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Importance of extra- and intracellular domains of TLR1 and TLR2 in NFkappa B signaling.

Sandor F, Latz E, Re F, Mandell L, Repik G, Golenbock DT, Espevik T, Kurt-Jones EA, Finberg RW - J. Cell Biol. (2003)

Bottom Line: Simultaneous cross-linking of both receptors resulted in ligand-independent signal transduction.Using chimeric TLRs, we found that expression of the extracellular domains along with simultaneous expression of the intracellular domains of both TLRs was necessary to achieve functional signaling.The domains from each receptor did not need to be contained within a single contiguous protein.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Massachusetts Medical Center, Worcester, MA 01605-2324, USA.

ABSTRACT
Recognition of ligands by toll-like receptor (TLR) 2 requires interactions with other TLRs. TLRs form a combinatorial repertoire to discriminate between the diverse microbial ligands. Diversity results from extracellular and intracellular interactions of different TLRs. This paper demonstrates that TLR1 and TLR2 are required for ara-lipoarabinomannan- and tripalmitoyl cysteinyl lipopeptide-stimulated cytokine secretion from mononuclear cells. Confocal microscopy revealed that TLR1 and TLR2 cotranslationally form heterodimeric complexes on the cell surface and in the cytosol. Simultaneous cross-linking of both receptors resulted in ligand-independent signal transduction. Using chimeric TLRs, we found that expression of the extracellular domains along with simultaneous expression of the intracellular domains of both TLRs was necessary to achieve functional signaling. The domains from each receptor did not need to be contained within a single contiguous protein. Chimeric TLR analysis further defined the toll/IL-1R domains as the area of crucial intracellular TLR1-TLR2 interaction.

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Heterologous expression of the extracellular domains of TLR1 and TLR2 together with heterologous expression of the TIR domains of TLR1 and TLR2 is sufficient for NFκB signal activation. (A) HEK293-CD14 cells were cotransfected with TLR [1–2] and TLR [2–1 TIR] DNA encoding chimeric proteins or wild-type TLR2 DNA. After 6 h of stimulation with 1 μg/ml araLAM, 100 ng/ml Pam3CSK4, or 10 μg/ml zymosan, cells were lysed and NFκB luciferase reporter gene activity was measured. IL-1β was used as a positive control. (B) A schematic model of cotransfection of TLR [1–2] and TLR [2–1 TIR] chimeric proteins. Co-transfection of TLR [1–2] chimeric protein containing the entire intracellular domain of TLR2 (including the TIR domain) with the TLR [2–1 TIR] chimeric protein containing the TIR domain of TLR1, confers responsiveness. The transfected cells express the extracellular domains of TLR1 and TLR2 (ligand recognition) as well as both the TIR domains of TLR1 and TLR2 (signal transduction). With TLR1 endogenously present, transfection with TLR2-WT alone is sufficient to confer responsiveness.
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fig7: Heterologous expression of the extracellular domains of TLR1 and TLR2 together with heterologous expression of the TIR domains of TLR1 and TLR2 is sufficient for NFκB signal activation. (A) HEK293-CD14 cells were cotransfected with TLR [1–2] and TLR [2–1 TIR] DNA encoding chimeric proteins or wild-type TLR2 DNA. After 6 h of stimulation with 1 μg/ml araLAM, 100 ng/ml Pam3CSK4, or 10 μg/ml zymosan, cells were lysed and NFκB luciferase reporter gene activity was measured. IL-1β was used as a positive control. (B) A schematic model of cotransfection of TLR [1–2] and TLR [2–1 TIR] chimeric proteins. Co-transfection of TLR [1–2] chimeric protein containing the entire intracellular domain of TLR2 (including the TIR domain) with the TLR [2–1 TIR] chimeric protein containing the TIR domain of TLR1, confers responsiveness. The transfected cells express the extracellular domains of TLR1 and TLR2 (ligand recognition) as well as both the TIR domains of TLR1 and TLR2 (signal transduction). With TLR1 endogenously present, transfection with TLR2-WT alone is sufficient to confer responsiveness.

Mentions: To analyze the role of the intracellular domain in signaling, another TLR fusion protein was generated (TLR [2–1 TIR]) in which the TIR domain of TLR2 was replaced with the TIR domain of TLR1. Transfection with the TLR [2–1 TIR] construct alone did not confer responsiveness to araLAM and zymosan (Table I). HEK293-CD14 cells cotransfected with TLR [1–2] fusion protein and TLR [2–1 TIR] fusion protein were activated in response to araLAM and Pam3CSK4, but not to zymosan (Fig. 7, A and B). These data suggest that the heterodimerization of the TIR domains of TLR1 and TLR2 is essential for the signaling in response to some (but not all) TLR2 ligands (i.e., araLAM and Pam3CSK4, but not zymosan).


Importance of extra- and intracellular domains of TLR1 and TLR2 in NFkappa B signaling.

Sandor F, Latz E, Re F, Mandell L, Repik G, Golenbock DT, Espevik T, Kurt-Jones EA, Finberg RW - J. Cell Biol. (2003)

Heterologous expression of the extracellular domains of TLR1 and TLR2 together with heterologous expression of the TIR domains of TLR1 and TLR2 is sufficient for NFκB signal activation. (A) HEK293-CD14 cells were cotransfected with TLR [1–2] and TLR [2–1 TIR] DNA encoding chimeric proteins or wild-type TLR2 DNA. After 6 h of stimulation with 1 μg/ml araLAM, 100 ng/ml Pam3CSK4, or 10 μg/ml zymosan, cells were lysed and NFκB luciferase reporter gene activity was measured. IL-1β was used as a positive control. (B) A schematic model of cotransfection of TLR [1–2] and TLR [2–1 TIR] chimeric proteins. Co-transfection of TLR [1–2] chimeric protein containing the entire intracellular domain of TLR2 (including the TIR domain) with the TLR [2–1 TIR] chimeric protein containing the TIR domain of TLR1, confers responsiveness. The transfected cells express the extracellular domains of TLR1 and TLR2 (ligand recognition) as well as both the TIR domains of TLR1 and TLR2 (signal transduction). With TLR1 endogenously present, transfection with TLR2-WT alone is sufficient to confer responsiveness.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172862&req=5

fig7: Heterologous expression of the extracellular domains of TLR1 and TLR2 together with heterologous expression of the TIR domains of TLR1 and TLR2 is sufficient for NFκB signal activation. (A) HEK293-CD14 cells were cotransfected with TLR [1–2] and TLR [2–1 TIR] DNA encoding chimeric proteins or wild-type TLR2 DNA. After 6 h of stimulation with 1 μg/ml araLAM, 100 ng/ml Pam3CSK4, or 10 μg/ml zymosan, cells were lysed and NFκB luciferase reporter gene activity was measured. IL-1β was used as a positive control. (B) A schematic model of cotransfection of TLR [1–2] and TLR [2–1 TIR] chimeric proteins. Co-transfection of TLR [1–2] chimeric protein containing the entire intracellular domain of TLR2 (including the TIR domain) with the TLR [2–1 TIR] chimeric protein containing the TIR domain of TLR1, confers responsiveness. The transfected cells express the extracellular domains of TLR1 and TLR2 (ligand recognition) as well as both the TIR domains of TLR1 and TLR2 (signal transduction). With TLR1 endogenously present, transfection with TLR2-WT alone is sufficient to confer responsiveness.
Mentions: To analyze the role of the intracellular domain in signaling, another TLR fusion protein was generated (TLR [2–1 TIR]) in which the TIR domain of TLR2 was replaced with the TIR domain of TLR1. Transfection with the TLR [2–1 TIR] construct alone did not confer responsiveness to araLAM and zymosan (Table I). HEK293-CD14 cells cotransfected with TLR [1–2] fusion protein and TLR [2–1 TIR] fusion protein were activated in response to araLAM and Pam3CSK4, but not to zymosan (Fig. 7, A and B). These data suggest that the heterodimerization of the TIR domains of TLR1 and TLR2 is essential for the signaling in response to some (but not all) TLR2 ligands (i.e., araLAM and Pam3CSK4, but not zymosan).

Bottom Line: Simultaneous cross-linking of both receptors resulted in ligand-independent signal transduction.Using chimeric TLRs, we found that expression of the extracellular domains along with simultaneous expression of the intracellular domains of both TLRs was necessary to achieve functional signaling.The domains from each receptor did not need to be contained within a single contiguous protein.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Massachusetts Medical Center, Worcester, MA 01605-2324, USA.

ABSTRACT
Recognition of ligands by toll-like receptor (TLR) 2 requires interactions with other TLRs. TLRs form a combinatorial repertoire to discriminate between the diverse microbial ligands. Diversity results from extracellular and intracellular interactions of different TLRs. This paper demonstrates that TLR1 and TLR2 are required for ara-lipoarabinomannan- and tripalmitoyl cysteinyl lipopeptide-stimulated cytokine secretion from mononuclear cells. Confocal microscopy revealed that TLR1 and TLR2 cotranslationally form heterodimeric complexes on the cell surface and in the cytosol. Simultaneous cross-linking of both receptors resulted in ligand-independent signal transduction. Using chimeric TLRs, we found that expression of the extracellular domains along with simultaneous expression of the intracellular domains of both TLRs was necessary to achieve functional signaling. The domains from each receptor did not need to be contained within a single contiguous protein. Chimeric TLR analysis further defined the toll/IL-1R domains as the area of crucial intracellular TLR1-TLR2 interaction.

Show MeSH