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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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NFκB signal activation requires both extracellular and intracellular domains of TLR1 and TLR2. (A) HEK293-CD14 cells were cotransfected with TLR [1–2] and TLR [2–1] 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] chimeric proteins. Co-transfection of both chimeric proteins confers responsiveness as a result of concomitant expression of both intracellular and extracellular domains of TLR1 and TLR2. With TLR1 endogenously present, transfection with TLR2-WT alone is sufficient to confer responsiveness.
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fig6: NFκB signal activation requires both extracellular and intracellular domains of TLR1 and TLR2. (A) HEK293-CD14 cells were cotransfected with TLR [1–2] and TLR [2–1] 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] chimeric proteins. Co-transfection of both chimeric proteins confers responsiveness as a result of concomitant expression of both intracellular and extracellular domains of TLR1 and TLR2. With TLR1 endogenously present, transfection with TLR2-WT alone is sufficient to confer responsiveness.

Mentions: Neither the TLR [1–2] nor the TLR [2–1] chimeric proteins alone were sufficient to confer responsiveness to TLR2 ligands. Therefore, we examined the ability of combinations of these chimeric fusion proteins to signal. HEK293-CD14 cells were cotransfected with TLR [1–2] and TLR [2–1] fusion proteins, and the response of cells to araLAM, Pam3CSK4, and zymosan was tested. This combination of chimeric receptors was sufficient to confer responsiveness to araLAM and Pam3CSK4. Interestingly, the combination of TLR [1–2] and TLR [2–1] did not confer responsiveness to zymosan (Fig. 6, A and B). These results suggest that both intracellular and extracellular domains of TLR1 and TLR2 are required in recognition of araLAM and Pam3CSK4. We hypothesize that within the intracellular domains of both receptors, dimerization of TIR domains is essential for subsequent signal activation.


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)

NFκB signal activation requires both extracellular and intracellular domains of TLR1 and TLR2. (A) HEK293-CD14 cells were cotransfected with TLR [1–2] and TLR [2–1] 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] chimeric proteins. Co-transfection of both chimeric proteins confers responsiveness as a result of concomitant expression of both intracellular and extracellular domains of TLR1 and TLR2. 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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fig6: NFκB signal activation requires both extracellular and intracellular domains of TLR1 and TLR2. (A) HEK293-CD14 cells were cotransfected with TLR [1–2] and TLR [2–1] 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] chimeric proteins. Co-transfection of both chimeric proteins confers responsiveness as a result of concomitant expression of both intracellular and extracellular domains of TLR1 and TLR2. With TLR1 endogenously present, transfection with TLR2-WT alone is sufficient to confer responsiveness.
Mentions: Neither the TLR [1–2] nor the TLR [2–1] chimeric proteins alone were sufficient to confer responsiveness to TLR2 ligands. Therefore, we examined the ability of combinations of these chimeric fusion proteins to signal. HEK293-CD14 cells were cotransfected with TLR [1–2] and TLR [2–1] fusion proteins, and the response of cells to araLAM, Pam3CSK4, and zymosan was tested. This combination of chimeric receptors was sufficient to confer responsiveness to araLAM and Pam3CSK4. Interestingly, the combination of TLR [1–2] and TLR [2–1] did not confer responsiveness to zymosan (Fig. 6, A and B). These results suggest that both intracellular and extracellular domains of TLR1 and TLR2 are required in recognition of araLAM and Pam3CSK4. We hypothesize that within the intracellular domains of both receptors, dimerization of TIR domains is essential for subsequent signal activation.

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