Abstract
Caveolin-1 associates with store-operated cation channels (SOC) in endothelial cells. We examined the role of the caveolin-1 scaffolding domain (CSD) in regulating the SOC [i.e., transient receptor potential channel-1 (TRPC1)] in human pulmonary artery endothelial cells (HPAECs). We used the cellpermeant antennapedia (AP)-conjugated CSD peptide, which competes for protein binding partners with caveolin-1, to assess the interactions of caveolin-1 with TRPC1 and its consequences on thrombin-induced Ca2+ influx. We observed that AP-CSD peptide markedly reduced thrombin-induced Ca2+ influx via SOC in HPAECs in contrast to control peptide. AP-CSD also suppressed thapsigargin-induced Ca2+ influx. Streptavidin-bead pull-down assay indicated strong binding of biotin-labeled AP-CSD peptide to TRPC1. Immunoprecipitation studies demonstrated an interaction between endogenous TRPC1 and ectopically expressed hemagglutinin-tagged CSD. Analysis of the deduced TRPC1 amino acid sequence revealed the presence of CSD binding consensus sequence in the TRPC1 C terminus. We also observed that an AP-TRPC1 peptide containing the CSD binding sequence markedly reduced the thrombin-induced Ca2+ influx. We identified the interaction between biotin-labeled AP-TRPC1 C terminus peptide and caveolin-1. Thus, these results demonstrate a crucial role of caveolin-1 scaffolding domain interaction with TRPC1 in regulating Ca2+ influx via SOC.
Footnotes
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This work was supported by National Institutes of Health Grants GM58531, P01 HL077806, and T32HL007829.
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ABBREVIATIONS: SOC, store-operated cation channel; Cav-1, caveolin-1; TRP, transient receptor potential; TRPC1, transient receptor potential channel-1; CSD, caveolin-1 scaffolding domain; HPAEC, human pulmonary artery endothelial cell; AP, antennapedia; Ab, antibody; HA, hemagglutinin; ER, endoplasmic reticulum; eNOS, endothelial nitric-oxide synthase; HBSS, Hanks' balanced salt solution; FBS, fetal bovine serum; AM, acetoxymethyl ester; mAb, monoclonal antibody; MS, mass spectrometry; HMEC, human dermal microvessel endothelial cell line; SELDI-TOF, surface-enhanced laser/desorption ionization-time of flight; PAGE, polyacrylamide gel electrophoresis; WT, wild-type; PCR, polymerase chain reaction; IP3, inositol 1,4,5-trisphosphate.
- Received December 15, 2005.
- Accepted July 5, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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