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Critical role of PIP5KI{gamma}87 in InsP3-mediated Ca(2+) signaling.

Wang YJ, Li WH, Wang J, Xu K, Dong P, Luo X, Yin HL - J. Cell Biol. (2004)

Bottom Line: Using RNA interference (RNAi), we identified the short splice variant of type I phosphatidylinositol 4-phosphate 5-kinase gamma (PIP5KIgamma87) as the major contributor of the PIP(2) pool that supports G protein-coupled receptor (GPCR)-mediated IP(3) generation.PIP5KIgamma87 RNAi decreases the histamine-induced IP(3) response and Ca(2+) flux by 70%.Therefore, PIP5KIgamma87's PIP(2) pool that supports GPCR-mediated Ca(2+) signaling is functionally compartmentalized from those generated by the other PIP5KIs.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390, USA.

ABSTRACT
Phosphatidylinositol 4,5-bisphosphate (PIP(2)) is the obligatory precursor of inositol 1,4,5-trisphosphate (InsP(3) or IP(3)) and is therefore critical to intracellular Ca(2+) signaling. Using RNA interference (RNAi), we identified the short splice variant of type I phosphatidylinositol 4-phosphate 5-kinase gamma (PIP5KIgamma87) as the major contributor of the PIP(2) pool that supports G protein-coupled receptor (GPCR)-mediated IP(3) generation. PIP5KIgamma87 RNAi decreases the histamine-induced IP(3) response and Ca(2+) flux by 70%. Strikingly, RNAi of other PIP5KI isoforms has minimal effect, even though some of these isoforms account for a larger percent of total PIP(2) mass and have previously been implicated in receptor mediated endocytosis or focal adhesion formation. Therefore, PIP5KIgamma87's PIP(2) pool that supports GPCR-mediated Ca(2+) signaling is functionally compartmentalized from those generated by the other PIP5KIs.

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PIP5KIγpan siRNA attenuates intracellular Ca2+ signaling. Cells loaded with fura2-AM in randomly chosen microscopic fields were ratio imaged (F340/F380) to obtain baseline Ca2+ values. Histamine was added in the absence of extracellular Ca2+, and the ratio image was recorded as a function of time. (A) Ca2+ response to 100 μM histamine (indicated by the arrow). Representative tracings for each type of RNAi are shown. Values in bar graphs are expressed as percent (mean ± SEM) of control. (B) In vivo rescue of intracellular Ca2+ signaling in PIP5KIγpan siRNA-treated cells by Shuttle PIP2. Top panels are representative tracings. (Bottom) The Ca2+ flux of the transients elicited by the second histamine addition was plotted and 10 cells were analyzed per condition.
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fig3: PIP5KIγpan siRNA attenuates intracellular Ca2+ signaling. Cells loaded with fura2-AM in randomly chosen microscopic fields were ratio imaged (F340/F380) to obtain baseline Ca2+ values. Histamine was added in the absence of extracellular Ca2+, and the ratio image was recorded as a function of time. (A) Ca2+ response to 100 μM histamine (indicated by the arrow). Representative tracings for each type of RNAi are shown. Values in bar graphs are expressed as percent (mean ± SEM) of control. (B) In vivo rescue of intracellular Ca2+ signaling in PIP5KIγpan siRNA-treated cells by Shuttle PIP2. Top panels are representative tracings. (Bottom) The Ca2+ flux of the transients elicited by the second histamine addition was plotted and 10 cells were analyzed per condition.

Mentions: Consistent with a decrease in IP3 production, PIP5KIγpan RNAi also attenuates histamine-induced Ca2+ signaling. In control cells, 100 μM histamine induces a rapid and transient rise in intracellular Ca2+ concentration ([Ca2+]i) in 95% of the cells examined (Fig. 3 A). PIP5KIγ87 RNAi reduces the percent of responding cells slightly (by 13%), decreases the amplitude of the first Ca2+ peak more [by 36%; from 1.14 ± 0.07 (n = 19) to 0.73 ± 0.08 (n = 10) fluorescence ratio unit], and has the most impact on Ca2+ flux [76% decrease; from 0.33 ± 0.03 (n = 19) to 0.08 ± 0.01 (n = 10) unit/s] (Fig. 3 A). Because Ca2+ flux correlates with the open probability of the IP3Rs and hence the rate of IP3 generation (Johenning et al., 2004), our results establish that PIP5KIγ87 RNAi depletes the PIP2 pool used for IP3 generation. A similarly large decrease in Ca2+ flux was also observed when PIP2 was depleted by overexpressing the PIP2 phosphatase synaptojanin (Johenning et al., 2004).


Critical role of PIP5KI{gamma}87 in InsP3-mediated Ca(2+) signaling.

Wang YJ, Li WH, Wang J, Xu K, Dong P, Luo X, Yin HL - J. Cell Biol. (2004)

PIP5KIγpan siRNA attenuates intracellular Ca2+ signaling. Cells loaded with fura2-AM in randomly chosen microscopic fields were ratio imaged (F340/F380) to obtain baseline Ca2+ values. Histamine was added in the absence of extracellular Ca2+, and the ratio image was recorded as a function of time. (A) Ca2+ response to 100 μM histamine (indicated by the arrow). Representative tracings for each type of RNAi are shown. Values in bar graphs are expressed as percent (mean ± SEM) of control. (B) In vivo rescue of intracellular Ca2+ signaling in PIP5KIγpan siRNA-treated cells by Shuttle PIP2. Top panels are representative tracings. (Bottom) The Ca2+ flux of the transients elicited by the second histamine addition was plotted and 10 cells were analyzed per condition.
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Related In: Results  -  Collection

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fig3: PIP5KIγpan siRNA attenuates intracellular Ca2+ signaling. Cells loaded with fura2-AM in randomly chosen microscopic fields were ratio imaged (F340/F380) to obtain baseline Ca2+ values. Histamine was added in the absence of extracellular Ca2+, and the ratio image was recorded as a function of time. (A) Ca2+ response to 100 μM histamine (indicated by the arrow). Representative tracings for each type of RNAi are shown. Values in bar graphs are expressed as percent (mean ± SEM) of control. (B) In vivo rescue of intracellular Ca2+ signaling in PIP5KIγpan siRNA-treated cells by Shuttle PIP2. Top panels are representative tracings. (Bottom) The Ca2+ flux of the transients elicited by the second histamine addition was plotted and 10 cells were analyzed per condition.
Mentions: Consistent with a decrease in IP3 production, PIP5KIγpan RNAi also attenuates histamine-induced Ca2+ signaling. In control cells, 100 μM histamine induces a rapid and transient rise in intracellular Ca2+ concentration ([Ca2+]i) in 95% of the cells examined (Fig. 3 A). PIP5KIγ87 RNAi reduces the percent of responding cells slightly (by 13%), decreases the amplitude of the first Ca2+ peak more [by 36%; from 1.14 ± 0.07 (n = 19) to 0.73 ± 0.08 (n = 10) fluorescence ratio unit], and has the most impact on Ca2+ flux [76% decrease; from 0.33 ± 0.03 (n = 19) to 0.08 ± 0.01 (n = 10) unit/s] (Fig. 3 A). Because Ca2+ flux correlates with the open probability of the IP3Rs and hence the rate of IP3 generation (Johenning et al., 2004), our results establish that PIP5KIγ87 RNAi depletes the PIP2 pool used for IP3 generation. A similarly large decrease in Ca2+ flux was also observed when PIP2 was depleted by overexpressing the PIP2 phosphatase synaptojanin (Johenning et al., 2004).

Bottom Line: Using RNA interference (RNAi), we identified the short splice variant of type I phosphatidylinositol 4-phosphate 5-kinase gamma (PIP5KIgamma87) as the major contributor of the PIP(2) pool that supports G protein-coupled receptor (GPCR)-mediated IP(3) generation.PIP5KIgamma87 RNAi decreases the histamine-induced IP(3) response and Ca(2+) flux by 70%.Therefore, PIP5KIgamma87's PIP(2) pool that supports GPCR-mediated Ca(2+) signaling is functionally compartmentalized from those generated by the other PIP5KIs.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390, USA.

ABSTRACT
Phosphatidylinositol 4,5-bisphosphate (PIP(2)) is the obligatory precursor of inositol 1,4,5-trisphosphate (InsP(3) or IP(3)) and is therefore critical to intracellular Ca(2+) signaling. Using RNA interference (RNAi), we identified the short splice variant of type I phosphatidylinositol 4-phosphate 5-kinase gamma (PIP5KIgamma87) as the major contributor of the PIP(2) pool that supports G protein-coupled receptor (GPCR)-mediated IP(3) generation. PIP5KIgamma87 RNAi decreases the histamine-induced IP(3) response and Ca(2+) flux by 70%. Strikingly, RNAi of other PIP5KI isoforms has minimal effect, even though some of these isoforms account for a larger percent of total PIP(2) mass and have previously been implicated in receptor mediated endocytosis or focal adhesion formation. Therefore, PIP5KIgamma87's PIP(2) pool that supports GPCR-mediated Ca(2+) signaling is functionally compartmentalized from those generated by the other PIP5KIs.

Show MeSH
Related in: MedlinePlus