterial enhancer-binding protein that activates the transcription of at least five genes by a mechanism that does not require the RpoN RNA polymerase sigma factor. The nifR3-ntrB-ntrC operon in R. capsulatus codes for the nitrogen-sensing two component regulators NtrB and NtrC, as well as for NifR3, a protein of unknown function that is highly conserved in both prokaryotes and eukaryotes. Evidence of a unique translational control of NifR3 mediated directly by the NtrC enhancer-binding protein is reported. The nifR3-ntrB-ntrC operon is expressed from a single promoter upstream of nifR3 with the levels of transcript equivalent in wild-type and ntrC mutants under nitrogen-limited or nitrogen-sufficient conditions. LacZ reporter analyses of this operon and immunological quantitation of NifR3 and NtrC demonstrate that, unlike NtrC levels which remain constant, production of NifR3 is at least ten to 40-fold reduced in NtrC- strains. NifR3 is increased at least fivefold upon nitrogen limitation whereas NtrC production is constitutive. Surprisingly, the purified NtrC protein binds cooperatively to the nifR3 promoter region in vitro at two sets of tandem binding sites centered at +1 and -81 nucleotides relative to the transcriptional start site. Deletion analysis demonstrates that the upstream tandem sites are essential for nitrogen and NtrC-dependent production of NifR3 in vivo , but are not necessary for nifR3 transcription. These experiments indicate that NtrC stimulates the translation of the NifR3 messenger RNA while tethered to the promoter DNA. This is in contrast to five other promoters (nifA1, nifA2, glnB, mopA and anfA) in R. capsulatus which are transcriptionally activated by NtrC bound to one set of tandem binding sites that are centered greater than 100 bp upstream of the transcriptional start site., (Copyright 1998 Academic Press Limited.)

We have previously shown that addition of Ins(1,3,4,5)P4 to permeabilized L1210 cells increases the amount of Ca2+ mobilized by a submaximal concentration of Ins(2,4,5)P3, and we suggested that, in doing this, Ins(1,3,4,5)P4 is not working via an InsP3 receptor but indirectly via an InsP4 receptor [Loomis-Husselbee, Cullen, Dreikhausen, Irvine and Dawson (1996) Biochem. J. 314, 811-816]. Here we have investigated whether this effect might be mediated by GAP1(IP4BP), recently identified as a putative receptor for Ins(1,3, 4,5)P4. GAP1(IP4BP) is a protein that interacts with one or more monomeric G-proteins, so we sought evidence for involvement of monomeric G-proteins in the effects of Ins(1,3,4,5)P4 in permeabilized L1210 cells. Guanosine 5'-[gamma-thio]triphosphate (GTP[S]) enhanced the effect of Ins(1,3,4,5)P4 on Ins(2,4, 5)P3-stimulated Ca2+ mobilization, but had no effect on the action of Ins(2,4,5)P3 alone. A specific enhancement of only the action of Ins(1,3,4,5)P4 was also seen with GTP[S]-loaded R-Ras or Rap1a (two G-proteins known to interact with GAP1(IP4BP)), whereas H-Ras was inactive at similar concentrations. Guanosine 5'-[beta-thio]diphosphate (GDP[S]) did not alter the action of either Ins(2,4,5)P3 or Ins(1,3,4,5)P4. Finally, the addition of exogenous GAP1(IP4BP), purified from platelets, markedly enhanced the effect of Ins(1,3,4,5)P4, and again, the amount of Ca2+ mobilized by Ins(2,4,5)P3 alone was unaltered. We conclude that the increase in Ins(2,4,5)P3-stimulated Ca2+ mobilization by Ins(1,3,4, 5)P4 may be mediated by GAP1(IP4BP) or a closely related protein (such as GAP1(m)), and if so, the action of the GAP1 is not solely to regulate GTP loading of a G-protein, but rather it acts with a G-protein to cause its effect.

ADP-ribosylation factors (ARFs) are small GTP-binding proteins that are regulators of vesicle trafficking in eukaryotic cells [1]. ARNO is a member of the family of guanine nucleotide exchange factors for ARFs which includes cytohesin-1 and GRP-1 [2] [3-5]. Members of this family contain a carboxy-terminal pleckstrin homology (PH) domain which, in the case of GRP-1, has been shown to bind the second messenger phosphatidylinositol 3,4,5-trisphosphate (PIP3) in preference to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2) in vitro [3,4]. Here, we show that recombinant ARNO has the binding characteristics of a PIP3 receptor and that this activity is restricted to the PH domain. When expressed in murine 3T3 L1 adipocytes, ARNO tagged using green fluorescent protein (GFP) is localised exclusively in the cytoplasm. Stimulation with insulin, however, causes a rapid (< 50 second) PH-domain-dependent translocation of GFP-ARNO to the plasma membrane. This translocation is blocked by the PI(4,5)P2 3-kinase (PI 3-kinase) inhibitors wortmannin and LY294002, and by co-expression with a dominant-negative p85 mutant, suggesting that the translocation is a consequence of insulin stimulation of PI 3-kinase. Our data strongly suggest that ARNO binds PIP3 in vivo and that this interaction causes a translocation of ARNO to the plasma membrane where it might activate ARF6 and regulate subsequent plasma membrane cycling events.

Inositol 1,3,4,5-tetrakisphosphate (IP4), is a ubiquitous inositol phosphate that has been suggested to function as a second messenger. Recently, we purified and cloned a putative IP4 receptor, termed GAP1(IP4BP)[1], which is also a member of the GAP1 family of GTPase-activating proteins for the Ras family of GTPases. A homologue of GAP1(IP4BP), called GAP1(m), has been identified [2] and here we describe the cloning of a GAP1(m) cDNA from a human circulating-blood cDNA library. We found that a deletion mutant of GAP1(m), in which the putative phospholipid-binding domains (C2A and C2B) have been removed, binds to IP4 with a similar affinity and specificity to that of the corresponding GAP1(IP4BP) mutant. Expression studies of the proteins in either COS-7 or HeLa cells showed that, whereas GAP1(IP4BP) is located solely at the plasma membrane, GAP1(m) seems to have a distinct perinuclear localisation. By mutational analysis, we have shown that the contrast in subcellular distribution of these two closely related proteins may be a function of their respective pleckstrin homology (PH) domains. This difference in localisation has fundamental significance for our understanding of the second messenger functions of IP4.

To begin to characterize biochemically the transcriptional activation systems in photosynthetic bacteria, the Rhodobacter capsulatus RNA polymerase (RNAP) that contains the sigma70 factor (R. capsulatus RNAP/sigma70) was purified and characterized using two classical sigma70 type promoters, the bacteriophage T7A1 and the RNA I promoters. Transcription from these promoters was sensitive to rifampicin, RNase, and monoclonal antibody 2G10 (directed against the Escherichia coli sigma70 subunit). Specific transcripts were detected in vitro for R. capsulatus cytochrome c2 (cycA) and fructose-inducible (fruB) promoters and genes induced in photosynthesis (puf and puc) and bacteriochlorophyll biosynthesis (bchC). Alignment of these natural promoters activated by R. capsulatus RNAP/sigma70 indicated a preference for the sequence TTGAC at the -35 region for strong in vitro transcription. To test the -35 recognition pattern, the R. capsulatus nifA1 promoter, which exhibits only three of the five consensus nucleotides at the -35 region, was mutated to four and five of the consensus nucleotides. Although the nifA1 wild type promoter showed no transcription, the double mutated promoter exhibited high levels of in vitro transcription by the purified R. capsulatus RNAP/sigma70 enzyme. Similarities and differences between the RNAPs and the promoters of R. capsulatus and E. coli are discussed.

We have previously found that for permeabilized L1210 cells, low micromolar concentrations of Ins(1,3,4,5)P4 added prior to Ins(2,4,5)P3 enhance the effects of suboptimal concentrations of Ins(2,4,5)P3 in causing Ca2+ release from InsP3-sensitive Ca2+ stores [Cullen, Irvine and Dawson (1990) Biochem J. 271, 549-553]. If this was due either to some conversion of added Ins(1,3,4,5)P4 into Ins(1,4,5)P3 by the 3-phosphatase, or to Ins(1,3,4,5)P4 acting as a weak (or partial) agonist on the InsP3 receptor it would be expected that,in the presence of thimerosal to sensitize the InsP3 receptor, the dose-response curve to Ins(1,3,4,5)P4 would be left-shifted by the same extent as that of Ins(1,4,5)P3. This was found not to be the case; the dose-response curve to Ins(1,3,4,5)P4 was not shifted at all by thimerosal. Furthermore, L-Ins(1,3,4,5)P4, which can displace radiolabelled D-Ins(1,3,4,5)P4 but not D-Ins(1,4,5)P3 from their respective high-affinity binding sites, mimicked the effects of D-Ins(1,3,4,5)P4 in enhancing the slow phase of Ins(2,4,5)P3-stimulated Ca2+ release. Ins(1,3,4,5)P4 caused an increase in magnitude of the slow phase of InsP3-stimulated Ca2+ release leaving the magnitude of the fast phase unaltered, in contrast to increasing Ins(2,4,5)P3 concentrations which increased the size of both phases. In addition, Ins(1,3,4,5)P4 decreased the rate constant for the slow phase of Ca2+ release. These findings point strongly to the conclusion that InsP4 is not working directly via the InsP3 receptor but indirectly via an InsP4 receptor.

Enhancer-dependent transcription in enteric bacteria depends upon an activator protein that binds DNA far upstream from the promoter and an alternative sigma factor (sigma 54) that binds with the core RNA polymerase at the promoter. In the photosynthetic bacterium Rhodobacter capsulatus, the NtrB and NtrC proteins (RcNtrB and RcNtrC) are putative members of a two-component system that is novel because the enhancer-binding RcNtrC protein activates transcription of sigma 54-independent promoters. To reconstitute this putative two-component system in vitro, the ReNtrB protein was overexpressed in Escherichia coli and purified as a maltose-binding protein fusion (MBP-RcNtrB). MBP-RcNtrB autophosphorylates in vitro to the same steady state level and with the same stability as the Salmonella typhimurium NtrB (StNtrB) protein but at a lower initial rate. MBP-RcNtrB autophosphorylates the S.typhimurium NtrC (St-NtrC) and RcNtrC proteins in vitro. The enteric NtrC protein is also phosphorylated in vivo by RcNtrB because plasmids that encode either RcNtrB or MBP-Rc-NtrB activate transcription of an NtrC-dependent nifL-lacZ fusion. The rate of phosphotransfer to RcNtrC and autophosphatase activity of phosphorylated RcNtrC (RcNtrC---P) are comparable to the StNtrC protein. However, the RcNtrC protein appears to be a specific RcNtrB P phosphatase since RcNtrC is not phosphorylated by small molecular weight phosphate compounds or by the StNtrB protein. RcNtrC forms a dimer in solution, and RcNtrC - P binds the upstream tandem binding sites of the g1nB promoter 4-fold better than the unphos-phorylated RcNtrC protein, presumably due to oligomerization of RcNtrC -P. Therefore, the R. capsulatus NtrB and NtrC proteins form a two-component system similar to other NtrC-like systems, where specific Rc- NtrB phosphotransfer to the RcNtrC protein results in increased oligoinerization at the enhancer but with subsequent activation of a sigma 54-independent promoter.

Inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) is produced rapidly from inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) in stimulated cells. Despite extensive experimentation, no clearly defined cellular function has yet been described for this inositol phosphate. Binding sites specific for Ins(1,3,4,5)P4 have been identified in several tissues, and we have purified one such protein to homogeneity. Its high affinity for Ins(1,3,4,5)P4, and its exquisite specificity for this isomeric configuration, suggest it may be an Ins(1,3,4,5)P4 receptor. Here we report the cloning and characterization of this protein as a GTPase-activating protein, specifically a member of the GAP1 family. In vitro it shows GAP activity against both Rap and Ras, but only the Ras GAP activity is inhibited by phospholipids and is specifically stimulated by Ins(1,3,4,5)P4.

The specificity of the inositol 1,3,4,5-tetrakisphosphate binding protein purified from porcine platelets [Cullen et al. (1995) Biochem. J. 305, 139-143] was examined using all the isomers of myo-inositol tetrakisphosphate. From the relative potencies of these compounds it appears that phosphorylation of the 1, 3 and 5 positions is essential for high affinity binding, that there is some tolerance of phosphorylation of the 6-hydroxyl, but none of a phosphate in the 2-position, and that phosphorylation of the 4-hydroxyl has very little influence. The binding of Ins(1,3,4,5)P4 was not appreciably altered by physiological Mg2+ concentrations, and the pH dependence of binding under physiological conditions showed a decline from pH 5.5 to pH 9.0.

A novel Ins(1,3,4,5)P4-binding protein has been purified to apparent homogeneity from solubilized membranes derived from pig platelets. It has a high affinity for Ins(1,3,4,5)P4 (Kd 6.3 +/- 0.4 nM), a Bmax of 2.5-6.0 nmol/mg of protein, and a high specificity for Ins(1,3,4,5)P4 [Kd values for Ins(1,3,4,5,6)P5, InsP6, GroPtdIns(3,4,5)P3, Ins(1,4,5)P3, Ins(3,4,5,6)P4 and L-Ins(1,3,4,5)P4 of 85.0 +/- 4.1 nM, 800.0 +/- 20.2 nM, 65.6 +/- 2.6 nM, > 10 microM, 793.3 +/- 55.6 nM and 81.0 +/- 5.9 nM respectively]. The protein has an apparent molecular mass of 104 kDa, suggesting that this peripheral tissue protein may be different from Ins(1,3,4,5)P4 binding proteins previously isolated from neuronal tissues.

The enteric NtrC (NRI) protein has been the paradigm for a class of bacterial enhancer-binding proteins (EBPs) that activate transcription of RNA polymerase containing the sigma 54 factor. Activators in the NtrC class are characterized by essentially three properties: (i) they bind to sites distant from the promoters that they activate (> 100 bp upstream of the transcriptional start site), (ii) they contain a conserved nucleotide-binding fold and exhibit ATPase activity that is required for activation, and (iii) they activate the sigma 54 RNA polymerase. We have characterized the NtrC protein from a photosynthetic bacterium, Rhodobacter capsulatus, which represents a metabolically versatile group of bacteria found in aquatic environments. We have shown that the R. capsulatus NtrC protein (RcNtrC) binds to two tandem sites that are distant from promoters that it activates, nifA1 and nifA2. These tandem binding sites are shown to be important for RcNtrC-dependent nitrogen regulation in vivo. Moreover, the conserved nucleotide-binding fold of RcNtrC is required to activate nifA1 and nifA2 but is not required for DNA binding of RcNtrC to upstream activation sequences. However, nifA1 and nifA2 genes do not require the sigma 54 for activation and do not contain the highly conserved nucleotides that are present in all sigma 54-type, EBP-activated promoters. Thus, the NtrC from this photosynthetic bacterium represents a novel member of the class of bacterial EBPs. It is probable that this class of EBPs is more versatile in prokaryotes than previously envisioned.

In the present study we describe the characterization and localization of Ins(1,3,4,5)P4-binding sites in human platelet membranes. Specific binding sites for Ins(1,3,4,5)P4 have been identified on mixed, plasma and intracellular membranes from neuraminidase-treated platelets using highly purified carrier-free [32P]Ins(1,3,4,5)P4. The displacement of Ins(1,3,4,5)P4 from these sites by Ins(1,4,5)P3 and InsP6 occurs at greater than two orders of magnitude higher concentrations and with Ins(1,3,4,5,6)P5 at about 40-fold higher concentrations than with Ins(1,3,4,5)P4. The membranes were further separated by free-flow electrophoresis into plasma and intracellular membranes. The Ins(1,3,4,5)P4-binding sites separated with plasma membranes, and showed similar affinities and specificities as mixed membranes, whereas Ins(1,4,5)P3-binding sites were predominantly in the intracellular membranes. These results suggest a predominantly plasma membrane location for putative Ins(1,3,4,5)P4 receptors in human platelets.

The alternative sigma factor, RpoN (sigma 54) is responsible for recruiting core RNA polymerase to the promoters of genes required for diverse physiological functions in a variety of eubacterial species. The RpoN protein in Rhodobacter capsulatus is a putative sigma factor specific for nitrogen fixation (nif) genes. Insertional mutagenesis was used to define regions important for the function of the R. capsulatus RpoN protein. Insertions of four amino acids in the predicted helixturn-helix or in the highly conserved C-terminal eight amino acid residues (previously termed the RpoN box), and an in-frame deletion of the glutamine-rich N-terminus completely inactivated the R. capsulatus RpoN protein. Two separate insertions in the second hydrophobic heptad repeat, a putative leucine zipper, resulted in a partially functional RpoN protein. Eight other linkers in the rpoN open reading frame (ORF) resulted in a completely or partially functional RpoN protein. The rpoN gene in R. capsulatus is downstream from the nifHDKU2 genes, in a nifU2-rpoN operon. Results of genetic experiments on the nifU2-rpoN locus show that the rpoN gene is organized in a nifU2-rpoN superoperon. A primary promoter directly upstream of the rpoN ORF is responsible for the initial expression of rpoN. Deletion analysis and insertional mutagenesis were used to define the primary promoter to 50 bp, between 37 and 87 nucleotides upstream of the predicted rpoN translational start site. This primary promoter is expressed constitutively with respect to nitrogen, and it is necessary and sufficient for growth under nitrogen-limiting conditions typically used in the laboratory. A secondary promoter upstream of nifU2 is autoactivated by RpoN and NifA to increase the expression of rpoN, which ultimately results in higher expression of RpoN-dependent genes. Moreover, rpoN expression from this secondary promoter is physiologically beneficial under certain stressful conditions, such as nitrogen-limiting environments that contain high salt (> 50 mM NaCl) or low iron (< 400 nM FeSO4).

Transcription of Rhodobacter capsulatus genes encoding the nitrogenase polypeptides (nifHDK) is repressed by fixed nitrogen and oxygen. Regulatory genes required to sense and relay the nitrogen status of the cell are glnB, ntrB (nifR2), and ntrC (nifR1). R. capsulatus nifA1 and nifA2 require ntrC for activation when fixed nitrogen is limiting. The polypeptides encoded by nifA1 and nifA2 along with the alternate sigma factor RpoN activate nifHDK and the remaining nif genes in the absence of both fixed nitrogen and oxygen. In this study we report the sequence and genetic analysis of the previously identified nifR3-ntrB-ntrC regulatory locus. nifR3 is predicted to encode a 324-amino-acid protein with significant homology to an upstream open reading frame cotranscribed with the Escherichia coli regulatory gene, fis. Analysis of ntrC-lacZ fusions and complementation data indicate that nifR3 ntrBC constitute a single operon. nifR3-lacZ fusions are expressed only when lacZ is in the proper reading frame with the predicted nifR3 gene product. Tn5, a kanamycin-resistance cassette, and miniMu insertions in nifR3 are polar on ntrBC (required for nif transcription). This gene organization suggests that the nifR3 gene product may be involved in nitrogen regulation, although nifR3 is not stringently required for nitrogen fixation when ntrBC are present on a multicopy plasmid. In addition, a R. capsulatus strain with a 22-nucleotide insert in the chromosomal nifR3 gene was constructed. This nifR3 strain is able to fix nitrogen and activate nifA1 and nifA2 genes, again supporting the hypothesis that nifR3 is not stringently required for ntrC-dependent gene activation in R. capsulatus.

One hundred and eight inguinal hernias were treated following a standard surgical technique, using 3-mm thick loosely woven carbon-fibre patches. The average follow-up was five and a half years. There was one recurrence. Otherwise, excellent clinical results without pain were found in all but one patient and only a further three experienced a slight discomfort in the area of the implant, without any restriction on function. The technique is simple and permeation of the carbon-fibre patch by fibrous tissue showed a lasting repair.

1. Ins(1,3,4,5)P4 binding sites were studied in cerebellar and hepatic microsomes from rat, and in bovine adrenal-cortical microsomes. 2. At pH 7.0, all three tissues showed specific binding, with Ins(1,3,4,5)P4 being the most potent competing ligand of those tested [which included Ins(1,4,5)P3, Ins(1,3,4,5,6)P5 and InsP6] and Scatchard analysis suggested two sites; a site with high affinity and high specificity [Kd (1-6) x 10(-9) M] and a site with low affinity and low specificity [Kd (2-6) x 10(-7) M]. 3. At pH 5.5, cerebellar and bovine adrenal microsomes showed similar binding properties: two affinities with a similar specificity for Ins(1,3,4,5)P4 as at pH 7.0. 4. However, when assayed in a low-ionic strength acetate-based buffer at pH 5.0, cerebellar microsomes retain specific Ins(1,3,4,5)P4 binding sites, whereas bovine adrenal and hepatic microsomal binding sites lose much of their specificity, as InsP6 and Ins(1,3,4,5,6)P5 are equally as potent as Ins(1,3,4,5)P4. 5. Pi (25 mM), which is frequently included in Ins(1,3,4,5)P4 binding assays, had a small inhibitory effect on binding of cerebellar and adrenal microsomes at pH 5.5, but a large effect at pH 7.0, so that a considerable decrease occurs in the amount of specific binding at pH 5.5 compared with that at pH 7.0, if Pi is omitted from the binding assay. 6. Cerebellar and adrenal microsomes were used in a ligand-displacement mass assay (conducted under near-physiological conditions, at pH 7.0) on extracts of cerebral-cortex slices stimulated with agonists, and both preparations faithfully detected the increases in Ins(1,3,4,5)P4 that occurred, implying that Ins(1,3,4,5)P4 is the principal ligand on these binding sites in intact cells. 7. Apparent contradictions in the literature with regard to Ins(1,3,4,5)P4 binding sites in neuronal and peripheral tissues can be largely accounted for by the data, and the properties of the binding sites detected at physiological pH are consistent with the possibility that they are putative receptors for the proposed second-messenger role for Ins(1,3,4,5)P4.

1. In electroporated L1210 cells, Ins(1,3,4,5)P4 causes Ca2+ release, owing to its conversion into Ins(1,4,5)P3, but this does not happen in cells permeabilized by digitonin treatment [Cullen, Irvine, Drøbak & Dawson (1989) Biochem. J. 259, 931-933]. 2. If the assay medium is subjected to electroporation by using a commercially available electroporation apparatus and then the cells are added and permeabilized with digitonin, the cells behave as if they had been electroporated. 3. Electroporation causes the release of high concentrations of Al3+ into the experimental medium, and addition of these concentrations of Al3+ into the experimental medium mimics the effect of electroporation on the conversion of Ins(1,3,4,5)P4 into Ins(1,4,5)P3. 4. It is concluded that the difference between electroporated and digitonin-permeabilized L1210 cells in this experimental system can be attributed to dissolution of Al3+ from the electroporation cuvette. Al3+ contamination may thus be a serious problem when using this apparatus.

L1210 lymphoma cells were permeabilized with digitonin, and the ability of Ins(2,4,5)P3 and Ins(1,3,4,5)P4 to mobilize intracellular Ca2+ was studied. At high doses of Ins(2,4,5)P3 Ca2+ was rapidly released from intracellular stores, and prior or subsequent addition of Ins(1,3,4,5)P4 had no discernible effect. However, the Ca2(+)-mobilizing action of low (threshold or just above) concentrations of Ins(2,4,5)P3 was markedly enhanced by Ins(1,3,4,5)P4, which alone caused no mobilization of Ca2+; this phenomenon was shown not to be due to protection of Ins(2,4,5)P3 by the Ins(1,3,4,5)P4 against hydrolysis. The ability of the pre-addition of Ins(1,3,4,5)P4 to enhance subsequent Ins(2,4,5)P3-induced Ca2+ mobilization was always seen whether or not the free Ca2+ concentration was low (pCa = 7) or high (pCa = 6). However, at low Ca2+, Ins(1,3,4,5)P4 could cause a further mobilization if added after the Ins(2,4,5)P3, whereas at higher Ca2+ values Ins(1,3,4,5)P4 was only able to affect Ca2+ if added before Ins(2,4,5)P3. These effects of Ins(1,3,4,5)P4 were not, at the same concentration, mimicked by a random mixture of InsP4 isomers obtained by partial acid hydrolysis of phytic acid, by Ins(1,3,4)P3 or by Ins(1,3,4,5,6)P5, and they were shown not to be due to enzymic generation of Ins(1,4,5)P3 from Ins(1,3,4,5)P4 by (a) the absence of any detectable production of Ins(1,4,5)P3 if radiolabelled Ins(1,3,4,5)P4 was used, or (b) the observation that Ins(1,3,4,5,6)P5 could mimic Ins(1,3,4,5)P4 provided that higher doses were used; this inositol phosphate, when added radiolabelled, yielded only trace quantities of D/L-Ins(1,4,5,6)P4, which itself does not mobilize Ca2+. We interpret these results overall to mean that in these cells there is a small proportion of the Ins(2,4,5)P3-mobilizable Ca2+ pools which can only be mobilized in the presence of Ins(1,3,4,5)P4 [or at the least, Ins(1,3,4,5)P4 can help Ins(2,4,5)P3 to gain access to them]. The significance of this conclusion is discussed in the light of current concepts of the second messenger function of Ins(1,3,4,5)P4.

Thapsigargin, a tumor-promoting sesquiterpene lactone, discharges intracellular Ca2+ in rat hepatocytes, as it does in many vertebrate cell types. It appears to act intracellularly, as incubation of isolated rat liver microsomes with thapsigargin induces a rapid, dose-dependent release of stored Ca2+. The thapsigargin-releasable pool of microsomal Ca2+ includes the pools sensitive to inositol 1,4,5-trisphosphate and GTP. Thapsigargin pretreatment of microsomes blocks subsequent loading with 45Ca2+, suggesting that its target is the ATP-dependent Ca2+ pump of endoplasmic reticulum. This hypothesis is strongly supported by the demonstration that thapsigargin causes a rapid inhibition of the Ca2(+)-activated ATPase activity of rat liver microsomes, with an identical dose dependence to that seen in whole cell or isolated microsome Ca2+ discharge. The inhibition of the endoplasmic reticulum isoform of the Ca2(+)-ATPase is highly selective, as thapsigargin has little or no effect on the Ca2(+)-ATPases of hepatocyte or erythrocyte plasma membrane or of cardiac or skeletal muscle sarcoplasmic reticulum. These results suggest that thapsigargin increases the concentration of cytosolic free Ca2+ in sensitive cells by an acute and highly specific arrest of the endoplasmic reticulum Ca2+ pump, followed by a rapid Ca2+ leak from at least two pharmacologically distinct Ca2+ stores. The implications of this mechanism of action for the application of thapsigargin in the analysis of Ca2+ homeostasis and possible forms of Ca2+ control are discussed.

We present three cases of infection of the native aorta following angiography. The infection was an incidental finding at operation in two patients, while a third presented with fulminant sepsis. All had debridement of the retroperitoneum and underwent successful extraanatomic bypass. We feel caution is warranted in placing a retroperitoneal graft even in suspected aortic sepsis. Prophylactic antibiotics may be advisable to protect against infection of atherosclerotic plaque during angiography.