Article

Caveolin-1 scaffold domain interacts with TRPC1 and IP3R3 to regulate Ca2+ store release-induced Ca2+ entry in endothelial cells. Am J Physiol Cell Physiol 296:C403-C413

Dept. of Pharmacology (M/C 868) College of Medicine, Univ. of Illinois
AJP Cell Physiology (Impact Factor: 3.78). 03/2009; 296(3):C403-13. DOI: 10.1152/ajpcell.00470.2008
Source: PubMed
ABSTRACT
Caveolin-1 (Cav-1) regulates agonist-induced Ca2+ entry in endothelial cells; however, how Cav-1 regulates this process is poorly understood. Here, we describe that Cav-1 scaffold domain (NH2- terminal residues 82-101; CSD) interacts with transient receptor potential canonical channel 1 (TRPC1) and inositol 1,4,5-trisphosphate receptor 3 (IP 3R3) to regulate Ca2+ entry. We have shown previously that the TRPC1 COOH-terminal residues 781-789 bind to CSD. In the present study, we show that the TRPC1 COOH-terminal residues 781-789 truncated (TRPC1-CΔ781-789) mutant expression abolished Ca2+ store release-induced Ca2+ influx in human dermal microvascular endothelial cell line (HMEC) and human embryonic kidney (HEK-293) cells. To understand the basis of loss of Ca2+ influx, we determined TRPC1 binding to IP3R3. We observed that the wild-type (WT)-TRPC1 but not TRPC1-CΔ781-789 effectively interacted with IP3R3. Similarly, WT-TRPC1 interacted with Cav-1, whereas TRPC1-CΔ781-789 binding to Cav-1 was markedly suppressed. We also assessed the direct binding of Cav-1 with TRPC1 and observed that the WT-Cav-1 but not the Cav-1ΔCSD effectively interacted with TRPC1. Since the interaction between TRPC1 and Cav-1ΔCSD was reduced, we measured Ca2+ store release-induced Ca2+ influx in Cav-1ΔCSD-transfected cells. Surprisingly, Cav-1ΔCSD expression showed a gain-of-function in Ca2+ entry in HMEC and HEK-293 cells. We observed a similar gain-of-function in Ca2+ entry when Cav-1ΔCSD was expressed in lung endothelial cells of Cav-1 knockout mice. Immunoprecipitation results revealed that WT-Cav-1 but not Cav-1ΔCSD interacted with IP3R3. Furthermore, we observed using confocal imaging the colocalization of IP3R3 with WT-Cav-1 but not with Cav-1ΔCSD on Ca2+ store release in endothelial cells. These findings suggest that CSD interacts with TRPC1 and IP3R3 and thereby regulates Ca2+ store release-induced Ca2+ entry in endothelial cells.

Full-text preview

Available from: ajpcell.physiology.org
  • Source
    • "This effect of cholesterol is believed to be mediated by the increasing formation or stabilization of the lipid rafts/caveolae, presumably through the direct insertion of cholesterol into the plasma membranes of target cells [17, 18]. Lipid raft/ caveolae are thought to form platforms collecting assemblies of proteins involved in many key cellular functions, including signal transduction, membrane fusion, cytoskeleton organization, lipid sorting, protein trafficking, and the localization and activities of specific membrane chan- nels202122. Because BetA shares a similar chemical structure with cholesterol, and was reported to insert into the plasma membrane [23], it is rational to speculate that BetA changes the fluidity of the plasma membrane and modulates the signaling pathway associated with membrane microdomains. "
    [Show abstract] [Hide abstract] ABSTRACT: Background: TGF-β is a key modulator in the regulation of cell proliferation and migration, and is also involved in the process of cancer development and progression. Previous studies have indicated that TGF-β responsiveness is determined by TGF-β receptor partitioning between lipid raft/caveolae-mediated and clathrin-mediated endocytosis. Lipid raft/caveolae-mediated endocytosis facilitates TGF-β degradation and thus suppressing TGF-β responsiveness. By contrast, clathrin-mediated endocytosis results in Smad2/3-dependent endosomal signaling, thereby promoting TGF-β responsiveness. Because betulinic acid shares a similar chemical structure with cholesterol and has been reported to insert into the plasma membrane, we speculate that betulinic acid changes the fluidity of the plasma membrane and modulates the signaling pathway associated with membrane microdomains. We propose that betulinic acid modulates TGF-β responsiveness by changing the partitioning of TGF-β receptor between lipid-raft/caveolae and non-caveolae microdomain on plasma membrane. Methods: We employed sucrose-density gradient ultracentrifugation and confocal microscopy to determine membrane localization of TGF-β receptors and used a luciferase assay to examine the effects of betulinic acid in TGF-β-stimulated promoter activation. In addition, we perform western blotting to test TGF-β-induced Smad2 phosphorylation and fibronectin production. Results and conclusions: Betulinic acid induces translocation of TGF-β receptors from lipid raft/caveolae to non-caveolae microdomains without changing total level of TGF-β receptors. The betulinic acid-induced TGF-β receptors translocation is rapid and correlate with the TGF-β-induced PAI-1 reporter gene activation and growth inhibition in Mv1Lu cells.
    Full-text · Article · Feb 2016 · Journal of Biomedical Science
  • Source
    • "This effect of cholesterol is believed to be mediated by increasing formation of, or stabilization of, lipid rafts/caveolae, presumably via direct integration of cholesterol into the plasma membranes of target cells [22, 23]. Lipid rafts/caveolae are thought to form platforms for the aggregation for proteins complexes involved in many key cellular functions , including signal transduction, membrane fusion, cytoskeleton organization, lipid sorting, protein trafficking, and localization and activity of specific membrane channels25262728. In this study, euphol treatment induced segregation of TGF-β receptors to lipid rafts in Mv1Lu cells, as well as in the AGS and MKN45 gastric cancer cell lines (Fig 4A, 4B and 4C). "
    [Show abstract] [Hide abstract] ABSTRACT: Transforming growth factor-β (TGF-β) responsiveness in cultured cells can be modulated by TGF-β partitioning between lipid raft/caveolae- and clathrin-mediated endocytosis pathways. Lipid rafts are plasma membrane microdomains with an important role in cell survival signaling, and cholesterol is necessary for the lipid rafts' structure and function. Euphol is a euphane-type triterpene alcohol that is structurally similar to cholesterol and has a wide range of pharmacological properties, including anti-inflammatory and anti-cancer effects. In the present study, euphol suppressed TGF-β signaling by inducing TGF-β receptor movement into lipid-raft microdomains and degrading TGF-β receptors.
    Full-text · Article · Oct 2015 · PLoS ONE
  • Source
    • "endothelial nitric oxide synthase, eNOS) are a well-characterized players of caveolin-mediated signaling pathways [13,14] both in cultured cells and in vivo [14,15] . Furthermore it was demonstrated that caveo- lin-1 interacts with and regulates exocytic trafficking of a member of Transient Receptor Potential the calcium channel TRPC1 (transient receptor potential channel 1) [16,17]. Caveolin-1 is also a major protein component of lipid rafts [18], i.e. membrane microdomains enriched in cholesterol and saturated lipids [19] known to modulate the activity of several receptors [20]. "
    [Show abstract] [Hide abstract] ABSTRACT: Transient Receptor Potential Vanilloid 1 (TRPV1) is a non-selective cation channel that integrates several stimuli into nociception and neurogenic inflammation. Here we investigated the subtle TRPV1 interplay with candidate membrane partners in live cells by a combination of spatio-temporal fluctuation techniques and fluorescence resonance energy transfer (FRET) imaging. We show that TRPV1 is split into three populations with fairly different molecular properties: one binding to caveolin-1 and confined into caveolar structures, one actively guided by microtubules through selective binding, and one which diffuses freely and is not directly implicated in regulating receptor functionality. The emergence of caveolin-1 as a new interactor of TRPV1 evokes caveolar endocytosis as the main desensitization pathway of TRPV1 receptor, while microtubule binding agrees with previous data suggesting the receptor stabilization in functional form by these cytoskeletal components. Our results shed light on the hitherto unknown relationships between spatial organization and TRPV1 function in live-cell membranes.
    Full-text · Article · Mar 2015 · PLoS ONE
Show more