Involvement of protein kinase C-alpha and -epsilon in extracellular Ca(2+) signalling mediated by the calcium sensing receptor

The sensing of extracellular Ca(2+) concentration ([Ca(2+)](o)) and modulation of cellular processes associated with acute or sustained changes in [Ca(2+)](o) are cell-type specific and mediated by the calcium sensing receptor (CaR). [Ca(2+)](o) signalling requires protein kinase C (PKC), but the identity and role of PKC isoforms in CaR-mediated responses remain unclear. Here we show that high [Ca(2+)](o) activated PKC-alpha and PKC- in parathyroid cells and in human embryonic kidney (HEK293) cells overexpressing the CaR (HEK-CaR) and that this response correlated with the CaR-dependent activation of mitogen-activated protein kinases ERK1/2. Activation of ERK1/2 by acute high [Ca(2+)](o) required influx of Ca(2+)through Ni(2+)-sensitive Ca(2+)channels and phosphatidylinositol-dependent phospholipase C-beta activity. Inhibition of PKC by co-expression of dominant-negative (DN) mutants of PKC-alpha or - with the CaR attenuated sustained ERK1/2 activation. Overexpression of a PKC phosphorylation site (T888A) mutant CaR in HEK293 cells showed that this site was important for ERK1/2 activation at high [Ca(2+)](o). Activation of ERK1/2 by high [Ca(2+)](o) was not necessary for the [Ca(2+)](o)-regulated secretion of parathyroid hormone (PTH) in dispersed bovine parathyroid cells. These data suggest that the CaR-mediated [Ca(2+)](o) signal leading to regulated PTH secretion that requires diacylglycerol-responsive PKC isoforms is not mediated via the ERK pathway.     

Post-conditioning protects rat cardiomyocytes via PKCepsilon-mediated calcium-sensing receptors

Protein kinase C (PKC) plays a role in cardioprotection through reduction of intracellular Ca(2+) concentration [Ca(2+)](i) during ischemic preconditioning (IPC). Cardioprotection against ischemic post-conditioning (PC) could be associated with reduced [Ca(2+)](i) through PKC. The calcium-sensing receptor (CaR), G protein-coupled receptor, causes accumulation of inositol phosphate (IP) to increase the release of intracellular Ca(2+). However, this phenomenon can be negatively regulated by PKC through phosphorylation of Thr-888 of the CaR. This study tested the hypothesis that the prevention of cardiomyocyte damage by PC is associated with [Ca(2+)](i) reduction through an interaction of PKC with the CaR. Isolated rat hearts were subjected to 40min of ischemia followed by 90min of reperfusion. The hearts were post-conditioned after the 40min of ischemia by three cycles of 30s of reperfusion and 30s of re-ischemia applied before the 90min of reperfusion. Immunolocalization of PKCepsilon in the cell membrane was observed with IPC and PC, and in hearts exposed to GdCl(3) during PC. CaR was expressed in cardiac cell membrane and interacted with PKC in IPC, PC, and exposure to GdCl(3) during PC groups. On laser confocal microscopy, intracellular Ca(2+) was significantly decreased with IPC, PC, and exposure to GdCl(3) during PC compared with the I/R and PKC inhibitor groups, and cell structure was better preserved and promoted the recovery of cardiac function after reperfusion in the same groups. These results suggested that PKC is involved in cardioprotection against PC through negative feedback of a CaR-mediated reduction in [Ca(2+)](i).     

Protein kinase C-mediated phosphorylation of the calcium-sensing receptor is stimulated by receptor activation and attenuated by calyculin-sensitive phosphatase activity

The agonist sensitivity of the calcium-sensing receptor (CaR) can be altered by protein kinase C (PKC), with CaR residue Thr(888) contributing significantly to this effect. To determine whether CaR(T888) is a substrate for PKC and whether receptor activation modulates such phosphorylation, a phospho-specific antibody against this residue was raised (CaR(pT888)). In HEK-293 cells stably expressing CaR (CaR-HEK), but not in cells expressing the mutant receptor CaR(T888A), phorbol ester (PMA) treatment increased CaR(pT888) immunoreactivity as observed by immunoblotting and immunofluorescence. Raising extracellular Ca(2+) concentration from 0.5 to 2.5 mM increased CaR(T888) phosphorylation, an effect that was potentiated stereoselectively by the calcimimetic NPS R-467. These responses were mimicked by 5 mM extracellular Ca(2+) and abolished by the calcilytic NPS-89636 and also by PKC inhibition or chronic PMA pretreatment. Whereas CaR(T888A) did exhibit increased apparent agonist sensitivity, by converting intracellular Ca(2+) (Ca(2+)(i)) oscillations to sustained plateau responses in some cells, we still observed Ca(2+)(i) oscillations in a significant number of cells. This suggests that CaR(T888) contributes significantly to CaR regulation but is not the exclusive determinant of CaR-induced Ca(2+)(i) oscillations. Finally, dephosphorylation of CaR(T888) was blocked by the protein phosphatase 1/2A inhibitor calyculin, a treatment that also inhibited Ca(2+)(i) oscillations. In addition, calyculin/PMA cotreatment increased CaR(T888) phosphorylation in bovine parathyroid cells. Therefore, CaR(T888) is a substrate for receptor-induced, PKC-mediated feedback phosphorylation and can be dephosphorylated by a calyculin-sensitive phosphatase.     

Protein kinase C modulates agonist-sensitive release of Ca2+ from internal stores in HEK293 cells overexpressing the calcium sensing receptor

This study examined the mechanism of Ca2+ entry and the role of protein kinase C (PKC) in Ca2+ signaling induced by activation of the calcium sensing receptor (CaR) in HEK293 cells stably expressing the CaR. We demonstrate that influx of Ca2+ following CaR activation exhibits store-operated characteristics in being associated with Ca2+ store depletion and inhibited by 2-aminoethoxydiphenyl borate. Inhibition of PKC with GF109203X, Go6983, or Go6976 and down-regulation of PKC activity enhanced the release of Ca2+ from internal stores in response to the polyvalent cationic CaR agonist neomycin, whereas activation of PKC with acute 12-O-tetradecanoylphorbol-13-acetate treatment decreased the release. In contrast, overexpression of wild type PKC-alpha or -epsilon augmented the neomycin-induced release of Ca2+ from internal stores, whereas dominant negative PKC-epsilon strongly decreased the release, but dominant negative PKC-alpha had little effect. Prolonged treatment of cells with 12-O-tetradecanoylphorbol-13-acetate effectively down-regulated immunoreactive PKC-alpha but had little effect on the expression of PKC-epsilon. Together these results indicate that diacylglycerol-responsive PKC isoforms differentially influence CaR agonist-induced release of Ca2+ from internal stores. The fundamentally different results obtained when overexpressing or functionally down-regulating specific PKC isoforms as compared with pharmacological manipulation of PKC activity indicate the need for caution when interpreting data obtained with the latter approach.     

Ca2+-stimulated Ca2+ oscillations produced by the Ca2+-sensing receptor require negative feedback by protein kinase C

We examined the role of protein kinase C (PKC) in the mechanism and regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) oscillations elicited by an increase in the extracellular concentration of Ca(2+) ([Ca(2+)](e)) in human embryonic kidney 293 cells expressing the Ca(2+)-sensing receptor (CaR). Exposure to the PKC inhibitors bisindolylmaleimide I (GF I) or Ro-31-8220 converted oscillatory responses to transient, non-oscillatory responses, significantly reducing the percentage of cells that showed [Ca(2+)](i) oscillations but without decreasing the overall response to increase in [Ca(2+)](e). Exposure to 100 nm phorbol 12,13-dibutyrate, a direct activator of PKC, eliminated [Ca(2+)](i) oscillations. Addition of phorbol 12,13-dibutyrate at lower concentrations (3 and 10 nm) did not eliminate the oscillations but greatly reduced their frequency in a dose-dependent manner. Co-expression of CaR with constitutively active mutants of PKC (either epsilon or beta(1) isoforms) also reduced [Ca(2+)](i) oscillation frequency. Expression of a mutant CaR in which the major PKC phosphorylation site is altered by substitution of alanine for threonine (T888A) eliminated oscillatory behavior, producing [Ca(2+)](i) responses almost identical to those produced by the wild type CaR exposed to PKC inhibitors. These results support a model in which phosphorylation of the CaR at the inhibitory threonine 888 by PKC provides the negative feedback needed to cause [Ca(2+)](i) oscillations mediated by this receptor.     

Requirement of the TRPC1 cation channel in the generation of transient Ca2+ oscillations by the calcium-sensing receptor

The calcium-sensing receptor (CaR) is an allosteric protein that responds to extracellular Ca(2+) ([Ca(2+)](o)) and aromatic amino acids with the production of different patterns of oscillations in intracellular Ca(2+) concentration ([Ca(2+)](i)). An increase in [Ca(2+)](o) stimulates phospholipase C-mediated production of inositol 1,4,5-trisphosphate and causes sinusoidal oscillations in [Ca(2+)](i). Conversely, aromatic amino acid-induced CaR activation does not stimulate phospholipase C but engages an unidentified signaling mechanism that promotes transient oscillations in [Ca(2+)](i). We show here that the [Ca(2+)](i) oscillations stimulated by aromatic amino acids were selectively abolished by TRPC1 down-regulation using either a pool of small inhibitory RNAs (siRNAs) or two different individual siRNAs that targeted different coding regions of TRPC1. Furthermore, [Ca(2+)](i) oscillations stimulated by aromatic amino acids were also abolished by inhibition of TRPC1 function with an antibody that binds the pore region of the channel. We also show that aromatic amino acid-stimulated [Ca(2+)](i) oscillations can be prevented by protein kinase C (PKC) inhibitors or siRNA-mediated PKCalpha down-regulation and impaired by either calmodulin antagonists or by the expression of a dominant-negative calmodulin mutant. We propose a model for the generation of CaR-mediated transient [Ca(2+)](i) oscillations that integrates its stimulation by aromatic amino acids with TRPC1 regulation by PKC and calmodulin.     

Calcium sensing receptor activation by a calcimimetic suggests a link between cooperativity and intracellular calcium oscillations

Activation of the calcium sensing receptor (CaR) by small increments in extracellular calcium (Ca(2+)(e)) induces intracellular calcium (Ca(2+)(i)) oscillations that are dependent on thapsigargin-sensitive intracellular calcium stores. Phenylalkylamines such as NPS R-568 are allosteric modulators (calcimimetics) that activate CaR by increasing the apparent affinity of the receptor for calcium. We determined, by fluorescence imaging with fura-2, whether the calcimimetic NPS R-568 could activate Ca(2+)(i) oscillations in HEK-293 cells expressing human CaR. NPS R-568 was more potent than Ca(2+)(e) at eliciting Ca(2+)(i) oscillations, particularly at low [Ca(2+)](e) (as low as 0.1 mm). The oscillation frequencies elicited by NPS R-568 varied over a 2-fold range from peak to peak intervals of 60-70 to 30-45 s, depending upon the concentrations of both Ca(2+)(e) and NPS R-568. Finally, NPS R-568 induced sustained (>15 min after drug removal) Ca(2+)(i) oscillations, suggesting slow release of the drug from its binding site. We exploited the potency of NPS R-568 for eliciting Ca(2+)(i) oscillations for structural studies. Truncation of the CaR carboxyl terminus from 1077 to 886 amino acids had no effect on the ability of Ca(2+) or NPS R-568 to induce Ca(2+)(i) oscillations, but further truncation (to 868 amino acids) eliminated both highly cooperative Ca(2+)-dependent activation and regular Ca(2+)(i) oscillations. Alanine scanning within the amino acid sequence from Arg(873) to His(879) reveals a linkage between the cooperativity for Ca(2+)-dependent activation and establishment and maintenance of intracellular Ca(2+) oscillations. The amino acid residues critical to both functions of CaR may contribute to interactions with either G proteins or between CaR monomers within the functional dimer.     

Diacylglycerol mediates the T-cell receptor-driven Ca(2+) influx in T cells by a novel mechanism independent of protein kinase C activation

The mechanism of Ca(2+) influx in nonexcitable cells is not known yet. According to the capacitative hypothesis, Ca(2+) influx is triggered by IP(3)-mediated Ca(2+) release from the intracellular Ca(2+) stores. Conversely, many workers have reported a lack of association between release and influx. In this work, the role of diacylglycerol (DAG) as the mediator of T-cell receptor (TCR)-driven Ca(2+) influx in T cells was investigated. Stimulation of mouse splenic T cells with naturally occurring DAG caused Ca(2+) entry in a dose- and time-dependent manner. Such stimulation was blocked by Ni(2+), a divalent cation known to block Ca(2+) channels. Inhibition of protein kinase C (PKC) by calphostin C did not inhibit, but slightly enhanced, the DAG-stimulated Ca(2+) entry. However, inhibition of DAG metabolism by DAG kinase and lipase inhibitors enhanced the DAG-stimulated Ca(2+) entry. DAG lipase and kinase inhibitors also enhanced the Ca(2+) entry in T cells stimulated through TCR/CD3 complex with anti-CD3 antibody. Calphostin C did not affect the anti-CD3-stimulated Ca(2+) entry. These results showed that TCR-driven Ca(2+) influx in T cells is mediated by DAG through a novel mechanism(s) independent of PKC activation.     

Extracellular calcium-sensing receptor transactivates the epidermal growth factor receptor by a triple-membrane-spanning signaling mechanism

Activation of the extracellular calcium-sensing receptor (CaR) stimulates mitogen-activated protein kinases to upregulate the synthesis and secretion of parathyroid hormone related peptide (PTHrP) from cells expressing the CaR heterologously or endogenously. The current experiments demonstrate that this occurs because CaR activation "transactivates" the EGF receptor (EGFR). Time dependent increases in tyrosine phosphorylation of the EGFR after addition of extracellular calcium ([Ca2+]o, 3 mM) occurred in stably CaR-transfected HEK293 cells but not in non-transfected HEK293 cells. AG1478, an EGFR kinase inhibitor, prevented the CaR-mediated increases of pERK and PTHrP release, while AG1296, a PDGFR kinase inhibitor, had no effect. Inhibitors of matrix metalloproteinase and heparin bound-EGF prevented the CaR-mediated increases of pERK and PTHrP, consistent with a "triple-membrane-spanning signaling" requirement for transactivation of the EGFR by the CaR. Proximal and distal signal transduction cascades activated by the CaR may reflect transactivation of the EGFR by the extracellular calcium-sensing receptor.     

Three adjacent serines in the extracellular domains of the CaR are required for L-amino acid-mediated potentiation of receptor function

The extracellular calcium (Ca(2+)(o))-sensing receptor (CaR) is a key player in Ca(2+)(o) homeostasis. The activity of CaR can be potentiated by various l-amino acids. In this study, we examined whether conserved amino acid residues involved in the binding of glutamate to metabotropic glutamate receptors (mGluRs) also participate in the potentiation of the activity of CaR by l-phenylalanine. Ser-170 corresponding to Thr-188 in rat mGluR1a appears to be important for the modulating actions of phenylalanine. In the presence of phenylalanine, a mutant CaR with a single mutation S170A showed no significant decrease in its EC(50) for stimulation by Ca(2+)(o) and a modest increase in its maximal activity. In addition, mutating Ser-169 and Ser-171 together with Ser-170 yielded a more complete block of the phenylalanine modulation than did the single mutation. The presence of the triple mutation, S169A/S170A/S171A, also eliminated phenylalanine potentiation of the activities of heterodimeric receptors in which one of the monomeric receptors had intact triple serines (A877Stop). The putative amino acid binding site of the CaR is probably close to or structurally dependent on the Ca(2+)(o) binding sites of the receptor, because mutant CaRs with mutations in the putative amino acid binding site exhibited severely reduced responses to Ca(2+)(o).     

m-Calpain colocalizes with the calcium-sensing receptor (CaR) in caveolae in parathyroid cells and participates in degradation of the CaR

The calcium-sensing receptor (CaR) is a G protein-coupled, seven-transmembrane receptor and resides within caveolin-rich membrane domains in bovine parathyroid cells. The proenzyme of calpain 2 (m-calpain) is a heterodimeric calcium-dependent cysteine protease consisting of catalytic and regulatory subunits. The effects of calcium on the enzyme include activation, autolysis, and subunit dissociation. Here, we examine the potential role of caveolin-1 and caveolae in regulating the cellular distribution and function of m-calpain in parathyroid cells. We show that the inactive heterodimeric forms of m-calpain are concentrated in caveolin-rich membrane fractions prepared from parathyroid cells incubated with low extracellular calcium (Ca2+(o)). In contrast, in cells incubated with 3 mm Ca2+(o), which activates the CaR and increases intracellular calcium, there is a reduction in m-calpain in association with an increase in CaR protein and phosphorylated protein kinase C alpha and beta in caveolin-rich fractions. To assess the impact of activation of calpain on CaR protein in caveolar fractions, we analyzed the effects of m-calpain on the CaR. Activation of the CaR with high Ca2+(o) induced the release of lower molecular weight fragments of the receptor into the cell culture medium, and calpain inhibitors blocked this effect. Moreover, the fragments of the CaR as well as caveolin-1, m-calpain, and alkaline phosphatase were localized in membrane vesicles shed by parathyroid cells, supporting the association of these proteins in living cells. Treatment of CaR proteins in vitro with m-calpain also resulted in the appearance of lower molecular weight fragments of the CaR. Our data suggest that localization of m-calpain within caveolae may contribute to maintenance of the enzyme in an inactive state and that m-calpain may also contribute to the regulation of CaR levels.     

Spatial-temporal patterning of metabotropic glutamate receptor-mediated inositol 1,4,5-triphosphate, calcium, and protein kinase C oscillations: protein kinase C-dependent receptor phosphorylation is not required.

The metabotropic glutamate receptors (mGluR), mGluR1a and mGluR5a, are G protein-coupled receptors that couple via G(q) to the hydrolysis of phosphoinositides, the release of Ca(2+) from intracellular stores, and the activation of protein kinase C (PKC). We show here that mGluR1/5 activation results in oscillatory G protein coupling to phospholipase C thereby stimulating oscillations in both inositol 1,4,5-triphosphate formation and intracellular Ca(2+) concentrations. The mGluR1/5-stimulated Ca(2+) oscillations are translated into the synchronized repetitive redistribution of PKCbetaII between the cytosol and plasma membrane. The frequency at which mGluR1a and mGluR5a subtypes stimulate inositol 1,4,5-triphosphate, Ca(2+), and PKCbetaII oscillations is regulated by the charge of a single amino acid residue localized within their G protein-coupling domains. However, oscillatory mGluR signaling does not involve the repetitive feedback phosphorylation and desensitization of mGluR activity, since mutation of the putative PKC consensus sites within the first and second intracellular loops as well as the carboxyl-terminal tail does not prevent mGluR1a-stimulated PKCbetaII oscillations. Furthermore, oscillations in Ca(2+) continued in the presence of PKC inhibitors, which blocked PKCbetaII redistribution from the plasma membrane back into the cytosol. We conclude that oscillatory mGluR signaling represents an intrinsic receptor/G protein coupling property that does not involve PKC feedback phosphorylation.     

Ca2+ influx through reverse mode Na+/Ca2+ exchange is critical for vascular endothelial growth factor-mediated extracellular signal-regulated kinase (ERK) 1/2 activation and angiogenic functions of human endothelial cells

VEGF is a key angiogenic cytokine and a major target in anti-angiogenic therapeutic strategies. In endothelial cells (ECs), VEGF binds VEGF receptors and activates ERK1/2 through the phospholipase γ (PLCγ)-PKCα-B-Raf pathway. Our previous work suggested that influx of extracellular Ca(2+) is required for VEGF-induced ERK1/2 activation, and we hypothesized that this could occur through reverse mode (Ca(2+) in and Na(+) out) Na(+)-Ca(2+) exchange (NCX). However, the role of NCX activity in VEGF signaling and angiogenic functions of ECs had not previously been described. Here, using human umbilical vein ECs (HUVECs), we report that extracellular Ca(2+) is required for VEGF-induced ERK1/2 activation and that release of Ca(2+) from intracellular stores alone, in the absence of extracellular Ca(2+), is not sufficient to activate ERK1/2. Furthermore, inhibitors of reverse mode NCX suppressed the VEGF-induced activation of ERK1/2 in a time- and dose-dependent manner and attenuated VEGF-induced Ca(2+) transients. Knockdown of NCX1 (the main NCX isoform in HUVECs) by siRNA confirmed the pharmacological data. A panel of NCX inhibitors also significantly reduced VEGF-induced B-Raf activity and inhibited PKCα translocation to the plasma membrane and total PKC activity in situ. Finally, NCX inhibitors reduced VEGF-induced HUVEC proliferation, migration, and tubular differentiation in surrogate angiogenesis functional assays in vitro. We propose that Ca(2+) influx through reverse mode NCX is required for the activation and the targeting of PKCα to the plasma membrane, an essential step for VEGF-induced ERK1/2 phosphorylation and downstream EC functions in angiogenesis.     

Extracellular ATP-mediated phospholipase A(2) activation in rat thyroid FRTL-5 cells: regulation by a G(i)/G(o) protein, Ca(2+), and mitogen-activated protein kinase

We investigated the mechanism of phospholipase A(2) (PLA(2)) activation in response to the P2 receptor agonist ATP in rat thyroid FRTL-5 cells. The PLA(2) activity was determined by measuring the release of [(3)H]-arachidonic acid (AA) from prelabeled cells. ATP evoked a dose- and time-dependent AA release. This release was totally inhibited by pertussis toxin (PTX) treatment, indicating the involvement of a G(i)/G(o) protein. The AA release was also diminished by chelating extracellular Ca(2+) with EGTA or by inhibiting influx of Ca(2+) using Ni(2+). Although the activation of protein kinase C (PKC) by 12-phorbol 13-myristate acetate (PMA) alone did not induce any AA release, the ATP-evoked AA release was significantly reduced when PKC was inhibited by GF109203X or by a long incubation with PMA to downregulate PKC. Both the ATP-evoked AA release and the mitogen-activated protein kinase (MAP kinase) phosphorylation were decreased by the MAP kinase kinase (MEK) inhibitor PD98059. Furthermore, the ATP-evoked MAP kinase phosphorylation was also inhibited by GF109203X and by downregulation of PKC, suggesting a PKC-mediated activation of MAP kinase. Inhibiting Src-like kinases by PP1 attenuated both the MAP kinase phosphorylation and the AA release. These results suggest that these kinases are involved in the regulation of MAP kinase and PLA(2) activation. Elevation of intracellular cAMP by TSH or by dBucAMP did not induce a phosphorylation of MAP kinase. Furthermore, neither the ATP-evoked AA release nor the MAP kinase phosphorylation were attenuated by TSH or dBucAMP. Taken together, our results suggest that ATP regulates the activation of PLA(2) by a G(i)/G(o) protein-dependent mechanism. Moreover, Ca(2+), PKC, MAP kinase, and Src-like kinases are also involved in this regulatory process.     

Activation of the neurokinin-1 receptor in rat spinal astrocytes induces Ca2+ release from IP3-sensitive Ca2+ stores and extracellular Ca2+ influx through TRPC3

Substance P (SP) plays an important role in pain transmission through the stimulation of the neurokinin (NK) receptors expressed in neurons of the spinal cord, and the subsequent increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)) as a result of this stimulation. Recent studies suggest that spinal astrocytes also contribute to SP-related pain transmission through the activation of NK receptors. However, the mechanisms involved in the SP-stimulated [Ca(2+)](i) increase by spinal astrocytes are unclear. We therefore examined whether (and how) the activation of NK receptors evoked increase in [Ca(2+)](i) in rat cultured spinal astrocytes using a Ca(2+) imaging assay. Both SP and GR73632 (a selective agonist of the NK1 receptor) induced both transient and sustained increases in [Ca(2+)](i) in a dose-dependent manner. The SP-induced increase in [Ca(2+)](i) was significantly attenuated by CP-96345 (an NK1 receptor antagonist). The GR73632-induced increase in [Ca(2+)](i) was completely inhibited by pretreatment with U73122 (a phospholipase C inhibitor) or xestospongin C (an inositol 1,4,5-triphosphate (IP(3)) receptor inhibitor). In the absence of extracellular Ca(2+), GR73632 induced only a transient increase in [Ca(2+)](i). In addition, H89, an inhibitor of protein kinase A (PKA), decreased the GR73632-mediated Ca(2+) release from intracellular Ca(2+) stores, while bisindolylmaleimide I, an inhibitor of protein kinase C (PKC), enhanced the GR73632-induced influx of extracellular Ca(2+). RT-PCR assays revealed that canonical transient receptor potential (TRPC) 1, 2, 3, 4 and 6 mRNA were expressed in spinal astrocytes. Moreover, BTP2 (a general TRPC channel inhibitor) or Pyr3 (a TRPC3 inhibitor) markedly blocked the GR73632-induced sustained increase in [Ca(2+)](i). These findings suggest that the stimulation of the NK-1 receptor in spinal astrocytes induces Ca(2+) release from IP(3-)sensitive intracellular Ca(2+) stores, which is positively modulated by PKA, and subsequent Ca(2+) influx through TRPC3, which is negatively regulated by PKC.     

Intracellular calcium regulates the tyrosine kinase receptor encoded by the MET oncogene.

Previous work (Gandino, L., Di Renzo, M. F., Giordano, S., Bussolino, F., and Comoglio, P.M. (1990) Oncogene 5, 721-725) has shown that the tyrosine kinase activity of the receptor encoded by the MET protooncogene is negatively modulated by protein kinase C (PKC). We now show that an increase of intracellular Ca2+ has a similar inhibitory effect in vivo, via a PKC-independent mechanism. In GTL-16 cells the p145MET kinase is overexpressed and constitutively phosphorylated on tyrosine. A rapid and reversible decrease of p145MET tyrosine phosphorylation was induced by treatment with the calcium ionophores A23187 or ionomycin. Experiments performed with the ionophores in absence of extracellular calcium showed that a rise in cytoplasmic Ca2+ concentration to 450 nM (due to release from intracellular stores) resulted in a similar effect. These Ca2+ concentrations had no effect on p145MET autophosphorylation in an in vitro kinase assay. This suggests that the effect of Ca2+ on p145MET tyrosine phosphorylation is not direct but may be mediated by Ca(2+)-activated proteins(s). Involvement of Ca(2+)-dependent tyrosine phosphatases was ruled out by experiments carried out in presence of Na2VO4. In vivo labeling with [32P]orthophosphate showed that the rise of intracellular Ca2+ induces serine phosphorylation of p145MET on a specific phosphopeptide. This suggests that Ca2+ negatively modulates p145MET kinase through the phosphorylation of a critical serine residue by a Ca(2+)-activated serine kinase distinct from PKC.     

Human Ca(2+) receptor extracellular domain. Analysis of function of lobe I loop deletion mutants

The G protein-coupled Ca(2+) receptor (CaR) possesses an approximately 600-residue extracellular domain involved in ligand binding and receptor activation. Based on an alignment of the amino acid sequence of the CaR with that of bacterial periplasmic-binding proteins, the first approximately 530 residues of the extracellular domain are believed to form a domain resembling a bilobed Venus's flytrap (VFT). Four insertions in the CaR sequence that do not align with those of bacterial periplasmic-binding proteins correspond to four loops within lobe I of the VFT. We constructed a series of deletion mutants of these four loops and tested their ability to form fully processed CaR as well as their ability to be activated by Ca(2+). As many as 21 residues (365) of loop III could be deleted without impairing receptor expression or activation. Deletion of portions of either loops I (50) or IV (438) did not impair receptor expression but significantly reduced Ca(2+) activation. Deletion of the entire loop II (117) abolished receptor expression and function, but the replacement of even a single residue within this deletion mutant led to expression of a monomeric form of the receptor showing increased Ca(2+) sensitivity but reduced maximal activation. Our results reveal that certain residues within loops I and IV are dispensable in formation of the VFT domain but are critical for Ca(2+) activation of the receptor. In contrast, the residues in loop II are critical for maintaining the inactive state of the CaR. We discuss these results in light of the recently defined crystal structure of the homologous domain of the type 1 metabotropic glutamate receptor.     

Respiring mitochondria determine the pattern of activation and inactivation of the store-operated Ca(2+) current I(CRAC)

In eukaryotic cells, hormones and neurotransmitters that engage the phosphoinositide pathway evoke a biphasic increase in intracellular free Ca(2+) concentration: an initial transient release of Ca(2+) from intracellular stores is followed by a sustained phase of Ca(2+) influx. This influx is generally store dependent. Most attention has focused on the link between the endoplasmic reticulum and store-operated Ca(2+) channels in the plasma membrane. Here, we describe that respiring mitochondria are also essential for the activation of macroscopic store-operated Ca(2+) currents under physiological conditions of weak intracellular Ca(2+) buffering. We further show that Ca(2+)-dependent slow inactivation of Ca(2+) influx, a widespread but poorly understood phenomenon, is regulated by mitochondrial buffering of cytosolic Ca(2+). Thus, by enabling macroscopic store-operated Ca(2+) current to activate, and then by controlling its extent and duration, mitochondria play a crucial role in all stages of store-operated Ca(2+) influx. Store-operated Ca(2+) entry reflects a dynamic interplay between endoplasmic reticulum, mitochondria and plasma membrane.     

Cross-talk between protein kinase C and multifunctional Ca2+/calmodulin-dependent protein kinase.

Protein kinase C (PKC) exhibits both negative and positive cross-talk with multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) in PC12 cells. PKC effects negative cross-talk by inhibiting the mobilization of intracellular Ca2+ stores and by inhibiting Ca2+ influx through voltage-sensitive Ca2+ channels. In the absence of cross-talk, Ca2+ influx induced by depolarization with 56 mM K+ stimulates CaM kinase and its autophosphorylation and converts up to 50% of the enzyme to a Ca(2+)-independent or autonomous species. Acute treatment with phorbol myristate acetate (PMA) elicits a parallel reduction in depolarization-induced Ca2+ influx and in generation of autonomous CaM kinase. Negative cross-talk also occurs during stimulation of the phosphatidylinositol signaling system with bradykinin, which activates both PKC and CaM kinase. The extent of CaM kinase activation is attenuated by the simultaneous activation of PKC; it is enhanced by prior down-regulation of PKC. PKC also exhibits positive cross-talk with CaM kinase. Submaximal activation of CaM kinase by ionomycin is potentiated by concurrent activation of PKC with PMA. Such PMA treatment is found to increase the level of cytosolic calmodulin. Enhanced activation of CaM kinase by PKC may result from PKC-mediated phosphorylation of calmodulin-binding proteins, such as neuromodulin and MARCKS, and the subsequent increase in the availability of previously bound calmodulin for activation of CaM kinase.     

Calcium signalling in squid olfactory receptor neurons

Isolated squid olfactory receptor neurons respond to dopamine and betaine with hyperpolarizing conductances. We used Ca(2+) imaging techniques to determine if changes in intracellular Ca(2+) were involved in transducing the hyperpolarizing odor responses. We found that dopamine activated release of Ca(2+) from intracellular stores while betaine did not change internal Ca(2+) concentrations. Application of 10 mM caffeine also released Ca(2+) from intracellular stores, suggesting the presence of ryanodine-like receptors. Depletion of intracellular stores with 100 microM thapsigargin revealed the presence of a Ca(2+) store depletion-activated Ca(2+) influx. The influx of Ca(2+) through the store-operated channel was reversibly blocked by 10 mM Cd(2+). Taken together, these data suggest a novel odor transduction system in squid olfactory receptor neurons involving Ca(2+) release from intracellular stores. Copyright Copyright 1999 S. Karger AG, Basel