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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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TLR2 and TLR1 mutants inhibit NFκB activation in HEK 293 cells in stimulation with araLAM and zymosan. (A) The response of HEK293-CD14 cells to araLAM and zymosan is inhibited by a single point mutated TLR2 construct, TLR2-P681H. HEK293-CD14 cells were cotransfected with 5 ng TLR2-WT DNA and increasing amounts of TLR2-P681H (from 5 to 200 ng DNA). After 6 h of stimulation with 1 μg/ml araLAM and 10 μg/ml zymosan, cells were lysed and NFκB luciferase reporter gene activity was measured. IL-1β was used as a positive control. Luciferase activity is expressed in normalized RLU as the ratio of NFκB-dependent firefly luciferase activity to NFκB-independent renilla luciferase activity. Data shown are the mean ± SD of triplicate wells. (B) The response of HEK293-CD14 cells to araLAM and zymosan is inhibited by expression of a TLR2 mutant missing the TIR domain TLR2-ΔTIR. HEK293-CD14 cells were cotransfected with 5 ng TLR2-WT DNA and increasing amounts of TLR2-ΔTIR (from 5 to 200 ng DNA). After 6 h of stimulation with 1 μg/ml araLAM and 10 μg/ml zymosan, cells were lysed and NFκB luciferase reporter gene activity was measured. IL-1β was used as a positive control. (C) The response of HEK293-CD14 cells to araLAM and zymosan is inhibited by expression of a cytoplasmic deletion mutant of TLR1, TLR1-Δcyt. HEK293-CD14 cells were cotransfected with 5 ng TLR2-WT DNA and increasing amounts of TLR1-Δcyt (from 5 to 200 ng DNA). After 6 h of stimulation with 1 μg/ml araLAM and 10 μg/ml zymosan, cells were lysed and NFκB luciferase reporter gene activity was measured. IL-1β was used as a positive control.
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fig4: TLR2 and TLR1 mutants inhibit NFκB activation in HEK 293 cells in stimulation with araLAM and zymosan. (A) The response of HEK293-CD14 cells to araLAM and zymosan is inhibited by a single point mutated TLR2 construct, TLR2-P681H. HEK293-CD14 cells were cotransfected with 5 ng TLR2-WT DNA and increasing amounts of TLR2-P681H (from 5 to 200 ng DNA). After 6 h of stimulation with 1 μg/ml araLAM and 10 μg/ml zymosan, cells were lysed and NFκB luciferase reporter gene activity was measured. IL-1β was used as a positive control. Luciferase activity is expressed in normalized RLU as the ratio of NFκB-dependent firefly luciferase activity to NFκB-independent renilla luciferase activity. Data shown are the mean ± SD of triplicate wells. (B) The response of HEK293-CD14 cells to araLAM and zymosan is inhibited by expression of a TLR2 mutant missing the TIR domain TLR2-ΔTIR. HEK293-CD14 cells were cotransfected with 5 ng TLR2-WT DNA and increasing amounts of TLR2-ΔTIR (from 5 to 200 ng DNA). After 6 h of stimulation with 1 μg/ml araLAM and 10 μg/ml zymosan, cells were lysed and NFκB luciferase reporter gene activity was measured. IL-1β was used as a positive control. (C) The response of HEK293-CD14 cells to araLAM and zymosan is inhibited by expression of a cytoplasmic deletion mutant of TLR1, TLR1-Δcyt. HEK293-CD14 cells were cotransfected with 5 ng TLR2-WT DNA and increasing amounts of TLR1-Δcyt (from 5 to 200 ng DNA). After 6 h of stimulation with 1 μg/ml araLAM and 10 μg/ml zymosan, cells were lysed and NFκB luciferase reporter gene activity was measured. IL-1β was used as a positive control.

Mentions: HEK293 cells constitutively express a cytoplasmic pool of TLR1 that can be detected by fluorescent staining analysis of permeabilized cells and TLR1 mRNA-specific RT-PCR (unpublished data). Transfection of these cells with wild-type TLR2 is sufficient to confer responsiveness to both araLAM and zymosan (Kurt-Jones et al., 2002). To investigate the role of TLR2 and TLR1 in response to araLAM and zymosan, various mutants of TLR2 and TLR1 proteins were generated. Based on a dominant-negative mutation of the TIR domain of TLR4 found in the C3H/HeJ mice (Poltorak et al., 1998; Hoshino et al., 1999), the corresponding homologous conserved proline within the TIR domain of TLR2 protein was mutated to histidine TLR2 P681H. A second TLR2 mutant was generated in which a stop codon was introduced at aa 643, resulting in the deletion of the conserved intracellular TIR domain TLR2ΔTIR. Co-transfection experiments were performed using increasing amounts of TLR2 mutant proteins with a constant amount of TLR2 wild-type protein. The response of transfected cells to araLAM and zymosan was determined. Transient transfection of HEK293 cells stably expressing CD14 on the surface (HEK293-CD14) with increasing amounts of TLR2 mutants resulted in dose-dependent impairment of NFκB activation in response to both araLAM and zymosan (Fig. 4, A and B).


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)

TLR2 and TLR1 mutants inhibit NFκB activation in HEK 293 cells in stimulation with araLAM and zymosan. (A) The response of HEK293-CD14 cells to araLAM and zymosan is inhibited by a single point mutated TLR2 construct, TLR2-P681H. HEK293-CD14 cells were cotransfected with 5 ng TLR2-WT DNA and increasing amounts of TLR2-P681H (from 5 to 200 ng DNA). After 6 h of stimulation with 1 μg/ml araLAM and 10 μg/ml zymosan, cells were lysed and NFκB luciferase reporter gene activity was measured. IL-1β was used as a positive control. Luciferase activity is expressed in normalized RLU as the ratio of NFκB-dependent firefly luciferase activity to NFκB-independent renilla luciferase activity. Data shown are the mean ± SD of triplicate wells. (B) The response of HEK293-CD14 cells to araLAM and zymosan is inhibited by expression of a TLR2 mutant missing the TIR domain TLR2-ΔTIR. HEK293-CD14 cells were cotransfected with 5 ng TLR2-WT DNA and increasing amounts of TLR2-ΔTIR (from 5 to 200 ng DNA). After 6 h of stimulation with 1 μg/ml araLAM and 10 μg/ml zymosan, cells were lysed and NFκB luciferase reporter gene activity was measured. IL-1β was used as a positive control. (C) The response of HEK293-CD14 cells to araLAM and zymosan is inhibited by expression of a cytoplasmic deletion mutant of TLR1, TLR1-Δcyt. HEK293-CD14 cells were cotransfected with 5 ng TLR2-WT DNA and increasing amounts of TLR1-Δcyt (from 5 to 200 ng DNA). After 6 h of stimulation with 1 μg/ml araLAM and 10 μg/ml zymosan, cells were lysed and NFκB luciferase reporter gene activity was measured. IL-1β was used as a positive control.
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fig4: TLR2 and TLR1 mutants inhibit NFκB activation in HEK 293 cells in stimulation with araLAM and zymosan. (A) The response of HEK293-CD14 cells to araLAM and zymosan is inhibited by a single point mutated TLR2 construct, TLR2-P681H. HEK293-CD14 cells were cotransfected with 5 ng TLR2-WT DNA and increasing amounts of TLR2-P681H (from 5 to 200 ng DNA). After 6 h of stimulation with 1 μg/ml araLAM and 10 μg/ml zymosan, cells were lysed and NFκB luciferase reporter gene activity was measured. IL-1β was used as a positive control. Luciferase activity is expressed in normalized RLU as the ratio of NFκB-dependent firefly luciferase activity to NFκB-independent renilla luciferase activity. Data shown are the mean ± SD of triplicate wells. (B) The response of HEK293-CD14 cells to araLAM and zymosan is inhibited by expression of a TLR2 mutant missing the TIR domain TLR2-ΔTIR. HEK293-CD14 cells were cotransfected with 5 ng TLR2-WT DNA and increasing amounts of TLR2-ΔTIR (from 5 to 200 ng DNA). After 6 h of stimulation with 1 μg/ml araLAM and 10 μg/ml zymosan, cells were lysed and NFκB luciferase reporter gene activity was measured. IL-1β was used as a positive control. (C) The response of HEK293-CD14 cells to araLAM and zymosan is inhibited by expression of a cytoplasmic deletion mutant of TLR1, TLR1-Δcyt. HEK293-CD14 cells were cotransfected with 5 ng TLR2-WT DNA and increasing amounts of TLR1-Δcyt (from 5 to 200 ng DNA). After 6 h of stimulation with 1 μg/ml araLAM and 10 μg/ml zymosan, cells were lysed and NFκB luciferase reporter gene activity was measured. IL-1β was used as a positive control.
Mentions: HEK293 cells constitutively express a cytoplasmic pool of TLR1 that can be detected by fluorescent staining analysis of permeabilized cells and TLR1 mRNA-specific RT-PCR (unpublished data). Transfection of these cells with wild-type TLR2 is sufficient to confer responsiveness to both araLAM and zymosan (Kurt-Jones et al., 2002). To investigate the role of TLR2 and TLR1 in response to araLAM and zymosan, various mutants of TLR2 and TLR1 proteins were generated. Based on a dominant-negative mutation of the TIR domain of TLR4 found in the C3H/HeJ mice (Poltorak et al., 1998; Hoshino et al., 1999), the corresponding homologous conserved proline within the TIR domain of TLR2 protein was mutated to histidine TLR2 P681H. A second TLR2 mutant was generated in which a stop codon was introduced at aa 643, resulting in the deletion of the conserved intracellular TIR domain TLR2ΔTIR. Co-transfection experiments were performed using increasing amounts of TLR2 mutant proteins with a constant amount of TLR2 wild-type protein. The response of transfected cells to araLAM and zymosan was determined. Transient transfection of HEK293 cells stably expressing CD14 on the surface (HEK293-CD14) with increasing amounts of TLR2 mutants resulted in dose-dependent impairment of NFκB activation in response to both araLAM and zymosan (Fig. 4, A and B).

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
Related in: MedlinePlus