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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 blocks histamine-stimulated IP3 generation. Cells were stimulated with 100 μM histamine. (A) Effect of PIP5KIγpan siRNA on IP3 generation. Data shown are representative of five independent experiments. (B) Effect of PIP5KI RNAi on the initial IP3 peak response. Values are mean ± SEM of multiple independent experiments (n).
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fig2: PIP5KIγpan siRNA blocks histamine-stimulated IP3 generation. Cells were stimulated with 100 μM histamine. (A) Effect of PIP5KIγpan siRNA on IP3 generation. Data shown are representative of five independent experiments. (B) Effect of PIP5KI RNAi on the initial IP3 peak response. Values are mean ± SEM of multiple independent experiments (n).

Mentions: Histamine binding to the H1 type G protein–coupled receptor (GPCR) in HeLa cells (Tilly et al., 1990) initiates a PLCβ activation cascade that culminates in the hydrolysis of PIP2 to generate IP3 and diacylglycerol. When control cells were stimulated with a supramaximal histamine dose (100 μM), they generate a transient robust IP3 response (Fig. 2 A). PIP5KIγ87 RNAi decreases the initial IP3 peak by 69% [to 10 ± 3 pmol IP3/mg protein (n = 5)] (Fig. 2, A and B). Neither PIP5KIγ90 nor PIP5KIβ RNAi has any apparent effect, and PIP5KIα RNAi only decreases the initial IP3 peak slightly [by 11%; to 28 ± 2 pmol/mg protein (n = 3)] (Fig. 2 B). Although we cannot rule out a small contribution by PIP5KIβ or α because each is less completely knocked down than PIP5KIγ, we can conclude that PIP5KIγ87 is the dominant regulator.


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 blocks histamine-stimulated IP3 generation. Cells were stimulated with 100 μM histamine. (A) Effect of PIP5KIγpan siRNA on IP3 generation. Data shown are representative of five independent experiments. (B) Effect of PIP5KI RNAi on the initial IP3 peak response. Values are mean ± SEM of multiple independent experiments (n).
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Related In: Results  -  Collection

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fig2: PIP5KIγpan siRNA blocks histamine-stimulated IP3 generation. Cells were stimulated with 100 μM histamine. (A) Effect of PIP5KIγpan siRNA on IP3 generation. Data shown are representative of five independent experiments. (B) Effect of PIP5KI RNAi on the initial IP3 peak response. Values are mean ± SEM of multiple independent experiments (n).
Mentions: Histamine binding to the H1 type G protein–coupled receptor (GPCR) in HeLa cells (Tilly et al., 1990) initiates a PLCβ activation cascade that culminates in the hydrolysis of PIP2 to generate IP3 and diacylglycerol. When control cells were stimulated with a supramaximal histamine dose (100 μM), they generate a transient robust IP3 response (Fig. 2 A). PIP5KIγ87 RNAi decreases the initial IP3 peak by 69% [to 10 ± 3 pmol IP3/mg protein (n = 5)] (Fig. 2, A and B). Neither PIP5KIγ90 nor PIP5KIβ RNAi has any apparent effect, and PIP5KIα RNAi only decreases the initial IP3 peak slightly [by 11%; to 28 ± 2 pmol/mg protein (n = 3)] (Fig. 2 B). Although we cannot rule out a small contribution by PIP5KIβ or α because each is less completely knocked down than PIP5KIγ, we can conclude that PIP5KIγ87 is the dominant regulator.

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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Related in: MedlinePlus