Isolation, cloning, and localization of rat PV-1, a novel endothelial caveolar protein

By using an immunoisolation procedure (Stan, R.-V., W.G. Roberts, K. Ihida, D. Predescu, L. Saucan, L. Ghitescu, and G.E. Palade. 1997. Mel. Biol. Cel l. 8:595-605) developed in our laboratory, we have isolated a caveolar subf raction from rat lung endothelium and we have partially characterized the p roteins of this subfraction which include an apparently caveolae-specific g lycoprotein 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 leng th PV-1 cDNA led to the following conclusions: (a) PV-1 is a novel single s pan type TT integral membrane protein (438 amino acids long) which forms: h omodimers in situ; (b) the transmembrane domain of PV-1 is near the NH2 ter minus defining a short cytoplasmic endodomain and a large COOH-terminal ect odomain ex-posed to the blood plasma; (c) PV-1 is N-glycosylated and its gl ycan antennae bear terminal nonreducing galactosyl residues in alpha 1-3 li nkage. 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 low er molecular weight forms were detected in brain. Immunocytochemical studie s carried out by immunodiffusion on rat lung with an anti-PV-1 polyclonal a ntibody directed against a COOH-terminal epitope reveal a specific localiza tion of PV-1 to the stomatal diaphragms of rat lung endothelial caveolae an d confirm the extracellular orientation of the PV-1 COOH terminus.