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. 1999 Jun 14;145(6):1189-98.
doi: 10.1083/jcb.145.6.1189.

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

R V Stan  1 , L Ghitescu, B S Jacobson, G E Palade
Affiliations

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

R V Stan et al. J Cell Biol. .

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 alpha1-3 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.

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Figures

Figure 1
Figure 1
PV-1 isolation. (a) Schematic of the isolation procedure of PV-1 from rat lung. (b–g) Immunoblotting with anti–PV-1 21D5 mAb of equivalent volumes of fractions from different steps in the isolation procedure. (b) Rat lung homogenate separation into cytosol and total membranes. Rat plasma proteins (20 μg) were resolved on the same gel to demonstrate the blood plasma origin of the lower (∼45–50 kD) band recognized by the 21D5 mAb in the cytosolic fraction; (c) high-pH soluble and insoluble material resulted from extraction of rat lung total membranes with 0.1 M Na2CO3, pH 11; (d) Triton X-100 soluble and insoluble material resulted from the extraction of high-pH insoluble material in 2% Triton X-100; and (e) fractions resulted from the IEF separation of the Triton X-100 extract in a Rotofor cell. (f) Equivalent amounts of fractions resulted from GS I chromatography of the PV-1–containing Rotofor fractions (fractions 11–14 from e). (g) DEAE chromatography of the glycoproteins eluted from GS I column (see Materials and Methods). To monitor PV-1 losses on the DEAE column, the column was washed with 10 ml HBS-T followed by 10 ml of HBS-T adjusted to 0.5 M NaCl final concentration. The proteins of the two washes were collected by TCA precipitation and solubilized in 0.5 ml 2× reducing SDS-PAGE sample buffer. Equivalent volumes of the flow-through (FT), 0.15 M NaCl, and 0.5 M NaCl wash were resolved by 8% SDS-PAGE and immunoblotted with the anti–PV-1 21D5 mAb. (h) Silver staining of the 8% SDS-PAGE resolved proteins unbound to the DEAE-Sephacel column. The position of the reduced PV-1 is indicated by the arrow.
Figure 2
Figure 2
(a) Sequence of the full length rat PV-1 cDNA and protein. The positions of the peptides obtained by protein microsequencing (square boxes) and the putative transmembrane domain (oval box) are indicated. The portion of the PV-1 message that was obtained by rescreening of the rat lung library with a DNA probe (see Materials and Methods) is underlined. These sequence data are available from GenBank/EMBL/DDBJ under accession number AF154831. (b) Hydrophilicity plot and (c) schematic of the protein sequence. The positions of the consensus phosphorylation sites (*), N-glycosylation sites (Y), cysteines ( ), the 21D5 mAb epitope (filled box), and the proline-rich region (PRR) are marked. The number of amino acids in between cysteines is indicated.
Figure 3
Figure 3
Northern blotting of rat tissues. Rat lung total RNA (20 μg) was probed with a 428-bp 32P-labeled PV-1 DNA fragment as described in Materials and Methods. A single band corresponding to the PV-1 mRNA was obtained at ∼2000 nt in heart (H), spleen (S), lung (L), liver (Li), muscle (M), and kidney (K). No message was obtained in the testis (Te). Exposure time: upper panel, 4 h; lower panel, 12 h.
Figure 4
Figure 4
Anti–PV-1C chicken polyclonal antibody specificity. (a) Immunoblotting of rat lung total membrane proteins containing strips with 1 μg of each 21D5 mAb, preimmune IgY, and anti–PV-1C polyclonal antibody as indicated in the figure. (b) Immunoblotting of rat blood plasma (Pl), rat lung cytosol (C), and rat lung membranes (M) (10 μg each) in nonreducing (left) and reducing (right) conditions. (c) Competition of the anti–PV-1C pAb by the PV-1C peptide compared with an irrelevant (caveolin) peptide. (d) Immunoprecipitation of rat PV-1 using the anti–PV-1 C pAb followed by immunoblotting of equivalent amounts of the immunoprecipitated (IP) and the unbound (NB) material using the 21D5 mAb. (e) Immunoprecipitation of rat lung PV-1 with anti–PV-1C pAb as described in Materials and Methods. The immunoprecipitated proteins by anti–PV-1C pAb (lane 1), preimmune total IgY control (lane 2), and anti–chicken IgY-agarose control (lane 3) were resolved by 12% SDS-PAGE in nonreducing conditions and silver stained. The asterisk marks the position of the PV-1 dimer.
Figure 5
Figure 5
Western blotting of different rat tissues using the anti–PV-1C pAb. Proteins from total membranes from different rat tissues were prepared as in Materials and Methods and immunoblotted with the anti–PV-1C pAb (1:500 dilution) followed by an HRP-conjugated anti–chicken IgY reporter antibody and the signal was detected by enhanced chemiluminescence. B, brain; H, heart; M, muscle; K, kidney; Li, liver; Lu, lung; S, spleen; Te, testis.
Figure 6
Figure 6
Immunolocaliza-tion of PV-1 in rat lung by preembedding immunocytochemistry. Rat lung specimens prepared as described in Materials and Methods were reacted with anti–PV-1C pAb followed by a 5 nm gold-conjugated reporter antibody. The label (arrowheads) was found associated specifically with the necks and stomatal diaphragms of endothelial caveolae (a–e) at both fronts of the endothelial cell (a, b, and e). Most caveolar stomatal diaphragms are marked by a single gold particle and occasionally by a cluster of particles (a, d, and e). No label was found on either plasmalemma proper (a–e) or coated pits (cp in d). Bars, 100 nm.
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