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Differential requirement for LAT and SLP-76 in GPVI versus T cell receptor signaling.

Judd BA, Myung PS, Obergfell A, Myers EE, Cheng AM, Watson SP, Pear WS, Allman D, Shattil SJ, Koretzky GA - J. Exp. Med. (2002)

Bottom Line: Mice deficient in the adaptor Src homology 2 domain-containing leukocyte phosphoprotein of 76 kD (SLP-76) exhibit a bleeding disorder and lack T cells.Linker for activation of T cells (LAT)-deficient mice exhibit a similar T cell phenotype, but show no signs of hemorrhage.In addition, SLP-76 is involved in signaling mediated by integrin alphaIIbbeta3.

View Article: PubMed Central - PubMed

Affiliation: Program in Molecular Biology, University of Iowa, Iowa City, IA 52242, USA.

ABSTRACT
Mice deficient in the adaptor Src homology 2 domain-containing leukocyte phosphoprotein of 76 kD (SLP-76) exhibit a bleeding disorder and lack T cells. Linker for activation of T cells (LAT)-deficient mice exhibit a similar T cell phenotype, but show no signs of hemorrhage. Both SLP-76 and LAT are important for optimal platelet activation downstream of the collagen receptor, GPVI. In addition, SLP-76 is involved in signaling mediated by integrin alphaIIbbeta3. Because SLP-76 and LAT function coordinately in T cell signal transduction, yet their roles appear to differ in hemostasis, we investigated in detail the functional consequences of SLP-76 and LAT deficiencies in platelets. Previously we have shown that LAT(-/-) platelets exhibit defective responses to the GPVI-specific agonist, collagen-related peptide (CRP). Consistent with this, we find that surface expression of P-selectin in response to high concentrations of GPVI ligands is reduced in both LAT- and SLP-76-deficient platelets. However, platelets from LAT(-/-) mice, but not SLP-76(-/-) mice, aggregate normally in response to high concentrations of collagen and convulxin. Additionally, unlike SLP-76, LAT is not tyrosine phosphorylated after fibrinogen binding to integrin alphaIIbbeta3, and collagen-stimulated platelets deficient in LAT spread normally on fibrinogen-coated surfaces. Together, these findings indicate that while LAT and SLP-76 are equally required for signaling via the T cell antigen receptor (TCR) and pre-TCR, platelet activation downstream of GPVI and alphaIIbbeta3 shows a much greater dependency on SLP-76 than LAT.

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Structure/function analysis of SLP-76 by retroviral reconstitution. (A) Retroviral constructs used for coexpression of SLP-76 and GFP in transduced SLP-76−/− hematopoietic cells. Virus expressing GFP alone (MigR1), or GFP with WT-SLP-76, SLP-76 which cannot be tyrosine phosphorylated (Y3F-SLP-76), or SLP-76 which cannot bind Gads (Δ224–244-SLP-76) were used to transduce SLP-76–deficient stem cells followed by transfer to lethally irradiated Rag2−/− hosts. After ∼6 wk, mice were killed and tissues harvested for analysis. (B) Thymocytes were removed from the reconstituted mice, fixed, and stained for CD4 and CD8 followed by flow cytometric analysis. In the top two panels all thymocytes (All) from a nonmanipulated C57/Bl6 (a normal thymic profile) and Rag2−/− (a profile from mice deficient for T cells) mouse were analyzed. For retrovirally reconstituted mice, gates were set to distinguish GFP-positive cells (GFP) which were then analyzed for CD4 and CD8 expression. Numbers indicate the percentage of cells in each quadrant.
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fig5: Structure/function analysis of SLP-76 by retroviral reconstitution. (A) Retroviral constructs used for coexpression of SLP-76 and GFP in transduced SLP-76−/− hematopoietic cells. Virus expressing GFP alone (MigR1), or GFP with WT-SLP-76, SLP-76 which cannot be tyrosine phosphorylated (Y3F-SLP-76), or SLP-76 which cannot bind Gads (Δ224–244-SLP-76) were used to transduce SLP-76–deficient stem cells followed by transfer to lethally irradiated Rag2−/− hosts. After ∼6 wk, mice were killed and tissues harvested for analysis. (B) Thymocytes were removed from the reconstituted mice, fixed, and stained for CD4 and CD8 followed by flow cytometric analysis. In the top two panels all thymocytes (All) from a nonmanipulated C57/Bl6 (a normal thymic profile) and Rag2−/− (a profile from mice deficient for T cells) mouse were analyzed. For retrovirally reconstituted mice, gates were set to distinguish GFP-positive cells (GFP) which were then analyzed for CD4 and CD8 expression. Numbers indicate the percentage of cells in each quadrant.

Mentions: As the transgenic constructs are not expressed in platelets, we used a retroviral reconstitution system to study the structure/function relationships of SLP-76 in this lineage. The retroviral system makes use of an IRES for expression of the marker gene, GFP, and the gene of interest from the same transcript. Four viral constructs were used to transduce SLP-76–deficient bone marrow: GFP alone (MigR1), full length murine SLP-76 (WT-SLP-76), SLP-76 in which each of the three critical tyrosine residues have been mutated to phenylalanine (Y3F-SLP-76), and SLP-76 which cannot bind to Gads (Δ224–244-SLP-76) (Fig. 5 A). Lethally irradiated recombination activating gene (Rag)2−/− mice were reconstituted with the retrovirally transduced SLP-76−/− bone marrow and ∼6 wk later, mice were killed and tissues harvested for study.


Differential requirement for LAT and SLP-76 in GPVI versus T cell receptor signaling.

Judd BA, Myung PS, Obergfell A, Myers EE, Cheng AM, Watson SP, Pear WS, Allman D, Shattil SJ, Koretzky GA - J. Exp. Med. (2002)

Structure/function analysis of SLP-76 by retroviral reconstitution. (A) Retroviral constructs used for coexpression of SLP-76 and GFP in transduced SLP-76−/− hematopoietic cells. Virus expressing GFP alone (MigR1), or GFP with WT-SLP-76, SLP-76 which cannot be tyrosine phosphorylated (Y3F-SLP-76), or SLP-76 which cannot bind Gads (Δ224–244-SLP-76) were used to transduce SLP-76–deficient stem cells followed by transfer to lethally irradiated Rag2−/− hosts. After ∼6 wk, mice were killed and tissues harvested for analysis. (B) Thymocytes were removed from the reconstituted mice, fixed, and stained for CD4 and CD8 followed by flow cytometric analysis. In the top two panels all thymocytes (All) from a nonmanipulated C57/Bl6 (a normal thymic profile) and Rag2−/− (a profile from mice deficient for T cells) mouse were analyzed. For retrovirally reconstituted mice, gates were set to distinguish GFP-positive cells (GFP) which were then analyzed for CD4 and CD8 expression. Numbers indicate the percentage of cells in each quadrant.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2193740&req=5

fig5: Structure/function analysis of SLP-76 by retroviral reconstitution. (A) Retroviral constructs used for coexpression of SLP-76 and GFP in transduced SLP-76−/− hematopoietic cells. Virus expressing GFP alone (MigR1), or GFP with WT-SLP-76, SLP-76 which cannot be tyrosine phosphorylated (Y3F-SLP-76), or SLP-76 which cannot bind Gads (Δ224–244-SLP-76) were used to transduce SLP-76–deficient stem cells followed by transfer to lethally irradiated Rag2−/− hosts. After ∼6 wk, mice were killed and tissues harvested for analysis. (B) Thymocytes were removed from the reconstituted mice, fixed, and stained for CD4 and CD8 followed by flow cytometric analysis. In the top two panels all thymocytes (All) from a nonmanipulated C57/Bl6 (a normal thymic profile) and Rag2−/− (a profile from mice deficient for T cells) mouse were analyzed. For retrovirally reconstituted mice, gates were set to distinguish GFP-positive cells (GFP) which were then analyzed for CD4 and CD8 expression. Numbers indicate the percentage of cells in each quadrant.
Mentions: As the transgenic constructs are not expressed in platelets, we used a retroviral reconstitution system to study the structure/function relationships of SLP-76 in this lineage. The retroviral system makes use of an IRES for expression of the marker gene, GFP, and the gene of interest from the same transcript. Four viral constructs were used to transduce SLP-76–deficient bone marrow: GFP alone (MigR1), full length murine SLP-76 (WT-SLP-76), SLP-76 in which each of the three critical tyrosine residues have been mutated to phenylalanine (Y3F-SLP-76), and SLP-76 which cannot bind to Gads (Δ224–244-SLP-76) (Fig. 5 A). Lethally irradiated recombination activating gene (Rag)2−/− mice were reconstituted with the retrovirally transduced SLP-76−/− bone marrow and ∼6 wk later, mice were killed and tissues harvested for study.

Bottom Line: Mice deficient in the adaptor Src homology 2 domain-containing leukocyte phosphoprotein of 76 kD (SLP-76) exhibit a bleeding disorder and lack T cells.Linker for activation of T cells (LAT)-deficient mice exhibit a similar T cell phenotype, but show no signs of hemorrhage.In addition, SLP-76 is involved in signaling mediated by integrin alphaIIbbeta3.

View Article: PubMed Central - PubMed

Affiliation: Program in Molecular Biology, University of Iowa, Iowa City, IA 52242, USA.

ABSTRACT
Mice deficient in the adaptor Src homology 2 domain-containing leukocyte phosphoprotein of 76 kD (SLP-76) exhibit a bleeding disorder and lack T cells. Linker for activation of T cells (LAT)-deficient mice exhibit a similar T cell phenotype, but show no signs of hemorrhage. Both SLP-76 and LAT are important for optimal platelet activation downstream of the collagen receptor, GPVI. In addition, SLP-76 is involved in signaling mediated by integrin alphaIIbbeta3. Because SLP-76 and LAT function coordinately in T cell signal transduction, yet their roles appear to differ in hemostasis, we investigated in detail the functional consequences of SLP-76 and LAT deficiencies in platelets. Previously we have shown that LAT(-/-) platelets exhibit defective responses to the GPVI-specific agonist, collagen-related peptide (CRP). Consistent with this, we find that surface expression of P-selectin in response to high concentrations of GPVI ligands is reduced in both LAT- and SLP-76-deficient platelets. However, platelets from LAT(-/-) mice, but not SLP-76(-/-) mice, aggregate normally in response to high concentrations of collagen and convulxin. Additionally, unlike SLP-76, LAT is not tyrosine phosphorylated after fibrinogen binding to integrin alphaIIbbeta3, and collagen-stimulated platelets deficient in LAT spread normally on fibrinogen-coated surfaces. Together, these findings indicate that while LAT and SLP-76 are equally required for signaling via the T cell antigen receptor (TCR) and pre-TCR, platelet activation downstream of GPVI and alphaIIbbeta3 shows a much greater dependency on SLP-76 than LAT.

Show MeSH
Related in: MedlinePlus