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Caveolin-1 alters the pattern of cytoplasmic Ca2+ oscillations and Ca2+-dependent gene expression by enhancing leukotriene receptor desensitization.

Yeh YC, Tang MJ, Parekh AB - J. Biol. Chem. (2014)

Bottom Line: Here, we show that the scaffolding protein caveolin-1 has a profound effect on receptor-driven Ca(2+) signals and downstream gene expression.Mutagenesis studies revealed that these effects required a functional scaffolding domain within caveolin-1.Our results reveal that caveolin-1 is a bimodal regulator of receptor-dependent Ca(2+) signaling, which fine-tunes the spatial and temporal profile of the Ca(2+) rise and thereby its ability to activate the NFAT pathway.

View Article: PubMed Central - PubMed

Affiliation: From the Department of Physiology, Anatomy, and Genetics, University of Oxford, Parks Road, Oxford OX1 3PT, United Kingdom and.

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Mutations within the scaffolding domain abolish the stimulatory effect of caveolin-1-GFP (Cav1) on Ca2+ release.A, numerous Ca2+ oscillations are obtained in Ca2+-free solution in response to LTC4 when the mutated caveolin-1-GFP protein is expressed. B and C, the amplitude of the first Ca2+ oscillation (B) and rate of store-operated Ca2+ entry (C) are compared between wild type cells and those expressing mutant Cav1-GFP (each bar represents between 21 and 31 cells from four independent experiments). D, Ca2+ oscillations to LTC4 in the presence of external Ca2+ do not run down in the presence of mutant caveolin-1. E, the graph compares the number of oscillations/100-s bin between wild type cells and those expressing the mutant caveolin-1-GFP protein. Each point is between 30 and 60 cells. F, the size of the first oscillation in 2 mm external Ca2+ is compared for the conditions shown. G, the histogram compares the GFP fluorescence for all cells transfected with either caveolin-1-GFP (137 cells) or mutant caveolin-1-GFP (140 cells). H, the averaged GFP intensity is compared for the two conditions. I, Ca2+ responses in two cells that expressed very similar levels of GFP are compared. J, merged confocal images showing the presence of FLAG-tagged CysLT1 receptors and either Cav1-RFP or mutant Cav1-RFP. K, Ca2+ oscillations run down quickly when a Cav1 protein is expressed with a mutation in the tyrosine phosphorylation site (Y14F). L, aggregate data comparing the number of oscillations over the entire 600-s recording for the conditions described are shown (each bar represents the mean of 10 and 17 cells from two independent experiments). NS, nonsignificant; ***, p < 0.001.
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Figure 4: Mutations within the scaffolding domain abolish the stimulatory effect of caveolin-1-GFP (Cav1) on Ca2+ release.A, numerous Ca2+ oscillations are obtained in Ca2+-free solution in response to LTC4 when the mutated caveolin-1-GFP protein is expressed. B and C, the amplitude of the first Ca2+ oscillation (B) and rate of store-operated Ca2+ entry (C) are compared between wild type cells and those expressing mutant Cav1-GFP (each bar represents between 21 and 31 cells from four independent experiments). D, Ca2+ oscillations to LTC4 in the presence of external Ca2+ do not run down in the presence of mutant caveolin-1. E, the graph compares the number of oscillations/100-s bin between wild type cells and those expressing the mutant caveolin-1-GFP protein. Each point is between 30 and 60 cells. F, the size of the first oscillation in 2 mm external Ca2+ is compared for the conditions shown. G, the histogram compares the GFP fluorescence for all cells transfected with either caveolin-1-GFP (137 cells) or mutant caveolin-1-GFP (140 cells). H, the averaged GFP intensity is compared for the two conditions. I, Ca2+ responses in two cells that expressed very similar levels of GFP are compared. J, merged confocal images showing the presence of FLAG-tagged CysLT1 receptors and either Cav1-RFP or mutant Cav1-RFP. K, Ca2+ oscillations run down quickly when a Cav1 protein is expressed with a mutation in the tyrosine phosphorylation site (Y14F). L, aggregate data comparing the number of oscillations over the entire 600-s recording for the conditions described are shown (each bar represents the mean of 10 and 17 cells from two independent experiments). NS, nonsignificant; ***, p < 0.001.

Mentions: Subcellular distribution of caveolin-1 and CysLT1 receptors in RBL-1 cells. Cells were co-transfected with caveolin-1-RFP and FLAG-tagged CysLT1 receptor and then fixed 48 h later. A, confocal images for the conditions shown. Line scans are shown in the merged panel. B, fluorescence profiles from the line scans are shown. Caveolin-1-RFP distribution is shown in red, and FLAG-tagged CysLT1 receptors are in green. C, histogram compares Pearson's correlation coefficient for the conditions shown. CaV-mutant denotes caveolin-1 with point mutations in the scaffolding domain (see Fig. 4). Mem., membrane; Cyt., cytosol.


Caveolin-1 alters the pattern of cytoplasmic Ca2+ oscillations and Ca2+-dependent gene expression by enhancing leukotriene receptor desensitization.

Yeh YC, Tang MJ, Parekh AB - J. Biol. Chem. (2014)

Mutations within the scaffolding domain abolish the stimulatory effect of caveolin-1-GFP (Cav1) on Ca2+ release.A, numerous Ca2+ oscillations are obtained in Ca2+-free solution in response to LTC4 when the mutated caveolin-1-GFP protein is expressed. B and C, the amplitude of the first Ca2+ oscillation (B) and rate of store-operated Ca2+ entry (C) are compared between wild type cells and those expressing mutant Cav1-GFP (each bar represents between 21 and 31 cells from four independent experiments). D, Ca2+ oscillations to LTC4 in the presence of external Ca2+ do not run down in the presence of mutant caveolin-1. E, the graph compares the number of oscillations/100-s bin between wild type cells and those expressing the mutant caveolin-1-GFP protein. Each point is between 30 and 60 cells. F, the size of the first oscillation in 2 mm external Ca2+ is compared for the conditions shown. G, the histogram compares the GFP fluorescence for all cells transfected with either caveolin-1-GFP (137 cells) or mutant caveolin-1-GFP (140 cells). H, the averaged GFP intensity is compared for the two conditions. I, Ca2+ responses in two cells that expressed very similar levels of GFP are compared. J, merged confocal images showing the presence of FLAG-tagged CysLT1 receptors and either Cav1-RFP or mutant Cav1-RFP. K, Ca2+ oscillations run down quickly when a Cav1 protein is expressed with a mutation in the tyrosine phosphorylation site (Y14F). L, aggregate data comparing the number of oscillations over the entire 600-s recording for the conditions described are shown (each bar represents the mean of 10 and 17 cells from two independent experiments). NS, nonsignificant; ***, p < 0.001.
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Figure 4: Mutations within the scaffolding domain abolish the stimulatory effect of caveolin-1-GFP (Cav1) on Ca2+ release.A, numerous Ca2+ oscillations are obtained in Ca2+-free solution in response to LTC4 when the mutated caveolin-1-GFP protein is expressed. B and C, the amplitude of the first Ca2+ oscillation (B) and rate of store-operated Ca2+ entry (C) are compared between wild type cells and those expressing mutant Cav1-GFP (each bar represents between 21 and 31 cells from four independent experiments). D, Ca2+ oscillations to LTC4 in the presence of external Ca2+ do not run down in the presence of mutant caveolin-1. E, the graph compares the number of oscillations/100-s bin between wild type cells and those expressing the mutant caveolin-1-GFP protein. Each point is between 30 and 60 cells. F, the size of the first oscillation in 2 mm external Ca2+ is compared for the conditions shown. G, the histogram compares the GFP fluorescence for all cells transfected with either caveolin-1-GFP (137 cells) or mutant caveolin-1-GFP (140 cells). H, the averaged GFP intensity is compared for the two conditions. I, Ca2+ responses in two cells that expressed very similar levels of GFP are compared. J, merged confocal images showing the presence of FLAG-tagged CysLT1 receptors and either Cav1-RFP or mutant Cav1-RFP. K, Ca2+ oscillations run down quickly when a Cav1 protein is expressed with a mutation in the tyrosine phosphorylation site (Y14F). L, aggregate data comparing the number of oscillations over the entire 600-s recording for the conditions described are shown (each bar represents the mean of 10 and 17 cells from two independent experiments). NS, nonsignificant; ***, p < 0.001.
Mentions: Subcellular distribution of caveolin-1 and CysLT1 receptors in RBL-1 cells. Cells were co-transfected with caveolin-1-RFP and FLAG-tagged CysLT1 receptor and then fixed 48 h later. A, confocal images for the conditions shown. Line scans are shown in the merged panel. B, fluorescence profiles from the line scans are shown. Caveolin-1-RFP distribution is shown in red, and FLAG-tagged CysLT1 receptors are in green. C, histogram compares Pearson's correlation coefficient for the conditions shown. CaV-mutant denotes caveolin-1 with point mutations in the scaffolding domain (see Fig. 4). Mem., membrane; Cyt., cytosol.

Bottom Line: Here, we show that the scaffolding protein caveolin-1 has a profound effect on receptor-driven Ca(2+) signals and downstream gene expression.Mutagenesis studies revealed that these effects required a functional scaffolding domain within caveolin-1.Our results reveal that caveolin-1 is a bimodal regulator of receptor-dependent Ca(2+) signaling, which fine-tunes the spatial and temporal profile of the Ca(2+) rise and thereby its ability to activate the NFAT pathway.

View Article: PubMed Central - PubMed

Affiliation: From the Department of Physiology, Anatomy, and Genetics, University of Oxford, Parks Road, Oxford OX1 3PT, United Kingdom and.

Show MeSH
Related in: MedlinePlus