Abstract
Caveolae are organelles abundant in the plasma membrane of many specialized cells including endothelial cells (ECs), epithelial cells, and adipocytes, and in these cells, caveolin-1 (Cav-1) is the major coat protein essential for the formation of caveolae. To identify proteins that require Cav-1 for stable incorporation into membrane raft domains, a quantitative proteomics analysis using isobaric tagging for relative and absolute quantification was performed on rafts isolated from wild-type and Cav-1-deficient mice. In three independent experiments, 117 proteins were consistently identified in membrane rafts with the largest differences in the levels of Cav-2 and in the caveola regulatory proteins Cavin-1 and Cavin-2. Because the lung is highly enriched in ECs, we validated and characterized the role of the newly described protein Cavin-1 in several cardiovascular tissues and in ECs. Cavin-1 was highly expressed in ECs lining blood vessels and in cultured ECs. Knockdown of Cavin-1 reduced the levels of Cav-1 and -2 and weakly influenced the formation of high molecular weight oligomers containing Cav-1 and -2. Cavin-1 silencing enhanced basal nitric oxide release from ECs but blocked proangiogenic phenotypes such as EC proliferation, migration, and morphogenesis in vitro. Thus, these data support an important role of Cavin-1 as a regulator of caveola function in ECs.
Highlights
Caveolae are organelles abundant in the plasma membrane of many specialized cells including endothelial cells (ECs), epithelial cells, and adipocytes, and in these cells, caveolin-1 (Cav-1) is the major coat protein essential for the formation of caveolae
As caveolae and Cav-1 are present in the endothelium lining all blood vessels of the body [18, 19] and play an important role in the regulation of several EC functions (8, 20 –22), we examined the protein expression profile of proteins localized in detergentresistant membranes (DRMs) prepared from the lungs of wildtype (WT) and Cav-1 KO mice via isobaric tagging for relative and absolute quantification (iTRAQ) labeling and multidimensional LC and MS/MS analysis
Quantitative Proteomics Examining Proteins in Lipid Rafts Isolated from WT and Cav-1 KO Lungs—To examine the proteins that interact with and stabilize caveolin-1, DRMs were isolated using sucrose gradient fractionation from lung tissues prepared from WT and Cav-1 KO mice
Summary
Caveolae are organelles abundant in the plasma membrane of many specialized cells including endothelial cells (ECs), epithelial cells, and adipocytes, and in these cells, caveolin-1 (Cav-1) is the major coat protein essential for the formation of caveolae. Cavin-1 silencing enhanced basal nitric oxide release from ECs but blocked proangiogenic phenotypes such as EC proliferation, migration, and morphogenesis in vitro These data support an important role of Cavin-1 as a regulator of caveola function in ECs. Molecular & Cellular Proteomics 9:2109 –2124, 2010. The abbreviations used are: EC, endothelial cell; Cav, caveolin; PTRF, polymerase I and transcript release factor; DRM, detergentresistant membrane; SDPR, serum deprivation response protein; NS, negative nonsilencing control; HUVEC, human umbilical vein EC; iTRAQ, isobaric tagging for relative and absolute quantification; MBS, Mes-buffered saline; IPI, International Protein Index; eNOS, endothelial nitric-oxide synthase; RC, reconstitution; DSP, dithiobis(succinimidylpropionate); IP, immunoprecipitation
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