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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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Gene replacement strategy and Southern blot analysis of progeny from heterozygous  crosses. (a) The wild-type P-selectin allele, the  replacement vector, and the mutated allele are  shown. Exon TM encodes the transmembrane  domain and the first seven amino acids of the CT  of P-selectin. Using PCR amplification, two stop  codons and XhoI-EcoRI-XbaI restriction sites  were inserted after the first three amino acids of  the CT. A 620-bp fragment containing 3′-UTR of  the hGH gene and the PGKneo cassette were inserted into the EcoRI site introduced by PCR. A  320-bp fragment immediately following the TM  exon and before the XbaI site was deleted in the  targeting construct. A probe flanking the 3′-end of the vector was used for Southern blot analysis of ES cell clones and tail biopsies of the progeny. The probe detects a EcoRI fragment of >12 kb from the wild-type allele and a 6-kb fragment  from the targeted allele since an EcoRI site is introduced between the hGH and  PGKneo gene. (b) Representative Southern blot analysis of tail biopsies of progeny from heterozygous crosses. Genomic DNA was digested with EcoRI, electrophoresed, and then blotted. The fragments from wild-type and mutant alleles are  as indicated.
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Figure 1: Gene replacement strategy and Southern blot analysis of progeny from heterozygous crosses. (a) The wild-type P-selectin allele, the replacement vector, and the mutated allele are shown. Exon TM encodes the transmembrane domain and the first seven amino acids of the CT of P-selectin. Using PCR amplification, two stop codons and XhoI-EcoRI-XbaI restriction sites were inserted after the first three amino acids of the CT. A 620-bp fragment containing 3′-UTR of the hGH gene and the PGKneo cassette were inserted into the EcoRI site introduced by PCR. A 320-bp fragment immediately following the TM exon and before the XbaI site was deleted in the targeting construct. A probe flanking the 3′-end of the vector was used for Southern blot analysis of ES cell clones and tail biopsies of the progeny. The probe detects a EcoRI fragment of >12 kb from the wild-type allele and a 6-kb fragment from the targeted allele since an EcoRI site is introduced between the hGH and PGKneo gene. (b) Representative Southern blot analysis of tail biopsies of progeny from heterozygous crosses. Genomic DNA was digested with EcoRI, electrophoresed, and then blotted. The fragments from wild-type and mutant alleles are as indicated.

Mentions: A mouse genomic library made from the livers of Black Agouti 129Sv strain (gift of R. Jaenisch, Massachusetts Institute of Technology, Cambridge, MA) was screened with a mouse cDNA probe spanning CR8, transmembrane domain, and C1 and C2 exons obtained from D. Vestweber (Münster, Germany). The genomic clone containing the 3′ end of the P-selectin gene was subcloned into Bluescript KS vector (Stratagene, La Jolla, CA). Two stop codons and XhoI-EcoRI-XbaI restriction sites were inserted into the genomic clone by PCR amplification using primers complementary to a sequence within the intron after CR8 and the 3′ end of exon TM (see Fig. 1 a) which encoded the transmembrane domain and the first seven amino acids of the CT. The 3′ end of the human growth hormone (hGH) gene was then inserted into the EcoRI site introduced by PCR. A 1.7-kb neomycin resistance gene with the phosphoglycerate kinase promoter (PGKneo) was inserted immediately after the hGH gene fragment. The resulting fragment contained 5 and 2.5 kb of P-selectin genomic sequences upstream and downstream of the insertions, respectively, and was cloned into a Bluescript vector containing a herpes simplex virus-thymidine kinase (HSV-TK) cassette (see Fig. 1 a). The final construct was linearized with NotI for transfection.


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)

Gene replacement strategy and Southern blot analysis of progeny from heterozygous  crosses. (a) The wild-type P-selectin allele, the  replacement vector, and the mutated allele are  shown. Exon TM encodes the transmembrane  domain and the first seven amino acids of the CT  of P-selectin. Using PCR amplification, two stop  codons and XhoI-EcoRI-XbaI restriction sites  were inserted after the first three amino acids of  the CT. A 620-bp fragment containing 3′-UTR of  the hGH gene and the PGKneo cassette were inserted into the EcoRI site introduced by PCR. A  320-bp fragment immediately following the TM  exon and before the XbaI site was deleted in the  targeting construct. A probe flanking the 3′-end of the vector was used for Southern blot analysis of ES cell clones and tail biopsies of the progeny. The probe detects a EcoRI fragment of >12 kb from the wild-type allele and a 6-kb fragment  from the targeted allele since an EcoRI site is introduced between the hGH and  PGKneo gene. (b) Representative Southern blot analysis of tail biopsies of progeny from heterozygous crosses. Genomic DNA was digested with EcoRI, electrophoresed, and then blotted. The fragments from wild-type and mutant alleles are  as indicated.
© Copyright Policy
Related In: Results  -  Collection

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Figure 1: Gene replacement strategy and Southern blot analysis of progeny from heterozygous crosses. (a) The wild-type P-selectin allele, the replacement vector, and the mutated allele are shown. Exon TM encodes the transmembrane domain and the first seven amino acids of the CT of P-selectin. Using PCR amplification, two stop codons and XhoI-EcoRI-XbaI restriction sites were inserted after the first three amino acids of the CT. A 620-bp fragment containing 3′-UTR of the hGH gene and the PGKneo cassette were inserted into the EcoRI site introduced by PCR. A 320-bp fragment immediately following the TM exon and before the XbaI site was deleted in the targeting construct. A probe flanking the 3′-end of the vector was used for Southern blot analysis of ES cell clones and tail biopsies of the progeny. The probe detects a EcoRI fragment of >12 kb from the wild-type allele and a 6-kb fragment from the targeted allele since an EcoRI site is introduced between the hGH and PGKneo gene. (b) Representative Southern blot analysis of tail biopsies of progeny from heterozygous crosses. Genomic DNA was digested with EcoRI, electrophoresed, and then blotted. The fragments from wild-type and mutant alleles are as indicated.
Mentions: A mouse genomic library made from the livers of Black Agouti 129Sv strain (gift of R. Jaenisch, Massachusetts Institute of Technology, Cambridge, MA) was screened with a mouse cDNA probe spanning CR8, transmembrane domain, and C1 and C2 exons obtained from D. Vestweber (Münster, Germany). The genomic clone containing the 3′ end of the P-selectin gene was subcloned into Bluescript KS vector (Stratagene, La Jolla, CA). Two stop codons and XhoI-EcoRI-XbaI restriction sites were inserted into the genomic clone by PCR amplification using primers complementary to a sequence within the intron after CR8 and the 3′ end of exon TM (see Fig. 1 a) which encoded the transmembrane domain and the first seven amino acids of the CT. The 3′ end of the human growth hormone (hGH) gene was then inserted into the EcoRI site introduced by PCR. A 1.7-kb neomycin resistance gene with the phosphoglycerate kinase promoter (PGKneo) was inserted immediately after the hGH gene fragment. The resulting fragment contained 5 and 2.5 kb of P-selectin genomic sequences upstream and downstream of the insertions, respectively, and was cloned into a Bluescript vector containing a herpes simplex virus-thymidine kinase (HSV-TK) cassette (see Fig. 1 a). The final construct was linearized with NotI for transfection.

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