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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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Ligand-induced NF-κB activation are dependent on a three–amino acid motif that is required for membrane association. (A, 1) Amino acid sequences of Flag-NOD2 COOH-terminal substitution mutants. (2) Expression of these mutants was determined by Western blot analysis using NOD2 antiserum HM2563. (3) Fold increase of NF-κB activation was determined as described in Fig. 4. (B, 1) GFP-NOD2 wild type and the three main NOD2 mutations that are associated with CD (GFP-NOD2 3020insC, GFP-NOD2 R702W, and GFP-NOD2 G908R) were transfected in COS7 cells. Only NOD2 3020insC failed to colocalize with the plasma membrane, whereas the two other NOD2 mutant forms still showed membrane association (arrows). Bar, 20 μm. (2) Expression of NOD2 mutants and NF-κB activation were determined and compared with untransfected and nonstimulated HEK293 with MDP-LD, as described in Fig. 4. Error bars represent SEM of at least four separate experiments. *, P < 0.05.
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fig5: Ligand-induced NF-κB activation are dependent on a three–amino acid motif that is required for membrane association. (A, 1) Amino acid sequences of Flag-NOD2 COOH-terminal substitution mutants. (2) Expression of these mutants was determined by Western blot analysis using NOD2 antiserum HM2563. (3) Fold increase of NF-κB activation was determined as described in Fig. 4. (B, 1) GFP-NOD2 wild type and the three main NOD2 mutations that are associated with CD (GFP-NOD2 3020insC, GFP-NOD2 R702W, and GFP-NOD2 G908R) were transfected in COS7 cells. Only NOD2 3020insC failed to colocalize with the plasma membrane, whereas the two other NOD2 mutant forms still showed membrane association (arrows). Bar, 20 μm. (2) Expression of NOD2 mutants and NF-κB activation were determined and compared with untransfected and nonstimulated HEK293 with MDP-LD, as described in Fig. 4. Error bars represent SEM of at least four separate experiments. *, P < 0.05.

Mentions: To determine which amino acid residues in the WLR motif are required for MDP recognition, we constructed three additional mutants (Fig. 5 A). NF-κB activation showed that both W1017G (mutant 13) and L1018G (mutant 14) play vital roles in MDP-LD recognition because NF-κB activation is significantly decreased compared with wild-type NOD2. However, R1019G substitution (mutant 15) had no effect on NOD2 MDP-LD recognition. These results were confirmed by determining the amounts of IL-8 released in the supernatant of HEK293 cells that were transfected with these mutants (unpublished data). Substitutions W1017G and L1018G led only to a twofold decrease of NF-κB, whereas WLR1017GGG substitutions had impaired NF-κB activation, indicating that both W and L amino acid residues are important but not sufficient to completely abrogate NOD2 function. However, the two other NOD2 mutants that are associated with CD—NOD2 R702W and NOD2 G908R—still showed membrane association in transfected COS7 cells, whereas these two mutants showed decreased levels of NF-κB activation after MDP-LD stimulation. This suggested that these two amino acids are required for MDP-LD recognition but did not play any role in the membrane targeting of NOD2 (Fig. 5 B).


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)

Ligand-induced NF-κB activation are dependent on a three–amino acid motif that is required for membrane association. (A, 1) Amino acid sequences of Flag-NOD2 COOH-terminal substitution mutants. (2) Expression of these mutants was determined by Western blot analysis using NOD2 antiserum HM2563. (3) Fold increase of NF-κB activation was determined as described in Fig. 4. (B, 1) GFP-NOD2 wild type and the three main NOD2 mutations that are associated with CD (GFP-NOD2 3020insC, GFP-NOD2 R702W, and GFP-NOD2 G908R) were transfected in COS7 cells. Only NOD2 3020insC failed to colocalize with the plasma membrane, whereas the two other NOD2 mutant forms still showed membrane association (arrows). Bar, 20 μm. (2) Expression of NOD2 mutants and NF-κB activation were determined and compared with untransfected and nonstimulated HEK293 with MDP-LD, as described in Fig. 4. Error bars represent SEM of at least four separate experiments. *, P < 0.05.
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Related In: Results  -  Collection

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fig5: Ligand-induced NF-κB activation are dependent on a three–amino acid motif that is required for membrane association. (A, 1) Amino acid sequences of Flag-NOD2 COOH-terminal substitution mutants. (2) Expression of these mutants was determined by Western blot analysis using NOD2 antiserum HM2563. (3) Fold increase of NF-κB activation was determined as described in Fig. 4. (B, 1) GFP-NOD2 wild type and the three main NOD2 mutations that are associated with CD (GFP-NOD2 3020insC, GFP-NOD2 R702W, and GFP-NOD2 G908R) were transfected in COS7 cells. Only NOD2 3020insC failed to colocalize with the plasma membrane, whereas the two other NOD2 mutant forms still showed membrane association (arrows). Bar, 20 μm. (2) Expression of NOD2 mutants and NF-κB activation were determined and compared with untransfected and nonstimulated HEK293 with MDP-LD, as described in Fig. 4. Error bars represent SEM of at least four separate experiments. *, P < 0.05.
Mentions: To determine which amino acid residues in the WLR motif are required for MDP recognition, we constructed three additional mutants (Fig. 5 A). NF-κB activation showed that both W1017G (mutant 13) and L1018G (mutant 14) play vital roles in MDP-LD recognition because NF-κB activation is significantly decreased compared with wild-type NOD2. However, R1019G substitution (mutant 15) had no effect on NOD2 MDP-LD recognition. These results were confirmed by determining the amounts of IL-8 released in the supernatant of HEK293 cells that were transfected with these mutants (unpublished data). Substitutions W1017G and L1018G led only to a twofold decrease of NF-κB, whereas WLR1017GGG substitutions had impaired NF-κB activation, indicating that both W and L amino acid residues are important but not sufficient to completely abrogate NOD2 function. However, the two other NOD2 mutants that are associated with CD—NOD2 R702W and NOD2 G908R—still showed membrane association in transfected COS7 cells, whereas these two mutants showed decreased levels of NF-κB activation after MDP-LD stimulation. This suggested that these two amino acids are required for MDP-LD recognition but did not play any role in the membrane targeting of NOD2 (Fig. 5 B).

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