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


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.

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    • "In regard to endothelial cells as mentioned above, studies have shown that the caveolar microdomain organizes and compartmentalizes the SOCE channel complex [75] [77]. Additionally, it has been shown that Cav-1 scaffolding domain interacts with both TRPC1 and IP 3 R 3 and thereby it regulates Ca 2+ store release-induced Ca 2+ entry in endothelial cells [124]. This implies a relevant role of Cav-1 in SOCE functioning and Ca 2+ signaling. "
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    ABSTRACT: Acidic Ca2+ stores, particularly lysosomes, are newly discovered players in the well-orchestrated arena of Ca2+ signaling and we are at the verge of understanding how lysosomes accumulate Ca2+ and how they release it in response to different chemical, such as NAADP, and physical signals. Additionally, it is now clear that lysosomes play a key role in autophagy, a process that allows cells to recycle components or to eliminate damaged structures to ensure cellular well-being. Moreover, lysosomes are being unraveled as hubs that coordinate both anabolism via insulin signaling and catabolism via AMPK. These acidic vesicles have close contact with the ER and there is a bidirectional movement of information between these two organelles that exquisitely regulates cell survival. Lysosomes also connect with plasma membrane where caveolae are located as specialized regions involved in Ca2+ and insulin signaling. Alterations of all these signaling pathways are at the core of insulin resistance and diabetes.
    Cell Calcium 11/2014; 56:323-331. DOI:10.1016/j.ceca.2014.08.005 · 3.51 Impact Factor
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    • "Several TRPC proteins have been reported to partition into detergent-resistant membranes [44-50], and TPRC1 binds to caveolin-1, a lipid raft-associated protein [45,47-49,51-53]. Furthermore, STIM1 overexpression was reported to shift TRPC1 into lipid rafts, thereby altering its channel properties [52,54,55]. "
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    ABSTRACT: Background Transient receptor potential canonical (TRPC) channels are non-selective cation channels involved in receptor-mediated calcium signaling in diverse cells and tissues. The canonical transient receptor potential 6 (TRPC6) has been implicated in several pathological processes, including focal segmental glomerulosclerosis (FSGS), cardiac hypertrophy, and pulmonary hypertension. The two large cytoplasmic segments of the cation channel play a critical role in the proper regulation of channel activity, and are involved in several protein-protein interactions. Results Here we report that SNF8, a component of the endosomal sorting complex for transport-II (ESCRT-II) complex, interacts with TRPC6. The interaction was initially observed in a yeast two-hybrid screen using the amino-terminal cytoplasmic domain of TRPC6 as bait, and confirmed by co-immunoprecipitation from eukaryotic cell extracts. The amino-terminal 107 amino acids are necessary and sufficient for the interaction. Overexpression of SNF8 enhances both wild-type and gain-of-function mutant TRPC6-mediated whole-cell currents in HEK293T cells. Furthermore, activation of NFAT-mediated transcription by gain-of-function mutants is enhanced by overexpression of SNF8, and partially inhibited by RNAi mediated knockdown of SNF8. Although the ESCRT-II complex functions in the endocytosis and lysosomal degradation of transmembrane proteins, SNF8 overexpression does not alter the amount of TRPC6 present on the cell surface. Conclusion SNF8 is novel binding partner of TRPC6, binding to the amino-terminal cytoplasmic domain of the channel. Modulating SNF8 expression levels alters the TRPC6 channel current and can modulate activation of NFAT-mediated transcription downstream of gain-of-function mutant TRPC6. Taken together, these results identify SNF8 as a novel regulator of TRPC6.
    BMC Cell Biology 11/2012; 13(1):33. DOI:10.1186/1471-2121-13-33 · 2.34 Impact Factor
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    • "Several studies have confirmed the existence of TRPC channels in various vascular preparations [74, 78, 79]. Among the members of TRPC family of proteins, TRPC1, TRPC3, TRPC4 and TRPC6 have been studied extensively showing their abundant expression levels in pulmonary artery smooth muscle and intralobar PASMC [45, 80–82]. "
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    ABSTRACT: Pulmonary circulation is an important circulatory system in which the body brings in oxygen. Pulmonary arterial hypertension (PAH) is a progressive and fatal disease that predominantly affects women. Sustained pulmonary vasoconstriction, excessive pulmonary vascular remodeling, in situ thrombosis, and increased pulmonary vascular stiffness are the major causes for the elevated pulmonary vascular resistance (PVR) in patients with PAH. The elevated PVR causes an increase in afterload in the right ventricle, leading to right ventricular hypertrophy, right heart failure, and eventually death. Understanding the pathogenic mechanisms of PAH is important for developing more effective therapeutic approach for the disease. An increase in cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)) in pulmonary arterial smooth muscle cells (PASMC) is a major trigger for pulmonary vasoconstriction and an important stimulus for PASMC migration and proliferation which lead to pulmonary vascular wall thickening and remodeling. It is thus pertinent to define the pathogenic role of Ca(2+) signaling in pulmonary vasoconstriction and PASMC proliferation to develop new therapies for PAH. [Ca(2+)](cyt) in PASMC is increased by Ca(2+) influx through Ca(2+) channels in the plasma membrane and by Ca(2+) release or mobilization from the intracellular stores, such as sarcoplasmic reticulum (SR) or endoplasmic reticulum (ER). There are two Ca(2+) entry pathways, voltage-dependent Ca(2+) influx through voltage-dependent Ca(2+) channels (VDCC) and voltage-independent Ca(2+) influx through store-operated Ca(2+) channels (SOC) and receptor-operated Ca(2+) channels (ROC). This paper will focus on the potential role of VDCC, SOC, and ROC in the development and progression of sustained pulmonary vasoconstriction and excessive pulmonary vascular remodeling in PAH.
    09/2012; 2012(3):951497. DOI:10.1155/2012/951497
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