title: Caveolin-1 scaffold domain interacts with TRPC1 and IP3R3 to regulate Ca2+ store release-induced Ca2+ entry in endothelial cells.
abstract: Caveolin-1 (Cav-1) regulates agonist-induced Ca(2+) entry in endothelial cells; however, how Cav-1 regulates this process is poorly understood. Here, we describe that Cav-1 scaffold domain (NH(2)-terminal residues 82-101; CSD) interacts with transient receptor potential canonical channel 1 (TRPC1) and inositol 1,4,5-trisphosphate receptor 3 (IP(3)R3) to regulate Ca(2+) 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-CDelta781-789) mutant expression abolished Ca(2+) store release-induced Ca(2+) influx in human dermal microvascular endothelial cell line (HMEC) and human embryonic kidney (HEK-293) cells. To understand the basis of loss of Ca(2+) influx, we determined TRPC1 binding to IP(3)R3. We observed that the wild-type (WT)-TRPC1 but not TRPC1-CDelta781-789 effectively interacted with IP(3)R3. Similarly, WT-TRPC1 interacted with Cav-1, whereas TRPC1-CDelta781-789 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-1DeltaCSD effectively interacted with TRPC1. Since the interaction between TRPC1 and Cav-1DeltaCSD was reduced, we measured Ca(2+) store release-induced Ca(2+) influx in Cav-1DeltaCSD-transfected cells. Surprisingly, Cav-1DeltaCSD expression showed a gain-of-function in Ca(2+) entry in HMEC and HEK-293 cells. We observed a similar gain-of-function in Ca(2+) entry when Cav-1DeltaCSD was expressed in lung endothelial cells of Cav-1 knockout mice. Immunoprecipitation results revealed that WT-Cav-1 but not Cav-1DeltaCSD interacted with IP(3)R3. Furthermore, we observed using confocal imaging the colocalization of IP(3)R3 with WT-Cav-1 but not with Cav-1DeltaCSD on Ca(2+) store release in endothelial cells. These findings suggest that CSD interacts with TRPC1 and IP(3)R3 and thereby regulates Ca(2+) store release-induced Ca(2+) entry in endothelial cells.
authors.affiliation: Dept. of Pharmacology (M/C 868) College of Medicine, Univ. of Illinois; 835 South Wolcott Ave., Chicago, IL 60612, USA.
authors.forename: Premanand C
authors.initials: PC
authors.lastname: Sundivakkam
authors.forename: Angela M
authors.initials: AM
authors.lastname: Kwiatek
authors.forename: Tiffany T
authors.initials: TT
authors.lastname: Sharma
authors.forename: Richard D
authors.initials: RD
authors.lastname: Minshall
authors.forename: Asrar B
authors.initials: AB
authors.lastname: Malik
authors.forename: Chinnaswamy
authors.initials: C
authors.lastname: Tiruppathi
mesh_terms: D000818:Animals; D020013:Calcium Signaling; D051242:Caveolin 1 / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000378:metabolism*; D006801:Humans; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D018613:Microscopy, Confocal; D009154:Mutation; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D050052:TRPC Cation Channels / Q000235:genetics / Q000378:metabolism*; D013917:Thrombin / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
chemical_list: C495540:CAV1 protein, human; C495541:Cav1 protein, mouse; D051242:Caveolin 1; C506929:ITPR3 protein, human; D053496:Inositol 1,4,5-Trisphosphate Receptors; D050052:TRPC Cation Channels; C096351:transient receptor potential cation channel, subfamily C, member 1; D013917:Thrombin