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Role of P-selectin cytoplasmic domain in granular targeting in vivo and in early inflammatory responses.

Hartwell DW, Mayadas TN, Berger G, Frenette PS, Rayburn H, Hynes RO, Wagner DD - J. Cell Biol. (1998)

Bottom Line: The deletion did not affect the sorting of P-selectin into alpha-granules of platelets but severely compromised the storage of P-selectin in endothelial cells.Unstored P-selectin was proteolytically shed from the plasma membrane, resulting in increased levels of soluble P-selectin in the plasma.Our results suggest that different sorting mechanisms for P-selectin are used in platelets and endothelial cells and that the storage pool of P-selectin in endothelial cells is functionally important during early stages of inflammation.

View Article: PubMed Central - PubMed

Affiliation: Center for Blood Research, Harvard Medical School, Boston, Massachusetts 02115, USA.

ABSTRACT
P-selectin is an adhesion receptor for leukocytes expressed on activated platelets and endothelial cells. The cytoplasmic domain of P-selectin was shown in vitro to contain signals required for both the sorting of this protein into storage granules and its internalization from the plasma membrane. To evaluate in vivo the role of the regulated secretion of P-selectin, we have generated a mouse that expresses P-selectin lacking the cytoplasmic domain (DeltaCT mice). The deletion did not affect the sorting of P-selectin into alpha-granules of platelets but severely compromised the storage of P-selectin in endothelial cells. Unstored P-selectin was proteolytically shed from the plasma membrane, resulting in increased levels of soluble P-selectin in the plasma. The DeltaCT-P-selectin appeared capable of mediating cell adhesion as it supported leukocyte rolling in the mutant mice. However, a secretagogue failed to upregulate leukocyte rolling in the DeltaCT mice, indicating an absence of a releasable storage pool of P-selectin in the endothelium. Furthermore, the neutrophil influx into the inflamed peritoneum was only 30% of the wild-type level 2 h after stimulation. Our results suggest that different sorting mechanisms for P-selectin are used in platelets and endothelial cells and that the storage pool of P-selectin in endothelial cells is functionally important during early stages of inflammation.

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Localization of ΔCT–P-selectin in platelets by electron microscopy. Indirect immunogold labeling of P-selectin was performed  in resting platelets from wild-type (WT) and ΔCT mice. Ultrathin frozen sections were stained with a rabbit antibody against P-selectin  and visualized with a goat anti–rabbit IgG conjugated to 10-nm colloidal gold particles. The majority of the gold particles are associated  with α-granules (arrowheads) in both wild-type and ΔCT platelets. A small amount of labeling was seen on the plasma membrane in  both genotypes. Bars, 200 nm.
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Figure 5: Localization of ΔCT–P-selectin in platelets by electron microscopy. Indirect immunogold labeling of P-selectin was performed in resting platelets from wild-type (WT) and ΔCT mice. Ultrathin frozen sections were stained with a rabbit antibody against P-selectin and visualized with a goat anti–rabbit IgG conjugated to 10-nm colloidal gold particles. The majority of the gold particles are associated with α-granules (arrowheads) in both wild-type and ΔCT platelets. A small amount of labeling was seen on the plasma membrane in both genotypes. Bars, 200 nm.

Mentions: Immunofluorescence staining of blood smears revealed a granular staining pattern for P-selectin in ΔCT platelets which was similar to that of wild-type platelets (data not shown). Furthermore, immunolocalization by electron microscopy was performed to locate precisely the ΔCT– P-selectin molecules inside the platelets. Immunogold labeling of P-selectin displayed essentially a similar distribution of gold particles in ΔCT and wild-type platelets. P-selectin molecules without CT were preferentially associated with the α-granules as was the case for wild-type molecules (Fig. 5). This indicates that the cytoplasmic domain is not necessary for P-selectin localization to the α-granules.


Role of P-selectin cytoplasmic domain in granular targeting in vivo and in early inflammatory responses.

Hartwell DW, Mayadas TN, Berger G, Frenette PS, Rayburn H, Hynes RO, Wagner DD - J. Cell Biol. (1998)

Localization of ΔCT–P-selectin in platelets by electron microscopy. Indirect immunogold labeling of P-selectin was performed  in resting platelets from wild-type (WT) and ΔCT mice. Ultrathin frozen sections were stained with a rabbit antibody against P-selectin  and visualized with a goat anti–rabbit IgG conjugated to 10-nm colloidal gold particles. The majority of the gold particles are associated  with α-granules (arrowheads) in both wild-type and ΔCT platelets. A small amount of labeling was seen on the plasma membrane in  both genotypes. Bars, 200 nm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132959&req=5

Figure 5: Localization of ΔCT–P-selectin in platelets by electron microscopy. Indirect immunogold labeling of P-selectin was performed in resting platelets from wild-type (WT) and ΔCT mice. Ultrathin frozen sections were stained with a rabbit antibody against P-selectin and visualized with a goat anti–rabbit IgG conjugated to 10-nm colloidal gold particles. The majority of the gold particles are associated with α-granules (arrowheads) in both wild-type and ΔCT platelets. A small amount of labeling was seen on the plasma membrane in both genotypes. Bars, 200 nm.
Mentions: Immunofluorescence staining of blood smears revealed a granular staining pattern for P-selectin in ΔCT platelets which was similar to that of wild-type platelets (data not shown). Furthermore, immunolocalization by electron microscopy was performed to locate precisely the ΔCT– P-selectin molecules inside the platelets. Immunogold labeling of P-selectin displayed essentially a similar distribution of gold particles in ΔCT and wild-type platelets. P-selectin molecules without CT were preferentially associated with the α-granules as was the case for wild-type molecules (Fig. 5). This indicates that the cytoplasmic domain is not necessary for P-selectin localization to the α-granules.

Bottom Line: The deletion did not affect the sorting of P-selectin into alpha-granules of platelets but severely compromised the storage of P-selectin in endothelial cells.Unstored P-selectin was proteolytically shed from the plasma membrane, resulting in increased levels of soluble P-selectin in the plasma.Our results suggest that different sorting mechanisms for P-selectin are used in platelets and endothelial cells and that the storage pool of P-selectin in endothelial cells is functionally important during early stages of inflammation.

View Article: PubMed Central - PubMed

Affiliation: Center for Blood Research, Harvard Medical School, Boston, Massachusetts 02115, USA.

ABSTRACT
P-selectin is an adhesion receptor for leukocytes expressed on activated platelets and endothelial cells. The cytoplasmic domain of P-selectin was shown in vitro to contain signals required for both the sorting of this protein into storage granules and its internalization from the plasma membrane. To evaluate in vivo the role of the regulated secretion of P-selectin, we have generated a mouse that expresses P-selectin lacking the cytoplasmic domain (DeltaCT mice). The deletion did not affect the sorting of P-selectin into alpha-granules of platelets but severely compromised the storage of P-selectin in endothelial cells. Unstored P-selectin was proteolytically shed from the plasma membrane, resulting in increased levels of soluble P-selectin in the plasma. The DeltaCT-P-selectin appeared capable of mediating cell adhesion as it supported leukocyte rolling in the mutant mice. However, a secretagogue failed to upregulate leukocyte rolling in the DeltaCT mice, indicating an absence of a releasable storage pool of P-selectin in the endothelium. Furthermore, the neutrophil influx into the inflamed peritoneum was only 30% of the wild-type level 2 h after stimulation. Our results suggest that different sorting mechanisms for P-selectin are used in platelets and endothelial cells and that the storage pool of P-selectin in endothelial cells is functionally important during early stages of inflammation.

Show MeSH
Related in: MedlinePlus