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© The Rockefeller University Press, 0021-9525/1999//1189 $5.00
The Journal of Cell Biology, Volume 145, Number 6, , 1999 1189-1198

Isolation, Cloning, and Localization of Rat PV-1, a Novel Endothelial Caveolar Protein

Radu-Virgil Stan^*, Lucian Ghitescu, Bruce S. Jacobson, and George E. Palade^*

^* Division of Cellular and Molecular Medicine, University of California, San Diego, San Diego, California 92093; Department of Pathology and Cell Biology, University of Montreal, Montreal, Canada H3C 1J4; and Department of Biochemistry and Cell Biology, University of Massachusetts, Amherst, Massachusetts 01003

By using an immunoisolation procedure (Stan, R.-V., W.G. Roberts, K. Ihida, D. Predescu, L. Saucan, L. Ghitescu, and G.E. Palade. 1997. Mol. Biol. Cell. 8:595–605) developed in our laboratory, we have isolated a caveolar subfraction from rat lung endothelium and we have partially characterized the proteins of this subfraction which include an apparently caveolae-specific glycoprotein we propose to call PV-1 (formerly known as gp68). The isolation and partial sequencing of PV-1, combined with the cloning of the full length PV-1 cDNA led to the following conclusions: (a) PV-1 is a novel single span type II integral membrane protein (438 amino acids long) which forms homodimers in situ; (b) the transmembrane domain of PV-1 is near the NH₂ terminus defining a short cytoplasmic endodomain and a large COOH-terminal ectodomain exposed to the blood plasma; (c) PV-1 is N-glycosylated and its glycan antennae bear terminal nonreducing galactosyl residues in 1-3 linkage. PV-1 is expressed mostly in the lung but both the messenger RNA and the protein can be detected at lower levels also in kidney, spleen, liver, heart, muscle, and brain. No signal could be detected in testis and two lower molecular weight forms were detected in brain. Immunocytochemical studies carried out by immunodiffusion on rat lung with an anti–PV-1 polyclonal antibody directed against a COOH-terminal epitope reveal a specific localization of PV-1 to the stomatal diaphragms of rat lung endothelial caveolae and confirm the extracellular orientation of the PV-1 COOH terminus.

Key Words: plasmalemmal vesicles • Griffonia simplicifolia • lectin chromatography • rat lung • immunodiffusion

Abbreviations used in this paper: aa, amino acid; GS I, Griffonia simplicifolia I lectin; PBSA, 1% BSA in PBS; PEG, polyethylene glycol; PLP, paraformaldehyde-lysine-metaperiodate fixate; PV, plasmalemmal vesicles; PVDF, polyvinyldifluoride; RT, room temperature.

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