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.
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    • "Consistent with this, several Ca 2+ signaling components, including TRPC channels, localize within caveolar domains (Pani and Singh, 2009). TRPC1 associates with Cav1 which serves as scaffold to retain the channel in the plasma membrane region and facilitate its interaction with STIM1 (Pani et al., 2009; Sundivakkam et al., 2009). Recent studies also suggest a role of lipid rafts domains (LRD) and Cav1 in regulation of Orai1 (Sathish et al., 2012; Yu et al., 2010). "
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    ABSTRACT: Neurotransmitter regulation of salivary fluid-secretion is mediated via activation of Ca(2+) influx. The Ca(2+)-permeable channel TRPC1 is critical for fluid-secretion. However, the mechanism(s) involved in channel assembly and regulation are not completely understood. We report that Caveolin1 (Cav1) is critical for the assembly of functional TRPC1 channels in salivary glands (SG) in vivo, and thus regulates fluid-secretion. In Cav1(-/-) mouse SG, agonist-stimulated Ca(2+) entry and fluid-secretion are significantly reduced. Microdomain localization of TRPC1 and interaction with its regulatory protein, STIM1, are disrupted in Cav1(-/-) SG acinar cells, whereas Orai1-STIM1 interaction is not affected. Further, localization of AQP5, but not that of IP(3)R3 or Ca(2+)-activated K(+) channel (IK), in the apical region of acinar cell was altered in Cav1(-/-) SG. In addition, agonist-stimulated increase in surface expression of AQP5 required Ca(2+) influx via TRPC1 channels and was inhibited in Cav1(-/-) SG. Importantly, adenovirus-mediated expression of Cav1 in Cav1(-/-) SG restored interaction of STIM1 with TRPC1 and channel activation, apical targeting and regulated trafficking of AQP5, and neurotransmitter stimulated fluid-secretion. Together these findings demonstrate that, by directing cellular localization of TRPC1 and AQP5 channels and by selectively regulating the functional assembly TRPC1-STIM1 channels, Cav1 is a critical determinant of SG fluid secretion.
    Full-text · Article · Nov 2012 · Journal of Cell Science
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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.
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