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Membrane recruitment of NOD2 in intestinal epithelial cells is essential for nuclear factor-{kappa}B activation in muramyl dipeptide recognition.

Barnich N, Aguirre JE, Reinecker HC, Xavier R, Podolsky DK - J. Cell Biol. (2005)

Bottom Line: To gain insight into the molecular mechanisms of NOD2 function, we performed a functional analysis of deletion and substitution NOD2 mutants.Membrane targeting and subsequent NF-kappaB activation are mediated by two leucine residues and a tryptophan-containing motif in the COOH-terminal domain of NOD2.The membrane targeting of NOD2 is required for NF-kappaB activation after the recognition of bacterial muramyl dipeptide in intestinal epithelial cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Center for the Study of Inflammatory Bowel Disease, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA.

ABSTRACT
Nucleotide oligomerization domain (NOD) 2 functions as a mammalian cytosolic pathogen recognition molecule, and mutant forms have been genetically linked to Crohn's disease (CD). NOD2 associates with the caspase activation and recruitment domain of RIP-like interacting caspase-like apoptosis regulatory protein kinase (RICK)/RIP2 and activates nuclear factor (NF)-kappaB in epithelial cells and macrophages, whereas NOD2 mutant 3020insC, which is associated with CD, shows an impaired ability to activate NF-kappaB. To gain insight into the molecular mechanisms of NOD2 function, we performed a functional analysis of deletion and substitution NOD2 mutants. NOD2, but not NOD2 3020insC mutant, associated with cell surface membranes of intestinal epithelial cells. Membrane targeting and subsequent NF-kappaB activation are mediated by two leucine residues and a tryptophan-containing motif in the COOH-terminal domain of NOD2. The membrane targeting of NOD2 is required for NF-kappaB activation after the recognition of bacterial muramyl dipeptide in intestinal epithelial cells.

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NOD2 membrane association is COOH-terminal dependent. (A) Sequences of Flag-NOD2 COOH-terminal deletion and substitution mutants. (B) Caco-2 cells were transfected with Flag-NOD2 mutant constructs. Mutants 1–12 were detected by confocal microscopy using monoclonal anti-Flag antibody followed by fluorescein-conjugated anti–mouse IgG. Membrane association is still observed for some NOD2 mutants (arrows). (C) COS7 cells were transfected with GFP-NOD2 3020insC and were stained with calnexin (ER marker). Some of the observed vesicles colocalized with calnexin (arrows). Bars, 20 μm.
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fig3: NOD2 membrane association is COOH-terminal dependent. (A) Sequences of Flag-NOD2 COOH-terminal deletion and substitution mutants. (B) Caco-2 cells were transfected with Flag-NOD2 mutant constructs. Mutants 1–12 were detected by confocal microscopy using monoclonal anti-Flag antibody followed by fluorescein-conjugated anti–mouse IgG. Membrane association is still observed for some NOD2 mutants (arrows). (C) COS7 cells were transfected with GFP-NOD2 3020insC and were stained with calnexin (ER marker). Some of the observed vesicles colocalized with calnexin (arrows). Bars, 20 μm.

Mentions: The difference between NOD2 wild type and NOD2 3020insC mutant is the deletion of the final 33 COOH-terminal amino acid residues, indicating that this region can be responsible for membrane targeting. The domain of NOD2 that is responsible for membrane targeting was more specifically identified by serial deletion mutants (Fig. 3 A). Mutants 1 and 2 failed to localize to the cell membranes in Caco-2 cells (Fig. 3 B), indicating that the terminal six amino acid residues of NOD2 are required for membrane targeting. Mutants 3 and 4 were constructed by adding selected COOH-terminal amino acids (last 6 and 17 amino acid residues of NOD2) to the NOD2 3020insC mutant to determine whether these amino acids were sufficient to confer NOD2 membrane association. No membrane targeting in Caco-2 cells that were transfected with these two mutant forms was observed. Instead, these NOD2 mutants were targeted to an intracellular vesicle compartment (Fig. 3 B). Therefore, the last six amino acid residues of NOD2 are necessary but not sufficient to confer membrane targeting. Finally, the deletion of LERNDILE (mutant 5) and VWLRGNTF (mutant 6), which are located in the COOH-terminal domain of NOD2, also resulted in the loss of membrane targeting in transfected Caco-2 cells (Fig. 3 B). The identical distribution of all of these mutant forms of NOD2 was observed when transfected into COS7 cells, including the concomitant loss of membrane targeting (unpublished data) compared with wild-type NOD2, which indicates that the final 33 amino acids play a crucial role for NOD2 membrane recruitment.


Membrane recruitment of NOD2 in intestinal epithelial cells is essential for nuclear factor-{kappa}B activation in muramyl dipeptide recognition.

Barnich N, Aguirre JE, Reinecker HC, Xavier R, Podolsky DK - J. Cell Biol. (2005)

NOD2 membrane association is COOH-terminal dependent. (A) Sequences of Flag-NOD2 COOH-terminal deletion and substitution mutants. (B) Caco-2 cells were transfected with Flag-NOD2 mutant constructs. Mutants 1–12 were detected by confocal microscopy using monoclonal anti-Flag antibody followed by fluorescein-conjugated anti–mouse IgG. Membrane association is still observed for some NOD2 mutants (arrows). (C) COS7 cells were transfected with GFP-NOD2 3020insC and were stained with calnexin (ER marker). Some of the observed vesicles colocalized with calnexin (arrows). Bars, 20 μm.
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Related In: Results  -  Collection

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fig3: NOD2 membrane association is COOH-terminal dependent. (A) Sequences of Flag-NOD2 COOH-terminal deletion and substitution mutants. (B) Caco-2 cells were transfected with Flag-NOD2 mutant constructs. Mutants 1–12 were detected by confocal microscopy using monoclonal anti-Flag antibody followed by fluorescein-conjugated anti–mouse IgG. Membrane association is still observed for some NOD2 mutants (arrows). (C) COS7 cells were transfected with GFP-NOD2 3020insC and were stained with calnexin (ER marker). Some of the observed vesicles colocalized with calnexin (arrows). Bars, 20 μm.
Mentions: The difference between NOD2 wild type and NOD2 3020insC mutant is the deletion of the final 33 COOH-terminal amino acid residues, indicating that this region can be responsible for membrane targeting. The domain of NOD2 that is responsible for membrane targeting was more specifically identified by serial deletion mutants (Fig. 3 A). Mutants 1 and 2 failed to localize to the cell membranes in Caco-2 cells (Fig. 3 B), indicating that the terminal six amino acid residues of NOD2 are required for membrane targeting. Mutants 3 and 4 were constructed by adding selected COOH-terminal amino acids (last 6 and 17 amino acid residues of NOD2) to the NOD2 3020insC mutant to determine whether these amino acids were sufficient to confer NOD2 membrane association. No membrane targeting in Caco-2 cells that were transfected with these two mutant forms was observed. Instead, these NOD2 mutants were targeted to an intracellular vesicle compartment (Fig. 3 B). Therefore, the last six amino acid residues of NOD2 are necessary but not sufficient to confer membrane targeting. Finally, the deletion of LERNDILE (mutant 5) and VWLRGNTF (mutant 6), which are located in the COOH-terminal domain of NOD2, also resulted in the loss of membrane targeting in transfected Caco-2 cells (Fig. 3 B). The identical distribution of all of these mutant forms of NOD2 was observed when transfected into COS7 cells, including the concomitant loss of membrane targeting (unpublished data) compared with wild-type NOD2, which indicates that the final 33 amino acids play a crucial role for NOD2 membrane recruitment.

Bottom Line: To gain insight into the molecular mechanisms of NOD2 function, we performed a functional analysis of deletion and substitution NOD2 mutants.Membrane targeting and subsequent NF-kappaB activation are mediated by two leucine residues and a tryptophan-containing motif in the COOH-terminal domain of NOD2.The membrane targeting of NOD2 is required for NF-kappaB activation after the recognition of bacterial muramyl dipeptide in intestinal epithelial cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Center for the Study of Inflammatory Bowel Disease, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA.

ABSTRACT
Nucleotide oligomerization domain (NOD) 2 functions as a mammalian cytosolic pathogen recognition molecule, and mutant forms have been genetically linked to Crohn's disease (CD). NOD2 associates with the caspase activation and recruitment domain of RIP-like interacting caspase-like apoptosis regulatory protein kinase (RICK)/RIP2 and activates nuclear factor (NF)-kappaB in epithelial cells and macrophages, whereas NOD2 mutant 3020insC, which is associated with CD, shows an impaired ability to activate NF-kappaB. To gain insight into the molecular mechanisms of NOD2 function, we performed a functional analysis of deletion and substitution NOD2 mutants. NOD2, but not NOD2 3020insC mutant, associated with cell surface membranes of intestinal epithelial cells. Membrane targeting and subsequent NF-kappaB activation are mediated by two leucine residues and a tryptophan-containing motif in the COOH-terminal domain of NOD2. The membrane targeting of NOD2 is required for NF-kappaB activation after the recognition of bacterial muramyl dipeptide in intestinal epithelial cells.

Show MeSH
Related in: MedlinePlus