Identification of a Ras GTPase-activating protein regulated by receptor-mediated Ca2+ oscillations

Receptor-mediated increases in the concentration of intracellular free calcium ([Ca2+]i) are responsible for controlling a plethora of physiological processes including gene expression, secretion, contraction, proliferation, neural signalling, and learning. Increases in [Ca2+]i often occur as repetitive Ca2+ spikes or oscillations. Induced by electrical or receptor stimuli, these repetitive Ca2+ spikes increase their frequency with the amplitude of the receptor stimuli, a phenomenon that appears critical for the induction of selective cellular functions. Here we report the characterisation of RASAL, a Ras GTPase-activating protein that senses the frequency of repetitive Ca2+ spikes by undergoing synchronous oscillatory associations with the plasma membrane. Importantly, we show that only during periods of plasma membrane association does RASAL inactivate Ras signalling. Thus, RASAL senses the frequency of complex Ca2+ signals, decoding them through a regulation of the activation state of Ras. Our data provide a hitherto unrecognised link between complex Ca2+ signals and the regulation of Ras.     

The plasma membrane shuttling of CAPRI is related to regulation of mast cell activation

The Ca(2+)-promoted Ras inactivator (CAPRI), a Ras GTPase-activating protein, is involved in the inactivation of mitogen-activated protein kinase pathway. However, a precise role of CAPRI in immune responses is still unknown. Here we showed that overexpression of CAPRI suppresses antigen-induced degranulation and cytokine production in mast cells (RBL cells). Antigen elicited the translocation of CAPRI to the plasma membrane from the cytoplasm, which was concomitant with the increase in the intracellular Ca(2+) concentration. The nuclear import of extracellular signal-regulated kinase 2 (ERK2) occurred after the re-localization of CAPRI to the cytoplasm in the mast cells, suggesting that the early phase of ERK2 activation is eliminated. A mutant of GAP-related domain, CAPRI(R472S), showed a feeble translocation to the plasma membrane but did not affect the degranulation, ERK2 activation, and cytokine production. The results suggested that the translocation of CAPRI to the plasma membranes regulates crucially cellular responses in mast cells.     

GAP1 family members constitute bifunctional Ras and Rap GTPase-activating proteins

GAP1(IP4BP) is a member of the GAP1 family of Ras GTPase-activating proteins (Ras GAPs) that includes GAP1(m), CAPRI, and RASAL. Composed of a central Ras GAP domain, surrounded by amino-terminal C(2) domains and a carboxyl-terminal pleckstrin homology/Bruton's tyrosine kinase domain, GAP1(IP4BP) has previously been shown to possess an unexpected GAP activity on the Ras-related protein Rap, besides the predicted Ras GAP activity (Cullen, P. J., Hsuan, J. J., Truong, O., Letcher, A. J., Jackson, T. R., Dawson, A. P., and Irvine, R. F. (1995) Nature 376, 527-530). Here we have shown that GAP1(IP4BP) is indeed an efficient Ras/Rap GAP, having K(m)s of 213 and 42 microm and estimated k(cat)s of 48 and 16 s(-1) for Ras and Rap, respectively. For this dual activity, regions outside the Ras GAP domain are required, as the isolated domain (residues 291-569) retains a pronounced Ras GAP activity yet has very low activity toward Rap. Interestingly, mutagenesis of the Ras GAP arginine finger, and surrounding residues important in Ras binding, inhibit both Ras and Rap GAP activity of GAP1(IP4BP). Although the precise details by which GAP1(IP4BP) can function as a Rap GAP remain to be determined, these data are consistent with Rap associating with GAP1(IP4BP) through the Ras-binding site within the Ras GAP domain. Finally, we have established that such dual Ras/Rap GAP activity is not restricted to GAP1(IP4BP). Although GAP1(m) appears to constitute a specific Ras GAP, CAPRI and RASAL display dual activity. For CAPRI, its Rap GAP activity is modulated upon its Ca(2+)-induced association with the plasma membrane.     

Decoding Ca2+ signals: a question of timing

Receptor-stimulated Ca2+ signals come in several flavors. The Ca2+ signals can be decoded linearly or by integration of the response. How the duration of the signal conveyed by cytosolic Ca2+ concentration ([Ca2+]i) changes is regulated is not well understood. Liu et al. (Liu, Q., S.A. Walker, D. Gao, J.A. Taylor, Y.-F. Dai, R.S. Arkell, M.D. Bootman, H.L. Roderick, P.J. Cullen, and P.J. Lockyer. 2005. J. Cell Biol. 170:183-190) now report an example of decoding based on the differential regulation of Ras function by two Ca2+-sensitive Ras inhibitors: Ca2+-promoted Ras activator (CAPRI), which extends the duration of the effect of Ca2+ on Ras activity, and Ras GTPase activating-like protein (RASAL), which functions as a linear decoder of the Ca2+ signal.     

Specific translocation of protein kinase Calpha to the plasma membrane requires both Ca2+ and PIP2 recognition by its C2 domain

The C2 domain of protein kinase Calpha (PKCalpha) controls the translocation of this kinase from the cytoplasm to the plasma membrane during cytoplasmic Ca2+ signals. The present study uses intracellular coimaging of fluorescent fusion proteins and an in vitro FRET membrane-binding assay to further investigate the nature of this translocation. We find that Ca2+-activated PKCalpha and its isolated C2 domain localize exclusively to the plasma membrane in vivo and that a plasma membrane lipid, phosphatidylinositol-4,5-bisphosphate (PIP2), dramatically enhances the Ca2+-triggered binding of the C2 domain to membranes in vitro. Similarly, a hybrid construct substituting the PKCalpha Ca2+-binding loops (CBLs) and PIP2 binding site (beta-strands 3-4) into a different C2 domain exhibits native Ca2+-triggered targeting to plasma membrane and recognizes PIP2. Conversely, a hybrid containing the CBLs but lacking the PIP2 site translocates primarily to trans-Golgi network (TGN) and fails to recognize PIP2. Similarly, PKCalpha C2 domains possessing mutations in the PIP2 site target primarily to TGN and fail to recognize PIP2. Overall, these findings demonstrate that the CBLs are essential for Ca2+-triggered membrane binding but are not sufficient for specific plasma membrane targeting. Instead, targeting specificity is provided by basic residues on beta-strands 3-4, which bind to plasma membrane PIP2.     

Ca2+-dependent monomer and dimer formation switches CAPRI Protein between Ras GTPase-activating protein (GAP) and RapGAP activities

CAPRI is a member of the GAP1 family of GTPase-activating proteins (GAPs) for small G proteins. It is known to function as an amplitude sensor for intracellular Ca(2+) levels stimulated by extracellular signals and has a catalytic domain with dual RasGAP and RapGAP activities. Here, we have investigated the mechanism that switches CAPRI between its two GAP activities. We demonstrate that CAPRI forms homodimers in vitro and in vivo in a Ca(2+)-dependent manner. The site required for dimerization was pinpointed by deletion and point mutations to a helix motif that forms a hydrophobic face in the extreme C-terminal tail of the CAPRI protein. Deletion of this helix motif abolished dimer formation but did not affect translocation of CAPRI to the plasma membrane upon cell stimulation with histamine. We found that dimeric and monomeric CAPRI coexist in cells and that the ratio of dimeric to monomeric CAPRI increases upon cell stimulation with histamine. Free Ca(2+) at physiologically relevant concentrations was both necessary and sufficient for dimer formation. Importantly, the monomeric and dimeric forms of CAPRI exhibited differential GAP activities in vivo; the wild-type form of CAPRI had stronger RapGAP activity than RasGAP activity, whereas a monomeric CAPRI mutant showed stronger RasGAP than RapGAP activity. These results demonstrate that CAPRI switches between its dual GAP roles by forming monomers or homodimers through a process regulated by Ca(2+). We propose that Ca(2+)-dependent dimerization of CAPRI may serve to coordinate Ras and Rap1 signaling pathways.     

Control of astrocyte Ca(2+) oscillations and waves by oscillating translocation and activation of protein kinase C

BACKGROUND: Glutamate-induced Ca2+ oscillations and waves coordinate astrocyte signaling responses, which in turn regulate neuronal excitability. Recent studies have suggested that the generation of these Ca2+ oscillations requires a negative feedback that involves the activation of conventional protein kinase C (cPKC). Here, we use total internal reflection fluorescence (TIRF) microscopy to investigate if and how periodic plasma membrane translocation of cPKC is used to generate Ca2+ oscillations and waves. RESULTS: Glutamate stimulation of astrocytes triggered highly localized GFP-PKCgamma plasma membrane translocation events, induced rapid oscillations in GFP-PKCgamma translocation, and generated GFP-PKCgamma translocation waves that propagated across and between cells. These translocation responses were primarily mediated by the Ca2+-sensitive C2 domains of PKCgamma and were driven by localized Ca2+ spikes, by oscillations in Ca2+ concentration, and by propagating Ca(2+) waves, respectively. Interestingly, GFP-conjugated C1 domains from PKCgamma or PKCdelta that have been shown to bind diacylglycerol (DAG) also oscillated between the cytosol and the plasma membrane after glutamate stimulation, suggesting that PKC is repetitively activated by combined oscillating increases in Ca(2+) and DAG concentrations. The expression of C1 domains, which increases the DAG buffering capacity and thereby delays changes in DAG concentrations, led to a marked prolongation of Ca(2+) spikes, suggesting that PKC activation is involved in terminating individual Ca(2+) spikes and waves and in defining the time period between Ca(2+) spikes. CONCLUSIONS: Our study suggests that cPKCs have a negative feedback role on Ca(2+) oscillations and waves that is mediated by their repetitive activation by oscillating DAG and Ca(2+) concentrations. Periodic translocation and activation of cPKC can be a rapid and markedly localized signaling event that can limit the duration of individual Ca(2+) spikes and waves and can define the Ca(2+) spike and wave frequencies.     

CAPRI regulates Ca(2+)-dependent inactivation of the Ras-MAPK pathway.

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.     

Isoform-specific phosphorylation of metabotropic glutamate receptor 5 by protein kinase C (PKC) blocks Ca2+ oscillation and oscillatory translocation of Ca2+-dependent PKC

Prolonged activation of metabotropic glutamate receptor 5a (mGluR5a) causes synchronized oscillations in intracellular calcium, inositol 1,4,5-trisphosphate production, and protein kinase C (PKC) activation. Additionally, mGluR5 stimulation elicited cyclical translocations of myristoylated alanine-rich protein kinase C substrate, which were opposite to that of gammaPKC (i.e. from plasma membrane to cytosol) and dependent on PKC activity, indicating that myristoylated alanine-rich protein kinase C substrate is repetitively phosphorylated by oscillating gammaPKC on the plasma membrane. Mutation of mGluR5 Thr(840) to aspartate abolished the oscillation of gammaPKC, but the mutation to alanine (T840A) did not. Cotransfection of gammaPKC with betaIIPKC, another Ca2+-dependent PKC, resulted in synchronous oscillatory translocation of both classical PKCs. In contrast, cotransfection of deltaPKC, a Ca2+-independent PKC, abolished the oscillations of both gammaPKC and inositol 1,4,5-trisphosphate. Regulation of the oscillations was dependent on deltaPKC kinase activity but not on gammaPKC. Furthermore, the T840A-mGluR5-mediated oscillations were not blocked by the deltaPKC overexpression. These results revealed that activation of mGluR5 causes translocation of both gammaPKC and deltaPKC to the plasma membrane. deltaPKC, but not gammaPKC, phosphorylates mGluR5 Thr(840), leading to the blockade of both Ca2+ oscillations and gammaPKC cycling. This subtype-specific targeting proposes the molecular basis of the multiple functions of PKC.     

Oscillations of phospholipase C activity triggered by depolarization and Ca2+ influx in insulin-secreting cells

Phospholipase C (PLC) is a ubiquitous enzyme involved in the regulation of a variety of cellular processes. Its dependence on Ca2+ is well recognized, but it is not known how PLC activity is affected by physiological variations of the cytoplasmic Ca2+ concentration ([Ca2+](i)). Here, we applied evanescent wave microscopy to monitor PLC activity in parallel with [Ca2+](i) in individual insulin-secreting INS-1 cells using the phosphatidylinositol 4,5-bisphosphate- and inositol 1,4,5-trisphosphate-binding pleckstrin homology domain from PLCdelta(1) fused to green fluorescent protein (PH(PLCdelta1)-GFP) and the Ca2+ indicator fura red. In resting cells, PH(PLCdelta1)-GFP was located predominantly at the plasma membrane. Activation of PLC by muscarinic or purinergic receptor stimulation resulted in PH(PLCdelta1)-GFP translocation from the plasma membrane to the cytoplasm, detected as a decrease in evanescent wave-excited PH(PLCdelta1)-GFP fluorescence. Using this translocation as a measure of PLC activity, we found that depolarization by raising extracellular [K+] triggered activation of the enzyme. This effect could be attributed both to a rise of [Ca2+](i) and to depolarization per se, because some translocation persisted during depolarization in a Ca2+-deficient medium containing the Ca2+ chelator EGTA. Moreover, oscillations of [Ca2+](i) resulting from depolarization with Ca2+ influx evoked concentration-dependent periodic activation of PLC. We conclude that PLC activity is under tight dynamic control of [Ca2+](i). In insulin-secreting beta-cells, this mechanism provides a link between Ca2+ influx and release from intracellular stores that may be important in the regulation of insulin secretion.     

CAPS acts at a prefusion step in dense-core vesicle exocytosis as a PIP2 binding protein

CAPS-1 is required for Ca2+-triggered fusion of dense-core vesicles with the plasma membrane, but its site of action and mechanism are unknown. We analyzed the kinetics of Ca2+-triggered exocytosis reconstituted in permeable PC12 cells. CAPS-1 increased the initial rate of Ca2+-triggered vesicle exocytosis by acting at a rate-limiting, Ca2+-dependent prefusion step. CAPS-1 activity depended upon prior ATP-dependent priming during which PIP2 synthesis occurs. CAPS-1 activity and binding to the plasma membrane depended upon PIP2. Ca2+ was ineffective in triggering vesicle fusion in the absence of CAPS-1 but instead promoted desensitization to CAPS-1 resulting from decreased plasma membrane PIP2. We conclude that CAPS-1 functions following ATP-dependent priming as a PIP2 binding protein to enhance Ca2+-dependent DCV exocytosis. Essential prefusion steps in dense-core vesicle exocytosis involve sequential ATP-dependent synthesis of PIP2 and the subsequent PIP2-dependent action of CAPS-1. Regulation of PIP2 levels and CAPS-1 activity would control the secretion of neuropeptides and monoaminergic transmitters.     

Reversible intracellular translocation of KRas but not HRas in hippocampal neurons regulated by Ca2+/calmodulin

The Ras/MAPK pathway regulates synaptic plasticity and cell survival in neurons of the central nervous system. Here, we show that KRas, but not HRas, acutely translocates from the plasma membrane (PM) to the Golgi complex and early/recycling endosomes in response to neuronal activity. Translocation is reversible and mediated by the polybasic-prenyl membrane targeting motif of KRas. We provide evidence that KRas translocation occurs through sequestration of the polybasic-prenyl motif by Ca2+/calmodulin (Ca2+/CaM) and subsequent release of KRas from the PM, in a process reminiscent of GDP dissociation inhibitor-mediated membrane recycling of Rab and Rho GTPases. KRas translocation was accompanied by partial intracellular redistribution of its activity. We conclude that the polybasic-prenyl motif acts as a Ca2+/CaM-regulated molecular switch that controls PM concentration of KRas and redistributes its activity to internal sites. Our data thus define a novel signaling mechanism that differentially regulates KRas and HRas localization and activity in neurons.     

Role of Ca2+-dependent metalloprotease-2 in stimulating Ca2+ ATPase activity under peroxynitrite treatment in bovine pulmonary artery smooth muscle membrane

We have determined effect of the oxidant peroxynitrite (ONOO-) on Ca2+-dependent matrix metalloprotease-2 (MMP-2) activity and the role of the protease on Ca2+ ATPase activity in bovine pulmonary vascular smooth muscle plasma membrane under ONOO- -triggered conditions. The smooth muscle plasma membrane possesses a 72-kDa protease activity in a gelatin-containing zymogram. The 72-kDa protease activity has been found to be inhibited by tissue inhibitor of metalloprotease-2 (TIMP-2), indicating that the protease is the matrix metalloprotease-2 (MMP-2). Treatment of the membrane suspension with ONOO- caused stimulation of the MMP-2 activity (as evidenced by 14C-gelatin degradation) and also increased Ca2+ ATPase activity. The ONOO- -triggered protease activity and the Ca2+ ATPase activity were found to be inhibited by the antioxidants: vitamin E, thiourea, and mannitol. Pretreatment with catalase and superoxide dismutase did not significantly alter ONOO- -stimulated MMP-2 activity and Ca2+ATPase activity, indicating that peroxide and superoxide are not present in appreciable amount in ONOO-. Under both basal and ONOO- triggered conditions, the MMP-2 activity and the Ca2+ ATPase activity were also inhibited by EGTA, 1:10-phenanthroline, and TIMP-2. However, the ONOO- -stimulated MMP-2 activity and the Ca2+ ATPase activity were found to be insensitive to phenylmethylsulfonylfluoride, Bowman-Birk inhibitor, chymostatin, leupeptin, antipain, N-ethylmaleimide, and pepstatin. These results suggest that ONOO- caused stimulation of MMP-2 activity and that the increased MMP-2 activity subsequently played a pivotal role in stimulating Ca2+ ATPase activity in bovine pulmonary vascular smooth muscle plasma membrane.     

Identification of an alternatively spliced variant of Ca2+-promoted Ras inactivator as a possible regulator of RANKL shedding

The receptor activator of NF-kappaB ligand (RANKL), a critical regulator of osteoclastogenesis, is synthesized as a membrane-anchored protein and cleaved into a soluble form by ectodomain shedding. We developed an assay system to identify molecules regulating the RANKL shedding. Using this system, we found that a splice variant of Ca2+-promoted Ras inactivator (CAPRI), deltaCAPRI, which is expressed in primary osteoblasts, promoted the RANKL shedding. The wild type CAPRI is a member of the Ras GTPase-activating protein (GAP) family and suppresses Ca2+-dependent Ras activation, whereas deltaCAPRI, which lacks one exon in the GAP-related domain, activated the Ras pathway. Overexpression of deltaCAPRI or a constitutive active form of Ras up-regulated the expression level of matrix-metalloproteinase 14 (MMP14), which directly cleaves the ectodomain of RANKL, whereas Erk activation by expressing the constitutive active Mek1 did not affect the MMP14 expression or RANKL shedding. These results suggest that deltaCAPRI is a possible regulator of RANKL shedding by modulating MMP14 expression through Ras signaling cascades other than the Erk pathway.     

Conventional PKCs regulate the temporal pattern of Ca2+ oscillations at fertilization in mouse eggs

In mammalian eggs, sperm-induced Ca2+ oscillations at fertilization are the primary trigger for egg activation and initiation of embryonic development. Identifying the downstream effectors that decode this unique Ca2+ signal is essential to understand how the transition from egg to embryo is coordinated. Here, we investigated whether conventional PKCs (cPKCs) can decode Ca2+ oscillations at fertilization. By monitoring the dynamics of GFP-labeled PKCalpha and PKCgamma in living mouse eggs, we demonstrate that cPKCs translocate to the egg membrane at fertilization following a pattern that is shaped by the amplitude, duration, and frequency of the Ca2+ transients. In addition, we show that cPKC translocation is driven by the C2 domain when Ca2+ concentration reaches 1-3 microM. Finally, we present evidence that one physiological function of activated cPKCs in fertilized eggs is to sustain long-lasting Ca2+ oscillations, presumably via the regulation of store-operated Ca2+ entry.     

Synaptotagmins form a hierarchy of exocytotic Ca(2+) sensors with distinct Ca(2+) affinities

Synaptotagmins constitute a large family of membrane proteins implicated in Ca(2+)-triggered exocytosis. Structurally similar synaptotagmins are differentially localized either to secretory vesicles or to plasma membranes, suggesting distinct functions. Using measurements of the Ca(2+) affinities of synaptotagmin C2-domains in a complex with phospholipids, we now show that different synaptotagmins exhibit distinct Ca(2+) affinities, with plasma membrane synaptotagmins binding Ca(2+) with a 5- to 10-fold higher affinity than vesicular synaptotagmins. To test whether these differences in Ca(2+) affinities are functionally important, we examined the effects of synaptotagmin C2-domains on Ca(2+)-triggered exocytosis in permeabilized PC12 cells. A precise correlation was observed between the apparent Ca(2+) affinities of synaptotagmins in the presence of phospholipids and their action in PC12 cell exocytosis. This was extended to PC12 cell exocytosis triggered by Sr(2+), which was also selectively affected by high-affinity C2-domains of synaptotagmins. Together, our results suggest that Ca(2+) triggering of exocytosis involves tandem Ca(2+) sensors provided by distinct plasma membrane and vesicular synaptotagmins. According to this hypothesis, plasma membrane synaptotagmins represent high-affinity Ca(2+) sensors involved in slow Ca(2+)-dependent exocytosis, whereas vesicular synaptotagmins function as low-affinity Ca(2+) sensors specialized for fast Ca(2+)-dependent exocytosis.     

Ca2+-dependent translocation of hexose carrier in mouse fibroblast Swiss 3T3 cells

Ca2+-induced translocation of hexose carriers from microsomal membrane to plasma membrane was demonstrated in saponin-permeabilized Swiss 3T3 cells by a specific D-glucose-inhibitable cytochalasin B-binding assay. The number of hexose carriers in the plasma membrane and the hexose transport activity in intact cells were also compared. The incubation of permeabilized cells with 10 microM Ca2+ at 37 degrees C rapidly increased the number of D-glucose-inhibitable cytochalasin B-binding sites in the plasma membrane from 13 to 40 pmol/mg protein and concomitantly decreased that in the microsomal membrane from 66 to 36 pmol/mg protein, each with a half-time of approx. 2 min. Furthermore, when Ca2+-stimulated cells were exposed to 50 microM EGTA, the effect of Ca2+ on the translocation of D-glucose-inhibitable cytochalasin B-binding sites was reversed with a half-time of approx. 5 min. The concentration of Ca2+ required for the half-maximal effect was approx 500 nM. The magnitude of the stimulatory effect of D-glucose-inhibitable cytochalasin B-binding sites in the plasma membrane closely correlated with the magnitude of stimulatory action of Ca2+ on 3-O-methylglucose transport in the intact cells. These results suggest that Ca2+ regulates the activity of hexose transport across the plasma membrane through a rapid and reversible translocation of hexose carrier between microsomal and plasma membranes of mouse fibroblast Swiss 3T3 cells.     

Direct inhibition of the interaction between alpha-interaction domain and beta-interaction domain of voltage-dependent Ca2+ channels by Gem

The Ras-related small G-protein Gem regulates voltage-dependent Ca2+ channels (VDCCs) through interaction with the beta-subunit of the VDCC. This action of Gem is mediated by regulated alpha1-subunit expression at the plasma membrane. In the present study, we examined the mechanism of the inhibition of VDCC activity by Gem. The beta-interaction domain (BID) of the beta-subunit, which specifically interacts with the alpha-interaction domain (AID) of the alpha1-subunit, is shown to be essential for the interaction between Gem and beta-subunits. In addition, the AID peptide inhibited interaction between Gem and beta-subunits in a dose-dependent manner. GemS88N mutant, which has low binding affinity for guanine nucleotide, did not interact with beta-subunits, allowing alpha1-subunit expression at the plasma membrane. This inhibitory effect of wild-type Gem on VDCC activity was reduced in cells expressing GemS88N. The overexpression of wild-type Gem in pancreatic beta-cell line MIN6 cells suppressed Ca2+-triggered secretion, whereas overexpression of GemS88N induced Ca2+-triggered secretion to control level. These results suggest that GTPase activity of Gem is required for the binding of Gem to BID that regulates VDCC activity through interaction with AID.     

Stimulation of intracellular Ca2+ oscillations by diacylglycerol in human myometrial cells

Stimulation of G-protein coupled membrane receptors linked to phospholipase C results in production of the second messengers diacylglycerol and inositol-1,4,5-trisphosphate (IP3). IP3 releases Ca2+ from the endoplasmic reticulum, which triggers increased Ca2+ influx across the plasma membrane, so-called capacitative calcium entry. DAG can also activate plasma membrane calcium-permeable channels but the mechanism is still not fully understood. In the pregnant human myometrial cell line PHM1 and in primary myometrial cells, 1-oleoyl-2-acetyl-sn-glycerol (OAG), a membrane-permeant analogue of diacylglycerol, induced variable oscillatory patterns of intracellular free Ca2+. Similar behavior was seen with Sr2+ entry. The Ca2+ oscillations were not blocked by a broad spectrum of protein kinase C inhibitors, including chelerytrine, bisindolylmaleimide I and calphostin C, and were enhanced and prolonged by RHC-80267, an inhibitor of diacylglycerol lipase. The OAG-induced oscillatory response was not dependent on Ca2+ release from the endoplasmic reticulum but required extracellular Ca2+. Our results indicate that diacylglycerol directly activates cation channels in PHM1 and primary myometrial cells and promotes intracellular Ca2+ oscillations by actions independent of intracellular Ca2+ -ATPase activity and protein kinase C involvement.     

Sensing and refilling calcium stores in an excitable cell.

Inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ mobilization leads to depletion of the endoplasmic reticulum (ER) and an increase in Ca2+ entry. We show here for the gonadotroph, an excitable endocrine cell, that sensing of ER Ca2+ content can occur without the Ca2+ release-activated Ca2+ current (Icrac), but rather through the coupling of IP3-induced Ca2+ oscillations to plasma membrane voltage spikes that gate Ca2+ entry. Thus we demonstrate that capacitative Ca2+ entry is accomplished through Ca(2+)-controlled Ca2+ entry. We develop a comprehensive model, with parameter values constrained by available experimental data, to simulate the spatiotemporal behavior of agonist-induced Ca2+ signals in both the cytosol and ER lumen of gonadotrophs. The model combines two previously developed models, one for ER-mediated Ca2+ oscillations and another for plasma membrane potential-driven Ca2+ oscillations. Simulations show agreement with existing experimental records of store content, cytosolic Ca2+ concentration ([Ca2+]i), and electrical activity, and make a variety of new, experimentally testable predictions. In particular, computations with the model suggest that [Ca2+]i in the vicinity of the plasma membrane acts as a messenger for ER content via Ca(2+)-activated K+ channels and Ca2+ pumps in the plasma membrane. We conclude that, in excitable cells that do not express Icrac, [Ca2+]i profiles provide a sensitive mechanism for regulating net calcium flux through the plasma membrane during both store depletion and refilling.