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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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Neither the intracellular nor the extracellular domain of TLR2 is sufficient to confer NFκB signal activation. (A) HEK293-CD14 cells were transfected with either TLR [1–2] or TLR [2–1] DNA encoding chimeric protein and 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 separate TLR1 and TLR2 chimeric protein transfection. With TLR1 endogenously expressed, transfection only of the intracellular portion of TLR2 (TLR [1–2]) does not confer responsiveness as a result of missing TLR2 extracellular domain causing ligand recognition failure. Transfection with only the extracellular portion of TLR2 (TLR [2–1]) is not sufficient to confer responsiveness because the TLR1 intracellular domain lacks the TLR2 intracellular domain for effective initiation of signaling pathways. With TLR1 endogenously present, transfection of TLR2-WT protein confers responsiveness by providing both extra- and intracellular domains needed for ligand recognition and signal activation respectively.
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fig5: Neither the intracellular nor the extracellular domain of TLR2 is sufficient to confer NFκB signal activation. (A) HEK293-CD14 cells were transfected with either TLR [1–2] or TLR [2–1] DNA encoding chimeric protein and 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 separate TLR1 and TLR2 chimeric protein transfection. With TLR1 endogenously expressed, transfection only of the intracellular portion of TLR2 (TLR [1–2]) does not confer responsiveness as a result of missing TLR2 extracellular domain causing ligand recognition failure. Transfection with only the extracellular portion of TLR2 (TLR [2–1]) is not sufficient to confer responsiveness because the TLR1 intracellular domain lacks the TLR2 intracellular domain for effective initiation of signaling pathways. With TLR1 endogenously present, transfection of TLR2-WT protein confers responsiveness by providing both extra- and intracellular domains needed for ligand recognition and signal activation respectively.

Mentions: To further understand the role of TLR1 and TLR2 in ligand recognition, various types of TLR chimeric (fusion) proteins were generated by domain swapping of the extracellular and intracellular domains of TLR1, TLR2, and TLR4. Chimeric TLR proteins were transfected into HEK293-CD14 cells, and the response of the cells to TLR ligands was determined. The expression level of chimeric TLR protein constructs on the cell surface was comparable to the wild-type TLR protein expression (unpublished data). Cells transfected with a TLR [2–1] chimeric protein (consisting of the extracellular domain of the TLR2 fused to the intracellular domain of TLR1) did not respond to stimulation with araLAM, Pam3CSK4, and zymosan. Similarly, the reciprocal construct TLR [1–2] (with the extracellular domain of TLR1 fused to the intracellular domain of TLR2) was not able to elicit a response to any of these stimulants (Fig. 5, A and B). These results suggested that neither the extracellular domain of TLR2 nor the intracellular domain of TLR2 alone was sufficient to confer signaling in response to tested ligands. Similar results were obtained using TLR [1–4], TLR [2–3], TLR [4–1], and TLR [2–5] chimeric fusion proteins including their combinations (Table I).


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)

Neither the intracellular nor the extracellular domain of TLR2 is sufficient to confer NFκB signal activation. (A) HEK293-CD14 cells were transfected with either TLR [1–2] or TLR [2–1] DNA encoding chimeric protein and 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 separate TLR1 and TLR2 chimeric protein transfection. With TLR1 endogenously expressed, transfection only of the intracellular portion of TLR2 (TLR [1–2]) does not confer responsiveness as a result of missing TLR2 extracellular domain causing ligand recognition failure. Transfection with only the extracellular portion of TLR2 (TLR [2–1]) is not sufficient to confer responsiveness because the TLR1 intracellular domain lacks the TLR2 intracellular domain for effective initiation of signaling pathways. With TLR1 endogenously present, transfection of TLR2-WT protein confers responsiveness by providing both extra- and intracellular domains needed for ligand recognition and signal activation respectively.
© Copyright Policy
Related In: Results  -  Collection

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fig5: Neither the intracellular nor the extracellular domain of TLR2 is sufficient to confer NFκB signal activation. (A) HEK293-CD14 cells were transfected with either TLR [1–2] or TLR [2–1] DNA encoding chimeric protein and 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 separate TLR1 and TLR2 chimeric protein transfection. With TLR1 endogenously expressed, transfection only of the intracellular portion of TLR2 (TLR [1–2]) does not confer responsiveness as a result of missing TLR2 extracellular domain causing ligand recognition failure. Transfection with only the extracellular portion of TLR2 (TLR [2–1]) is not sufficient to confer responsiveness because the TLR1 intracellular domain lacks the TLR2 intracellular domain for effective initiation of signaling pathways. With TLR1 endogenously present, transfection of TLR2-WT protein confers responsiveness by providing both extra- and intracellular domains needed for ligand recognition and signal activation respectively.
Mentions: To further understand the role of TLR1 and TLR2 in ligand recognition, various types of TLR chimeric (fusion) proteins were generated by domain swapping of the extracellular and intracellular domains of TLR1, TLR2, and TLR4. Chimeric TLR proteins were transfected into HEK293-CD14 cells, and the response of the cells to TLR ligands was determined. The expression level of chimeric TLR protein constructs on the cell surface was comparable to the wild-type TLR protein expression (unpublished data). Cells transfected with a TLR [2–1] chimeric protein (consisting of the extracellular domain of the TLR2 fused to the intracellular domain of TLR1) did not respond to stimulation with araLAM, Pam3CSK4, and zymosan. Similarly, the reciprocal construct TLR [1–2] (with the extracellular domain of TLR1 fused to the intracellular domain of TLR2) was not able to elicit a response to any of these stimulants (Fig. 5, A and B). These results suggested that neither the extracellular domain of TLR2 nor the intracellular domain of TLR2 alone was sufficient to confer signaling in response to tested ligands. Similar results were obtained using TLR [1–4], TLR [2–3], TLR [4–1], and TLR [2–5] chimeric fusion proteins including their combinations (Table I).

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