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

Abstract

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 α1-3 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.

Endothelial Transcytotic Machinery Involves Supramolecular Protein–Lipid Complexes

“…It may be an artifact generated by membrane fragmentation during the mechanical disruption of the cells. Yet, the validity of this finding is supported by 1) the EM survey of the cytosolic fraction that failed to reveal any membrane fragments (our unpublished results); 2) the control experiments in which the rat lung cytosol was analyzed by immunoblotting for the presence of PV-1, an integral membrane protein, a new marker for endothelial plasmalemmal vesicles from rat lung (Stan, 1999). PV-1 was not detected in the cytosol or EMTCs.…”

PV1 Is a Key Structural Component for the Formation of the Stomatal and Fenestral Diaphragms

“…The extracellular domain contains four N-glycosylation sites near the membrane, a proline-rich region near the C-terminus, and two large coiled-coil domains, suggesting a rodlike protein (Stan et al, 2001). From the studies done so far, PV1 seems to be endothelium specific and moreover to be specifically associated with the SDs of caveolae and TEC and the FDs, at both fronts of ECs (Stan et al, 1999a(Stan et al, , 1999b. PV1 forms homodimers in situ as well as in culture (Stan, 2003).…”

“…IgY purification was done as before (Stan et al, 1999a). The anti-huPV1C pAb was affinity-purified on the huPV1C peptide coupled to Affi-Gel10 followed by extensive cross-absorption on a BSAagarose column (Stan et al, 1999a). Mouse anti-FLAG mAb (clone M2), anti-FLAG-agarose, and anti-␤-actin mAb (clone AC15) were from Sigma (St. Louis, MO).…”

“…The hPV1C peptide consisted of the last 12 aa from hPV1 C terminus, to which a glycine and a lysine (in bold) were added for increased solubility and coupling purposes, respectively. IgY purification was done as before (Stan et al, 1999a). The anti-huPV1C pAb was affinity-purified on the huPV1C peptide coupled to Affi-Gel10 followed by extensive cross-absorption on a BSAagarose column (Stan et al, 1999a).…”

“…Recently, we have shown that the SDs of caveolae and TEC and the FDs of fenestrae share at least one protein, namely PV1 (Stan et al, 1999a(Stan et al, , 1999b. PV1 is a single-span, 60-kDa, type II membrane N-glycosylated glycoprotein with a short intracellular tail and a long extracellular domain (Stan et al, 1999b).…”