Ca2+/calmodulin independent inositol 1,4,5-trisphosphate 3-kinase activity in guinea pig peritoneal macrophages

1. The Ca2+/calmodulin (CaM) independent activity of inositol 1,4,5-trisphosphate (InsP3) 3-kinase in macrophages could be separated from the dependent activity by serial column chromatography, gel filtration, Orange A and DEAE-5PW. 2. An InsP3 analog which has an aminobenzoyl group on the 2nd carbon of the inositol ring inhibited the conversion of [3H]InsP3 to [3H]InsP4 (inositol 1,3,4,5-tetrakisphosphate) in a dose-dependent manner. The concentration required for half-maximal inhibition (IC50) with the Ca2+/CaM independent enzyme activity was also dependent on the free Ca2+ concentration, as with the dependent activity. 3. These results suggest that a conformational change in the enzyme occurs in response to a change in free Ca2+ concentration, and thus the potency to recognize the InsP3 analog would change, even when the Ca2+/CaM independent enzyme activity was used.     

Release of Ca2+ from a non-mitochondrial store site in peritoneal macrophages treated with saponin by inositol 1,4,5-trisphosphate.

The effects of inositol 1,4,5-trisphosphate, prepared from human erythrocyte ghosts, on Ca2+ release from intracellular store sites were studied in saponin-treated guinea pig peritoneal macrophages. Micromolar concentrations of inositol 1,4,5-trisphosphate released Ca2+ within 1 min from store sites which had accumulated Ca2+ in the presence of 10 mM-NaN3. In the presence of 10 mM-NaN3, the Ca2+ accumulated in the presence of oxalate was seen in the endoplasmic reticulum of saponin-treated macrophages by electron microscopy, indicating that the site of Ca2+ released by inositol 1,4,5-trisphosphate may be endoplasmic reticulum-like membranes. When the concentrations of free Ca2+ were over 3.5 X 10(-6) M, the release of Ca2+ by this agent was inhibited. This inhibition may be due to either the higher concentration of extra-vesicular free Ca2+ or the larger accumulation of Ca2+ into the store site or perhaps both effects. MgCl2 also had an inhibitory effect on the Ca2+ release. Inositol 1,4,5-trisphosphate also released Ca2+ from cardiac sarcoplasmic reticulum, but not from erythrocyte inside-out vesicles.     

Activation by calmodulin of inositol-1,4,5-trisphosphate 3-kinase in guinea pig peritoneal macrophages

The activity of inositol-1,4,5-trisphosphate 3-kinase in the cytosol fraction of guinea pig macrophages was assayed with special reference to the dependence on the free Ca2+ concentration. The enzyme activity, as assessed by the production of inositol 1,3,4,5-tetrakisphosphate was reversibly activated by free Ca2+ concentrations ranging from 10(-7) to 10(-6)M. The calmodulin antagonists, W-7 and chlorpromazine, inhibited the Ca2+-activated enzyme activity in a dose-dependent fashion, thereby indicating that calmodulin may be involved in the activation by Ca2+. The content of calmodulin in the cytosol fraction (about 2.8 micrograms/mg of cytosol protein) was markedly reduced to less than 0.03 microgram/mg of proteins by subfractionation by ammonium sulfate, followed by an anion-exchange chromatography. The subfraction obtained by the chromatography showed no Ca2+ dependence in the enzyme activity, while an exogenous addition of calmodulin with 10(-6)M Ca2+ increased the enzyme activity. The enzyme activity was retained on a calmodulin-affinity column in the presence of Ca2+, and was eluted from the column by lowering the free Ca2+ concentration by adding ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid. These results clearly indicate that calmodulin activates the inositol-1,4,5-trisphosphate 3-kinase activity.     

Stereospecific mobilization of intracellular Ca2+ by inositol 1,4,5-triphosphate. Comparison with inositol 1,4,5-trisphosphorothioate and inositol 1,3,4-trisphosphate.

The isolated activation segment of pig procarboxypeptidase A binds two Tb3+ ions in a strong and specific way. In contrast, the binding of Ca2+, Cd2+ and Mg2+ is weak. The binding of Tb3+ increases the resistance of the isolated activation segment against proteolysis and competes for the binding of the carbocyanine dye Stains-All. This dye forms complexes with the activation segment showing spectral properties similar to those observed with EF-hand structures. The presented results support a previous hypothesis on the existence of two regions in the activation segment of pancreatic procarboxypeptidases structurally related to Ca2+-binding domains of the EF-hand protein family.     

Amplification of Ca2+ signaling by diacylglycerol-mediated inositol 1,4,5-trisphosphate production

Stimulation of various cell surface receptors leads to the production of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) through phospholipase C (PLC) activation, and the IP3 and DAG in turn trigger Ca2+ release through IP3 receptors and protein kinase C activation, respectively. The amount of IP(3) produced is particularly critical to determining the spatio-temporally coordinated Ca(2+)-signaling patterns. In this paper, we report a novel signal cross-talk between DAG and the IP3-mediated Ca(2+)-signaling pathway. We found that a DAG derivative, 1-oleoyl-2-acyl-sn-glycerol (OAG), induces Ca2+ oscillation in various types of cells independently of protein kinase C activity and extracellular Ca2+. The OAG-induced Ca2+ oscillation was completely abolished by depletion of Ca2+ stores or inhibition of PLC and IP3 receptors, indicating that OAG stimulates IP3 production through PLC activation and thereby induces IP3-induced Ca2+ release. Furthermore, intracellular accumulation of endogenous DAG by a DAG-lipase inhibitor greatly increased the number of cells responding to agonist stimulation at low doses. These results suggest a novel physiological function of DAG, i.e. amplification of Ca2+ signaling by enhancing IP3 production via its positive feedback effect on PLC activity.     

Requirement of Ca2+ for the production and degradation of inositol 1,4,5-trisphosphate in macrophages

The requirement of Ca2+ for the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) or the accumulation of inositol 1,4,5-trisphosphate (InsP3) in macrophages stimulated with fMet-Leu-Phe was examined using [32P]Pi or [3H]inositol-labeled cells. The dependence on Ca2+ of inositol-trisphosphate phosphatase was also examined. The application of 1 X 10(-8) M fMet-Leu-Phe caused a rapid decrease in the amount of PtdInsP2 to 70% of the control within 10 s, and the decrease was reverted to the control level by prolonged incubation. The decrease in the amount of PtdInsP2 accompanied the accumulation of phosphatidic acid and of InsP3, indicating that the loss of PtdInsP2 is due to phosphodiesteric breakdown. The dose-dependence of fMet-Leu-Phe or its analog on the hydrolysis of PtdInsP2 was much the same as that of the increase in intracellular free Ca2+ concentration in macrophages. The loss of PtdInsP2 as induced by fMet-Leu-Phe was similarly observed in macrophages treated with ionophore A23187 in the absence of external Ca2+ for 10 min. InsP3 was degraded by the particulate or cytosol fraction prepared from macrophages, and the activity of inositol-trisphosphate phosphatase in the particulate fraction was higher than that in the cytosol fraction. The enzyme in the cytosol fraction required Mg2+ for activity, and was activated by free Ca2+ concentrations ranging from 10(-7) to 10(-6) M in the presence of 1 mM MgCl2.     

An inositol 1,4,5-trisphosphate receptor-gated intracellular Ca(2+) store is involved in regulating sperm hyperactivated motility.

Hyperactivated motility, a swimming pattern displayed by mammalian sperm in the oviduct around the time of ovulation, is essential to fertilization. Ca(2+) has been shown to be crucial for the initiation and maintenance of hyperactivated motility. Nevertheless, how Ca(2+) reaches the axoneme in the core of the flagellum to switch on hyperactivation is unknown. Ca(2+)-releasing agents were used to determine whether an intracellular store provides Ca(2+) to the axoneme. Hyperactivation was induced immediately in bull sperm by thapsigargin, caffeine, and thimerosal. The responses were dose-dependent and were induced in both capacitated and uncapacitated sperm. When external Ca(2+) was buffered below 50 nM with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, the response to caffeine was significantly reduced; however, the responses to thapsigargin and thimerosal were not affected. This indicates caffeine-induced hyperactivation depends on external Ca(2+) influx, whereas hyperactivation by thapsigargin and thimerosal do not. Acrosome reactions were not induced by these treatments; therefore, an acrosomal store was probably not involved. Indirect immunofluorescence labeling showed type I inositol 1,4,5-trisphosphate receptors (IP(3)R) in the acrosome and neck region, but no ryanodine receptors (RyR) were found using anti-RyR antibodies or BODIPY FL-X ryanodine. These data indicate that there is an IP(3)R-gated Ca(2+) store in the neck region of sperm that regulates hyperactivated motility.     

Ca2+ depletion and inositol 1,4,5-trisphosphate-evoked activation of Ca2+ entry in single guinea pig hepatocytes

Store-operated Ca(2+) entry was investigated by monitoring the Ca(2+)-dependent K(+) permeability in voltage-clamped guinea pig hepatocytes. In physiological conditions, intracellular Ca(2+) stores are discharged following agonist stimulation, but depletion of this stores can be achieved using Ca(2+)-Mg(2+)-ATPase inhibitors such as 2,5-di(tert-butyl)-1,4-benzohydroquinone and thapsigargin. The effect of internal Ca(2+) store depletion on Ca(2+) influx was tested in single cells using inositol 1,4,5-trisphosphate (InsP(3)) release from caged InsP(3) after treatment of the cells with 2, 5-di(tert-butyl)-1,4-benzohydroquinone or thapsigargin in Ca(2+)-free solutions. We show that the photolytic release of 1-d-myo-inositol 1,4-bisphosphate 5-phosphorothioate, a stable analog of InsP(3), and Ca(2+) store depletion have additive effects to activate a high level of Ca(2+) entry in single guinea pig hepatocytes. These results suggest that there is a direct functional interaction between InsP(3) receptors and Ca(2+) channels in the plasma membrane, although the nature of these Ca(2+) channels in hepatocytes is unclear.     

The size of inositol 1,4,5-trisphosphate-sensitive Ca2+ stores depends on inositol 1,4,5-trisphosphate concentration.

An explanation of the complex effects of hormones on intracellular Ca2+ requires that the intracellular actions of Ins(1,4,5)P3 and the relationships between intracellular Ca2+ stores are fully understood. We have examined the kinetics of 45Ca2+ efflux from pre-loaded intracellular stores after stimulation with Ins(1,4,5)P3 or the stable phosphorothioate analogue, Ins(1,4,5)P3[S]3, by simultaneous addition of one of them with glucose/hexokinase to rapidly deplete the medium of ATP. Under these conditions, a maximal concentration of either Ins(1,4,5)P3 or Ins(1,4,5)P3[S]3 evoked rapid efflux of about half of the accumulated 45Ca2+, and thereafter the efflux was the same as occurred under control conditions. Submaximal concentrations of Ins(1,4,5)P3 or Ins(1,4,5)P3[S]3 caused a smaller rapid initial efflux of 45Ca2+, after which the efflux was similar whatever the concentration of Ins(1,4,5)P3 or Ins(1,4,5)P3[S]3 present. The failure of submaximal concentrations of Ins(1,4,5)P3 and Ins(1,4,5)P3[S]3 to mobilize fully the Ins(1,4,5)P3-sensitive Ca2+ stores despite prolonged incubation was not due either to inactivation of Ins(1,4,5)P3 or to desensitization of the Ins(1,4,5)P3 receptor. The results suggest that the size of the Ins(1,4,5)P3 sensitive Ca2+ stores depends upon the concentration of Ins(1,4,5)P3.     

Polycystin 2 interacts with type I inositol 1,4,5-trisphosphate receptor to modulate intracellular Ca2+ signaling

Autosomal dominant polycystic kidney disease, a common cause of renal failure, arises from mutations in either the PKD1 or the PKD2 gene. The precise function of both PKD gene products polycystins (PCs) 1 and 2 remain controversial. PC2 has been localized to numerous cellular compartments, including the endoplasmic reticulum, plasma membrane, and cilia. It is unclear what pools are the most relevant to its physiological function as a putative Ca2+ channel. We employed a Xenopus oocyte Ca2+ imaging system to directly investigate the role of PC2 in inositol 1,4,5-trisphosphate (IP3)-dependent Ca2+ signaling. Cytosolic Ca2+ signals were recorded following UV photolysis of caged IP3 in the absence of extracellular Ca2+. We demonstrated that overexpression of PC2, as well as type I IP3 receptor (IP3R), significantly prolonged the half-decay time (t1/2) of IP3-induced Ca2+ transients. However, overexpressing the disease-associated PC2 mutants, the point mutation D511V, and the C-terminally truncated mutation R742X did not alter the t1/2. In addition, we found that D511V overexpression significantly reduced the amplitude of IP3-induced Ca2+ transients. Interestingly, overexpression of the C terminus of PC2 not only significantly reduced the amplitude but also prolonged the t1/2. Co-immunoprecipitation assays indicated that PC2 physically interacts with IP3R through its C terminus. Taken together, our data suggest that PC2 and IP3R functionally interact and modulate intracellular Ca2+ signaling. Therefore, mutations in either PC1 or PC2 could result in the misregulation of intracellular Ca2+ signaling, which in turn could contribute to the pathology of autosomal dominant polycystic kidney disease.     

Inositol 1,4,5-trisphosphate and inositol 1,3,4-trisphosphate formation in Ca2+-mobilizing-hormone-activated cells.

The inositol trisphosphate liberated on stimulation of guinea-pig hepatocytes, pancreatic acinar cells and dimethyl sulphoxide-differentiated human myelomonocytic HL-60 leukaemia cells is composed of two isomers, the 1,4,5-trisphosphate and the 1,3,4-trisphosphate. Inositol 1,4,5-trisphosphate was released rapidly, with no measurable latency on hormone stimulation, and, consistent with its proposed role as an intracellular messenger for Ca2+ mobilization, there was good temporal correlation between its formation and Ca2+-mediated events in these tissues. There was a definite latency before an increase in the formation of inositol 1,3,4-trisphosphate could be detected. In all of these tissues, however, it formed a substantial proportion of the total inositol trisphosphate by 1 min of stimulation. In guinea-pig hepatocytes, where inositol trisphosphate increases for at least 30 min after hormone application, inositol 1,3,4-trisphosphate made up about 90% of the total inositol trisphosphate by 5-10 min. In pancreatic acinar cells, pretreatment with 20 mM-Li+ caused an increase in hormone-induced inositol trisphosphate accumulation. This increase was accounted for by a rise in inositol 1,3,4-trisphosphate; inositol 1,4,5-trisphosphate was unaffected. This finding is consistent with the observation that Li+ has no effect on Ca2+-mediated responses in these cells. The role, if any, of inositol 1,3,4-trisphosphate in cellular function is unknown.     

Caspase-3-truncated type 1 inositol 1,4,5-trisphosphate receptor enhances intracellular Ca2+ leak and disturbs Ca2+ signalling

Background information. The IP₃R (inositol 1,4,5‐trisphosphate receptor) is a tetrameric channel that accounts for a large part of the intracellular Ca²⁺ release in virtually all cell types. We have previously demonstrated that caspase‐3‐mediated cleavage of IP₃R1 during cell death generates a C‐terminal fragment of 95 kDa comprising the complete channel domain. Expression of this truncated IP₃R increases the cellular sensitivity to apoptotic stimuli, and it was postulated to be a constitutively active channel. Results. In the present study, we demonstrate that expression of the caspase‐3‐cleaved C‐terminus of IP₃R1 increased the rate of thapsigargin‐mediated Ca²⁺ leak and decreased the rate of Ca²⁺ uptake into the ER (endoplasmic reticulum), although it was not sufficient by itself to deplete intracellular Ca²⁺ stores. We detected the truncated IP₃R1 in different cell types after a challenge with apoptotic stimuli, as well as in aged mouse oocytes. Injection of mRNA corresponding to the truncated IP₃R1 blocked sperm factor‐induced Ca²⁺ oscillations and induced an apoptotic phenotype. Conclusions. In the present study, we show that caspase‐3‐mediated truncation of IP₃R1 enhanced the Ca²⁺ leak from the ER. We suggest a model in which, in normal conditions, the increased Ca²⁺ leak is largely compensated by enhanced Ca²⁺‐uptake activity, whereas in situations where the cellular metabolism is compromised, as occurring in aging oocytes, the Ca²⁺ leak acts as a feed‐forward mechanism to divert the cell into apoptosis.     

Ca2+-induced Ca2+ release by activation of inositol 1,4,5-trisphosphate receptors in primary pancreatic beta-cells

The effect of sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA) inhibition on the cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) was studied in primary insulin-releasing pancreatic beta-cells isolated from mice, rats and human subjects as well as in clonal rat insulinoma INS-1 cells. In Ca(2+)-deficient medium the individual primary beta-cells reacted to the SERCA inhibitor cyclopiazonic acid (CPA) with a slow rise of [Ca(2+)](i) followed by an explosive transient elevation. The [Ca(2+)](i) transients were preferentially observed at low intracellular concentrations of the Ca(2+) indicator fura-2 and were unaffected by pre-treatment with 100 microM ryanodine. Whereas 20mM caffeine had no effect on basal [Ca(2+)](i) or the slow rise in response to CPA, it completely prevented the CPA-induced [Ca(2+)](i) transients as well as inositol 1,4,5-trisphosphate-mediated [Ca(2+)](i) transients in response to carbachol. In striking contrast to the primary beta-cells, caffeine readily mobilized intracellular Ca(2+) in INS-1 cells under identical conditions, and such mobilization was prevented by ryanodine pre-treatment. The results indicate that leakage of Ca(2+) from the endoplasmic reticulum after SERCA inhibition is feedback-accelerated by Ca(2+)-induced Ca(2+) release (CICR). In primary pancreatic beta-cells this CICR is due to activation of inositol 1,4,5-trisphosphate receptors. CICR by ryanodine receptor activation may be restricted to clonal beta-cells.     

Temperature-dependence and conformational basis of inositol 1,4,5-trisphosphate receptor regulated by Ca2+

The inositol 1,4,5-trisphosphate (InsP₃) receptor was purified from bovine cerebellum and reconstituted in liposomes composed of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) (1:1) successfully. No effect of Ca²⁺ concentration on [³H]-InsP₃ binding to unreconstituted InsP₃ receptor could be observed either at 4°C or at 25°C, whereas the effect of [Ca²⁺] on reconstituted InsP₃ receptor depended on the temperature. The Ca²⁺ concentration outside the proteolipsome ([Ca²⁺]_o) had no detectable effect on InsP₃ binding to InsP₃ receptor at 4°C. In contrast, with increase of [Ca²⁺]_o from 0 to 100 nmol/L at 25°C, the InsP₃ binding activity increased gradually. Then the InsP₃ binding activity was decreased drastically at higher [Ca²⁺]_o and inhibited entirely at 50 μmol/L [Ca²⁺]_o. Conformational studies on intrinsic fluorescence of the reconstituted InsP₃ receptor and its quenching by KI and HB indicated that the global conformation of reconstituted InsP₃ receptor could not be affected by [Ca²⁺]_o at 4°C. While at 25°C, the effects of 10 μmol/L [Ca²⁺]_o on global, membrane and cytoplasmic conformation of the reconstituted InsP₃ receptor were different significantly from that of 100 nmol/L [Ca²⁺]_o.     

Temperature-dependence and conformational basis of inositol 1,4,5-trisphosphate receptor regulated by Ca2+

The inositol 1,4,5-trisphosphate (InsP3) receptor was purified from bovine cerebellum and reconstituted in liposomes composed of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) (1:1) successfully.No effect of Ca2+ concentration on [3H]-InsP3 binding to unreconstituted InsP3 receptor could be observed either at 4℃ or at 25℃,whereas the effect of [Ca2+] on reconstituted InsP3 receptor depended on the temperature.The Ca2+ concentration outside the proteolipsome ([Ca2+]o) had no detectable effect on InsP3 binding to InsP3 receptor at 4℃.In contrast,with increase of [Ca2+]o from 0 to 100 nmol/L at 25℃,the InsP3 binding activity increased gradually.Then the InsP3 binding activity was decreased drastically at higher [Ca2+]o and inhibited entirely at 50 mol/L [Ca2+]o.Conformational studies on intrinsic fluorescence of the reconstituted InsP3 receptor and its quenching by KI and HB indicated that the global conformation of reconstituted InsP3 receptor could not be affected by [Ca2+]o at 4℃.While at 25℃,the effects of 10 m mol/L [Ca2+]o on global,membrane and cytoplasmic conformation of the reconstituted InsP3 receptor were different significantly from that of 100 nmol/L [Ca2+]o.     

Caspase-3-truncated type 1 inositol 1,4,5-trisphosphate receptor enhances intracellular Ca2+ leak and disturbs Ca2+ signalling.

BACKGROUND INFORMATION: The IP(3)R (inositol 1,4,5-trisphosphate receptor) is a tetrameric channel that accounts for a large part of the intracellular Ca(2+) release in virtually all cell types. We have previously demonstrated that caspase-3-mediated cleavage of IP(3)R1 during cell death generates a C-terminal fragment of 95 kDa comprising the complete channel domain. Expression of this truncated IP(3)R increases the cellular sensitivity to apoptotic stimuli, and it was postulated to be a constitutively active channel. RESULTS: In the present study, we demonstrate that expression of the caspase-3-cleaved C-terminus of IP(3)R1 increased the rate of thapsigargin-mediated Ca(2+) leak and decreased the rate of Ca(2+) uptake into the ER (endoplasmic reticulum), although it was not sufficient by itself to deplete intracellular Ca(2+) stores. We detected the truncated IP(3)R1 in different cell types after a challenge with apoptotic stimuli, as well as in aged mouse oocytes. Injection of mRNA corresponding to the truncated IP(3)R1 blocked sperm factor-induced Ca(2+) oscillations and induced an apoptotic phenotype. CONCLUSIONS: In the present study, we show that caspase-3-mediated truncation of IP(3)R1 enhanced the Ca(2+) leak from the ER. We suggest a model in which, in normal conditions, the increased Ca(2+) leak is largely compensated by enhanced Ca(2+)-uptake activity, whereas in situations where the cellular metabolism is compromised, as occurring in aging oocytes, the Ca(2+) leak acts as a feed-forward mechanism to divert the cell into apoptosis.     

Ca2+ release in the endoplasmic reticulum of guinea pig peritoneal macrophages

Passive permeability of the endoplasmic reticulum of saponin-treated macrophages to Ca2+ was studied by the filtration method using 45Ca. The Ca2+ release from the endoplasmic reticulum of macrophages was enhanced by the presence of submicromolar concentrations of Ca2+ in the medium. The Ca2+ release was enhanced by caffeine, and suppressed by MgCl2. These phenomena are similar to the Ca2+-induced Ca2+ release reported for the sarcoplasmic reticulum of skeletal muscle. On the other hand, adenine suppressed the Ca2+ release from the endoplasmic reticulum, while it reportedly enhanced the Ca2+-induced Ca2+ release of the skeletal muscle. The threshold concentration of Ca2+ for the Ca2+-induced Ca2+ release was approximately 10(-8) M in the presence of 0.95 mM MgCl2 in macrophages. The spontaneous spreading of macrophages and spontaneous migration of macrophages were inhibited by adenine, and also by caffeine in spite of the enhancement of the Ca2+-induced Ca2+ release.     

Inositol 1,4,5-trisphosphate releases Ca2+ from intracellular store sites in skinned single cells of porcine coronary artery

Effects of inositol 1,4,5- trisphosphate , extracted from human erythrocyte ghosts, on Ca2+ release from intracellular store sites were studied in saponin-treated single muscle cells of the porcine coronary artery. Application of micromolar concentrations of inositol 1,4,5- trisphosphate released Ca2+ from the intracellular non-mitochondrial store sites, within 1 min. However, when the concentrations of free Ca2+ were over 1.5 X 10(-6) M, the release of Ca2+ by this agent was inhibited. The Ca2+ releasing mechanism differed from that seen with A23187, therefore this release of Ca2+ from store sites was not due to Ca2+ ionophore actions. This agent may play the role of messenger in increasing the cytosolic Ca2+, provoking pharmaco-mechanical coupling, and thus producing the contraction.     

Junctional membrane inositol 1,4,5-trisphosphate receptor complex coordinates sensitization of the silent EGF-induced Ca2+ signaling

Ca(2+) is a highly versatile intracellular signal that regulates many different cellular processes, and cells have developed mechanisms to have exquisite control over Ca(2+) signaling. Epidermal growth factor (EGF), which fails to mobilize intracellular Ca(2+) when administrated alone, becomes capable of evoking [Ca(2+)](i) increase and exocytosis after bradykinin (BK) stimulation in chromaffin cells. Here, we provide evidence that this sensitization process is coordinated by a macromolecular signaling complex comprised of inositol 1,4,5-trisphosphate receptor type I (IP(3)R1), cAMP-dependent protein kinase (PKA), EGF receptor (EGFR), and an A-kinase anchoring protein, yotiao. The IP(3)R complex functions as a focal point to promote Ca(2+) release in two ways: (1) it facilitates PKA-dependent phosphorylation of IP(3)R1 in response to BK-induced elevation of cAMP, and (2) it couples the plasmalemmal EGFR with IP(3)R1 at the Ca(2+) store located juxtaposed to the plasma membrane. Our study illustrates how the junctional membrane IP(3)R complex connects different signaling pathways to define the fidelity and specificity of Ca(2+) signaling.