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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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Western blot analysis of P-selectin expressed in platelets. Platelets from wild-type (WT), ΔCT, and P-selectin–knockout (KO) mice were lysed in SDS lysis buffer at 5 × 109/ml. 10 μl  of lysates were loaded in each lane of 7.5% SDS polyacrylamide  gels. (a) The blot was probed with polyclonal antibodies against  P-selectin. (b) A polyclonal antibody against a KLH-coupled  P-selectin cytoplasmic domain peptide sequence was used for  probing.
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Figure 3: Western blot analysis of P-selectin expressed in platelets. Platelets from wild-type (WT), ΔCT, and P-selectin–knockout (KO) mice were lysed in SDS lysis buffer at 5 × 109/ml. 10 μl of lysates were loaded in each lane of 7.5% SDS polyacrylamide gels. (a) The blot was probed with polyclonal antibodies against P-selectin. (b) A polyclonal antibody against a KLH-coupled P-selectin cytoplasmic domain peptide sequence was used for probing.

Mentions: The P-selectin protein produced from the mutated gene was first analyzed by Western blot analysis of platelet lysates. Using a polyclonal anti–P-selectin antibody, a band at ∼140 kD was detected in both wild-type and ΔCT platelet lysates whereas it was absent in P-selectin– platelets (Fig. 3 a). A parallel probing using the polyclonal antibody against a peptide of the P-selectin CT revealed no signal at the molecular mass of P-selectin in ΔCT platelets whereas it was present in the wild-type (Fig. 3 b). Similar Western blot analysis of lung tissue lysates indicated that the endothelium-derived P-selectin in the mutant mice did not contain the CT either (data not shown). It is noteworthy that the mutant P-selectin detected in platelets and lung tissues was only slightly smaller than full-length P-selectin, entirely consistent with its being the ΔCT form predicted.


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)

Western blot analysis of P-selectin expressed in platelets. Platelets from wild-type (WT), ΔCT, and P-selectin–knockout (KO) mice were lysed in SDS lysis buffer at 5 × 109/ml. 10 μl  of lysates were loaded in each lane of 7.5% SDS polyacrylamide  gels. (a) The blot was probed with polyclonal antibodies against  P-selectin. (b) A polyclonal antibody against a KLH-coupled  P-selectin cytoplasmic domain peptide sequence was used for  probing.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Western blot analysis of P-selectin expressed in platelets. Platelets from wild-type (WT), ΔCT, and P-selectin–knockout (KO) mice were lysed in SDS lysis buffer at 5 × 109/ml. 10 μl of lysates were loaded in each lane of 7.5% SDS polyacrylamide gels. (a) The blot was probed with polyclonal antibodies against P-selectin. (b) A polyclonal antibody against a KLH-coupled P-selectin cytoplasmic domain peptide sequence was used for probing.
Mentions: The P-selectin protein produced from the mutated gene was first analyzed by Western blot analysis of platelet lysates. Using a polyclonal anti–P-selectin antibody, a band at ∼140 kD was detected in both wild-type and ΔCT platelet lysates whereas it was absent in P-selectin– platelets (Fig. 3 a). A parallel probing using the polyclonal antibody against a peptide of the P-selectin CT revealed no signal at the molecular mass of P-selectin in ΔCT platelets whereas it was present in the wild-type (Fig. 3 b). Similar Western blot analysis of lung tissue lysates indicated that the endothelium-derived P-selectin in the mutant mice did not contain the CT either (data not shown). It is noteworthy that the mutant P-selectin detected in platelets and lung tissues was only slightly smaller than full-length P-selectin, entirely consistent with its being the ΔCT form predicted.

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