Quantitative Proteomics of Caveolin-1-regulated Proteins

• DOI: 10.1074/mcp.m110.001289
• Publication Date: 2010-06-29T04:39:11Z
• Authors: Alberto Dávalos; Carlos Fernández-...; Grzegorz Sowa; Behrad Derakhshan; Michelle I. Lin; Ji Y. Lee; Hongyu Zhao; Ruiyan Luo; Christopher Colan...; William C. Sessa
• Keywords: Mice, Knockout; Proteomics; 0301 basic medicine; Base Sequence; Blotting, Western; Caveolin 1; Chromatography, Ion Exchange; DNA Polymerase I; Nitric Oxide; Mass Spectrometry; Cell Line; Mice; 03 medical and health sciences; Microscopy, Fluorescence; Cell Movement; Animals; Humans; Gene Silencing; RNA, Small Interfering; Cell Proliferation

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.