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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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Confocal imaging of live HEK cells expressing fluorescent protein–tagged TLR1, TLR2, and TLR4. (A) Confocal microscopy of HEK cells stably expressing TLR1YFP, TLR2YFP, or TLR4YFP. HEK cells stably expressing either TLR1YFP, TLR2YFP, or TLR4YFP were grown on glass-bottom tissue culture dishes, and living cells were analyzed by confocal microscopy at 37°C without any manipulation. Shown are confocal sections in representative cells. (B) TLR1 colocalizes with TLR2 in resting HEK cells stably expressing both TLR1YFP and TLR2CFP. HEK cells stably expressing both TLR1YFP and TLR2CFP were grown on glass-bottom tissue culture dishes, and living cells were analyzed as described in A. Left, Localization of TLR2; center, distribution of TLR1; right, overlay of the left and middle images. In these cells without any manipulation and without ligand stimulation, TLR1 colocalizes with TLR2 (arrows and arrowheads). Representative confocal sections of cells are shown. (C) Surface cross-linking of TLR2CFP by antibody leads to coaggregation of TLR1YFP on the cell surface. Double transfectants of TLR2CFP and TLR1YFP were surface stained with anti-TLR2 antibody (clone TL2.1), and further cross-linked with Alexa® 647–conjugated polyclonal rabbit anti–mouse antibody (2° AbAlexa674, shown in blue) on ice. After incubation for 10 min at 37°C to induce capping, living cells were analyzed by confocal microscopy, and representative cells are shown. Left, distribution (green) and surface labeling of TLR2CFP with anti-TLR2 + 2° AbAlexa674 (blue); middle, TLR1YFP distribution (red) and the overlay image of all three colors. The transmitted light image and the fluorescence intensities of a representative section of the overlay image is shown in the right panels. Surface cross-linking of TLR2CFP by antibody (blue) induced coaggregation of TLR2CFP and TLR1YFP. (D) Antibody-induced capping of MHC I does not induce coaggregation with TLR2CFP or TLR1YFP. HEK cells stably expressing TLR2CFP and TLR1YFP were stained for MHC I using a mouse monoclonal anti-HLA I antibody, counterstained and processed as described in B. Distribution of TLR2CFP (green) and surface labeling of MHC class I with anti-MHC I + 2° AbAlexa674 (blue) are shown in the left panels; the middle panels demonstrate TLR1YFP localization (red) and the overlay of all three colors. The transmitted light image and fluorescence intensities of a representative section of the overlay image are displayed in the right panels. MHC I cross-linking (blue) does not co-patch either TLR2CFP or TLR1YFP. (E) Antibody-induced capping of TLR2 does not coaggregate TLR4. Stably transfected HEK-TLR4YFP cells (green) were transiently transfected with TLR2. Antibody-induced capping of TLR2 was induced by sequentially incubating the cells with anti-TLR2 (clone TL2.1) and anti-mouse IgG (Alexa® 647 conjugated; red). Aggregation of TLR2 did not influence the expression pattern of TLR4.
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fig3: Confocal imaging of live HEK cells expressing fluorescent protein–tagged TLR1, TLR2, and TLR4. (A) Confocal microscopy of HEK cells stably expressing TLR1YFP, TLR2YFP, or TLR4YFP. HEK cells stably expressing either TLR1YFP, TLR2YFP, or TLR4YFP were grown on glass-bottom tissue culture dishes, and living cells were analyzed by confocal microscopy at 37°C without any manipulation. Shown are confocal sections in representative cells. (B) TLR1 colocalizes with TLR2 in resting HEK cells stably expressing both TLR1YFP and TLR2CFP. HEK cells stably expressing both TLR1YFP and TLR2CFP were grown on glass-bottom tissue culture dishes, and living cells were analyzed as described in A. Left, Localization of TLR2; center, distribution of TLR1; right, overlay of the left and middle images. In these cells without any manipulation and without ligand stimulation, TLR1 colocalizes with TLR2 (arrows and arrowheads). Representative confocal sections of cells are shown. (C) Surface cross-linking of TLR2CFP by antibody leads to coaggregation of TLR1YFP on the cell surface. Double transfectants of TLR2CFP and TLR1YFP were surface stained with anti-TLR2 antibody (clone TL2.1), and further cross-linked with Alexa® 647–conjugated polyclonal rabbit anti–mouse antibody (2° AbAlexa674, shown in blue) on ice. After incubation for 10 min at 37°C to induce capping, living cells were analyzed by confocal microscopy, and representative cells are shown. Left, distribution (green) and surface labeling of TLR2CFP with anti-TLR2 + 2° AbAlexa674 (blue); middle, TLR1YFP distribution (red) and the overlay image of all three colors. The transmitted light image and the fluorescence intensities of a representative section of the overlay image is shown in the right panels. Surface cross-linking of TLR2CFP by antibody (blue) induced coaggregation of TLR2CFP and TLR1YFP. (D) Antibody-induced capping of MHC I does not induce coaggregation with TLR2CFP or TLR1YFP. HEK cells stably expressing TLR2CFP and TLR1YFP were stained for MHC I using a mouse monoclonal anti-HLA I antibody, counterstained and processed as described in B. Distribution of TLR2CFP (green) and surface labeling of MHC class I with anti-MHC I + 2° AbAlexa674 (blue) are shown in the left panels; the middle panels demonstrate TLR1YFP localization (red) and the overlay of all three colors. The transmitted light image and fluorescence intensities of a representative section of the overlay image are displayed in the right panels. MHC I cross-linking (blue) does not co-patch either TLR2CFP or TLR1YFP. (E) Antibody-induced capping of TLR2 does not coaggregate TLR4. Stably transfected HEK-TLR4YFP cells (green) were transiently transfected with TLR2. Antibody-induced capping of TLR2 was induced by sequentially incubating the cells with anti-TLR2 (clone TL2.1) and anti-mouse IgG (Alexa® 647 conjugated; red). Aggregation of TLR2 did not influence the expression pattern of TLR4.

Mentions: Cells transfected with TLR2YFP or TLR4YFP fusion proteins alone displayed a predominant membrane localization of the receptors. In contrast, TLR1YFP-expressing cells displayed a diffuse pattern of TLR1 distribution (Fig. 3 A). Co-transfection of cells with TLR1YFP and TLR2CFP fusion proteins resulted in aggregation of TLR1 and TLR2 both on the surface and inside the cells (Fig. 3 B). Antibody-induced aggregation of TLR2 on the surface of the cells led to coaggregation of TLR1 (Fig. 3 C), whereas antibody-induced surface aggregation of MHC I did not coaggregate TLR1 or TLR2 (Fig. 3 D). As a further control, we surface aggregated TLR2 in TLR4YFP-expressing cells. Capping of TLR2 (Fig. 3 E, red) did not lead to coaggregation of TLR4 (Fig. 3 E, green), indicating the specificity of the observed coaggregation of TLR2 and TLR1. These results suggest that TLR1 and TLR2 are preassembled into heteromultimeric complexes on the cell surface.


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)

Confocal imaging of live HEK cells expressing fluorescent protein–tagged TLR1, TLR2, and TLR4. (A) Confocal microscopy of HEK cells stably expressing TLR1YFP, TLR2YFP, or TLR4YFP. HEK cells stably expressing either TLR1YFP, TLR2YFP, or TLR4YFP were grown on glass-bottom tissue culture dishes, and living cells were analyzed by confocal microscopy at 37°C without any manipulation. Shown are confocal sections in representative cells. (B) TLR1 colocalizes with TLR2 in resting HEK cells stably expressing both TLR1YFP and TLR2CFP. HEK cells stably expressing both TLR1YFP and TLR2CFP were grown on glass-bottom tissue culture dishes, and living cells were analyzed as described in A. Left, Localization of TLR2; center, distribution of TLR1; right, overlay of the left and middle images. In these cells without any manipulation and without ligand stimulation, TLR1 colocalizes with TLR2 (arrows and arrowheads). Representative confocal sections of cells are shown. (C) Surface cross-linking of TLR2CFP by antibody leads to coaggregation of TLR1YFP on the cell surface. Double transfectants of TLR2CFP and TLR1YFP were surface stained with anti-TLR2 antibody (clone TL2.1), and further cross-linked with Alexa® 647–conjugated polyclonal rabbit anti–mouse antibody (2° AbAlexa674, shown in blue) on ice. After incubation for 10 min at 37°C to induce capping, living cells were analyzed by confocal microscopy, and representative cells are shown. Left, distribution (green) and surface labeling of TLR2CFP with anti-TLR2 + 2° AbAlexa674 (blue); middle, TLR1YFP distribution (red) and the overlay image of all three colors. The transmitted light image and the fluorescence intensities of a representative section of the overlay image is shown in the right panels. Surface cross-linking of TLR2CFP by antibody (blue) induced coaggregation of TLR2CFP and TLR1YFP. (D) Antibody-induced capping of MHC I does not induce coaggregation with TLR2CFP or TLR1YFP. HEK cells stably expressing TLR2CFP and TLR1YFP were stained for MHC I using a mouse monoclonal anti-HLA I antibody, counterstained and processed as described in B. Distribution of TLR2CFP (green) and surface labeling of MHC class I with anti-MHC I + 2° AbAlexa674 (blue) are shown in the left panels; the middle panels demonstrate TLR1YFP localization (red) and the overlay of all three colors. The transmitted light image and fluorescence intensities of a representative section of the overlay image are displayed in the right panels. MHC I cross-linking (blue) does not co-patch either TLR2CFP or TLR1YFP. (E) Antibody-induced capping of TLR2 does not coaggregate TLR4. Stably transfected HEK-TLR4YFP cells (green) were transiently transfected with TLR2. Antibody-induced capping of TLR2 was induced by sequentially incubating the cells with anti-TLR2 (clone TL2.1) and anti-mouse IgG (Alexa® 647 conjugated; red). Aggregation of TLR2 did not influence the expression pattern of TLR4.
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fig3: Confocal imaging of live HEK cells expressing fluorescent protein–tagged TLR1, TLR2, and TLR4. (A) Confocal microscopy of HEK cells stably expressing TLR1YFP, TLR2YFP, or TLR4YFP. HEK cells stably expressing either TLR1YFP, TLR2YFP, or TLR4YFP were grown on glass-bottom tissue culture dishes, and living cells were analyzed by confocal microscopy at 37°C without any manipulation. Shown are confocal sections in representative cells. (B) TLR1 colocalizes with TLR2 in resting HEK cells stably expressing both TLR1YFP and TLR2CFP. HEK cells stably expressing both TLR1YFP and TLR2CFP were grown on glass-bottom tissue culture dishes, and living cells were analyzed as described in A. Left, Localization of TLR2; center, distribution of TLR1; right, overlay of the left and middle images. In these cells without any manipulation and without ligand stimulation, TLR1 colocalizes with TLR2 (arrows and arrowheads). Representative confocal sections of cells are shown. (C) Surface cross-linking of TLR2CFP by antibody leads to coaggregation of TLR1YFP on the cell surface. Double transfectants of TLR2CFP and TLR1YFP were surface stained with anti-TLR2 antibody (clone TL2.1), and further cross-linked with Alexa® 647–conjugated polyclonal rabbit anti–mouse antibody (2° AbAlexa674, shown in blue) on ice. After incubation for 10 min at 37°C to induce capping, living cells were analyzed by confocal microscopy, and representative cells are shown. Left, distribution (green) and surface labeling of TLR2CFP with anti-TLR2 + 2° AbAlexa674 (blue); middle, TLR1YFP distribution (red) and the overlay image of all three colors. The transmitted light image and the fluorescence intensities of a representative section of the overlay image is shown in the right panels. Surface cross-linking of TLR2CFP by antibody (blue) induced coaggregation of TLR2CFP and TLR1YFP. (D) Antibody-induced capping of MHC I does not induce coaggregation with TLR2CFP or TLR1YFP. HEK cells stably expressing TLR2CFP and TLR1YFP were stained for MHC I using a mouse monoclonal anti-HLA I antibody, counterstained and processed as described in B. Distribution of TLR2CFP (green) and surface labeling of MHC class I with anti-MHC I + 2° AbAlexa674 (blue) are shown in the left panels; the middle panels demonstrate TLR1YFP localization (red) and the overlay of all three colors. The transmitted light image and fluorescence intensities of a representative section of the overlay image are displayed in the right panels. MHC I cross-linking (blue) does not co-patch either TLR2CFP or TLR1YFP. (E) Antibody-induced capping of TLR2 does not coaggregate TLR4. Stably transfected HEK-TLR4YFP cells (green) were transiently transfected with TLR2. Antibody-induced capping of TLR2 was induced by sequentially incubating the cells with anti-TLR2 (clone TL2.1) and anti-mouse IgG (Alexa® 647 conjugated; red). Aggregation of TLR2 did not influence the expression pattern of TLR4.
Mentions: Cells transfected with TLR2YFP or TLR4YFP fusion proteins alone displayed a predominant membrane localization of the receptors. In contrast, TLR1YFP-expressing cells displayed a diffuse pattern of TLR1 distribution (Fig. 3 A). Co-transfection of cells with TLR1YFP and TLR2CFP fusion proteins resulted in aggregation of TLR1 and TLR2 both on the surface and inside the cells (Fig. 3 B). Antibody-induced aggregation of TLR2 on the surface of the cells led to coaggregation of TLR1 (Fig. 3 C), whereas antibody-induced surface aggregation of MHC I did not coaggregate TLR1 or TLR2 (Fig. 3 D). As a further control, we surface aggregated TLR2 in TLR4YFP-expressing cells. Capping of TLR2 (Fig. 3 E, red) did not lead to coaggregation of TLR4 (Fig. 3 E, green), indicating the specificity of the observed coaggregation of TLR2 and TLR1. These results suggest that TLR1 and TLR2 are preassembled into heteromultimeric complexes on the cell surface.

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