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Related leucine-based cytoplasmic targeting signals in invariant chain and major histocompatibility complex class II molecules control endocytic presentation of distinct determinants in a single protein.

Zhong G, Romagnoli P, Germain RN - J. Exp. Med. (1997)

Bottom Line: Some protein determinants, however, do not require Ii for effective class II presentation, although endocytic processing is still necessary.Elimination or mutation of this signal reduces the rate of re-entry of mature surface class II molecules into the endocytic pathway.This permits capture of protein fragments produced optimally under distinct conditions of pH and proteolytic activity.

View Article: PubMed Central - PubMed

Affiliation: Lymphocyte Biology Section, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, Maryland 20892-1892, USA.

ABSTRACT
Leucine-based signals in the cytoplasmic tail of invariant chain (Ii) control targeting of newly synthesized major histocompatibility complex class II molecules to the endocytic pathway for acquisition of antigenic peptides. Some protein determinants, however, do not require Ii for effective class II presentation, although endocytic processing is still necessary. Here we demonstrate that a dileucine-based signal in the cytoplasmic tail of the class II beta chain is critical for this Ii-independent presentation. Elimination or mutation of this signal reduces the rate of re-entry of mature surface class II molecules into the endocytic pathway. Antigen presentation controlled by this signal does not require newly synthesized class II molecules and appears to involve determinants requiring only limited proteolysis for exposure, whereas the opposite is true for li-dependent determinants. This demonstrates that related leucine-based trafficking signals in li and class II control the functional presentation of protein determinants with distinct processing requirements, suggesting that the peptide binding sites of newly synthesized versus mature class II molecules are made available for antigen binding in distinct endocytic compartments under the control of these homologous cytoplasmic signals. This permits capture of protein fragments produced optimally under distinct conditions of pH and proteolytic activity.

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Mutation of the  conserved LL sequence at the  COOH terminus of the Aβ  chain cytoplasmic tail eliminates  the ability of this protein to mediate Ii-independent presentation  of either the 34-45 or 116-129  determinants of HEL. RBLs stably transfected with constructs  encoding the indicated Aαk and  Aβk proteins with or without Ii  p31 were used as APCs for  mouse T hybridomas specific for  the indicated determinants. IL-2  production at 24 h is presented as  OD obtained using an IL-2–specific capture ELISA. The data  represent one of three independent experiments.
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Figure 4: Mutation of the conserved LL sequence at the COOH terminus of the Aβ chain cytoplasmic tail eliminates the ability of this protein to mediate Ii-independent presentation of either the 34-45 or 116-129 determinants of HEL. RBLs stably transfected with constructs encoding the indicated Aαk and Aβk proteins with or without Ii p31 were used as APCs for mouse T hybridomas specific for the indicated determinants. IL-2 production at 24 h is presented as OD obtained using an IL-2–specific capture ELISA. The data represent one of three independent experiments.

Mentions: In discussing their evidence that the class II β chain tail contributes to effective antigen presentation, Smiley et al. have noted the existence of a conserved dileucine in this segment of the protein, and suggested that it may be part of a targeting motif (32). We have independently compared the sequences of multiple class II β chains from various species, looking for highly conserved sequences and also for sequences resembling either the known leucine- or tyrosine-containing signals mediating endocytic protein targeting. As shown in Table 1, like Smiley et al., we identified the same conserved dileucine near the COOH terminus of the β chain, preceded by an invariant glycine, giving rise to the sequence GLL that closely resembles one of the two localization signals in Ii (GLI in the mouse). This sequence in class II is also spaced only one residue further from the end of the transmembrane domain as compared to the comparable Ii sequence; previous studies have shown that the distance of a signal from the membrane can play an important role in its traffic control function (46). To test directly whether these dileucines are a critical part of a functional targeting motif in the class II tail, a mutant cDNA was prepared that encodes a complete Aβk chain except for substitution of the two cytoplasmic leucines with alanines (βCTAA). This construct was stably transfected into RBLs along with plasmids encoding wild-type Aαk chains, with or without the construct for Ii. The ability of the transfected cells to present the three HEL determinants was compared to the wild-type Ak transfectants (Fig. 4). Like deletion of the entire β chain tail, substitution of the two leucines with alanine prevents the presentation of 34-45 and 116-129 in cells lacking Ii, and Ii coexpression with class II containing βCTAA can rescue presentation of 34-45 but not 116-129.


Related leucine-based cytoplasmic targeting signals in invariant chain and major histocompatibility complex class II molecules control endocytic presentation of distinct determinants in a single protein.

Zhong G, Romagnoli P, Germain RN - J. Exp. Med. (1997)

Mutation of the  conserved LL sequence at the  COOH terminus of the Aβ  chain cytoplasmic tail eliminates  the ability of this protein to mediate Ii-independent presentation  of either the 34-45 or 116-129  determinants of HEL. RBLs stably transfected with constructs  encoding the indicated Aαk and  Aβk proteins with or without Ii  p31 were used as APCs for  mouse T hybridomas specific for  the indicated determinants. IL-2  production at 24 h is presented as  OD obtained using an IL-2–specific capture ELISA. The data  represent one of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196034&req=5

Figure 4: Mutation of the conserved LL sequence at the COOH terminus of the Aβ chain cytoplasmic tail eliminates the ability of this protein to mediate Ii-independent presentation of either the 34-45 or 116-129 determinants of HEL. RBLs stably transfected with constructs encoding the indicated Aαk and Aβk proteins with or without Ii p31 were used as APCs for mouse T hybridomas specific for the indicated determinants. IL-2 production at 24 h is presented as OD obtained using an IL-2–specific capture ELISA. The data represent one of three independent experiments.
Mentions: In discussing their evidence that the class II β chain tail contributes to effective antigen presentation, Smiley et al. have noted the existence of a conserved dileucine in this segment of the protein, and suggested that it may be part of a targeting motif (32). We have independently compared the sequences of multiple class II β chains from various species, looking for highly conserved sequences and also for sequences resembling either the known leucine- or tyrosine-containing signals mediating endocytic protein targeting. As shown in Table 1, like Smiley et al., we identified the same conserved dileucine near the COOH terminus of the β chain, preceded by an invariant glycine, giving rise to the sequence GLL that closely resembles one of the two localization signals in Ii (GLI in the mouse). This sequence in class II is also spaced only one residue further from the end of the transmembrane domain as compared to the comparable Ii sequence; previous studies have shown that the distance of a signal from the membrane can play an important role in its traffic control function (46). To test directly whether these dileucines are a critical part of a functional targeting motif in the class II tail, a mutant cDNA was prepared that encodes a complete Aβk chain except for substitution of the two cytoplasmic leucines with alanines (βCTAA). This construct was stably transfected into RBLs along with plasmids encoding wild-type Aαk chains, with or without the construct for Ii. The ability of the transfected cells to present the three HEL determinants was compared to the wild-type Ak transfectants (Fig. 4). Like deletion of the entire β chain tail, substitution of the two leucines with alanine prevents the presentation of 34-45 and 116-129 in cells lacking Ii, and Ii coexpression with class II containing βCTAA can rescue presentation of 34-45 but not 116-129.

Bottom Line: Some protein determinants, however, do not require Ii for effective class II presentation, although endocytic processing is still necessary.Elimination or mutation of this signal reduces the rate of re-entry of mature surface class II molecules into the endocytic pathway.This permits capture of protein fragments produced optimally under distinct conditions of pH and proteolytic activity.

View Article: PubMed Central - PubMed

Affiliation: Lymphocyte Biology Section, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, Maryland 20892-1892, USA.

ABSTRACT
Leucine-based signals in the cytoplasmic tail of invariant chain (Ii) control targeting of newly synthesized major histocompatibility complex class II molecules to the endocytic pathway for acquisition of antigenic peptides. Some protein determinants, however, do not require Ii for effective class II presentation, although endocytic processing is still necessary. Here we demonstrate that a dileucine-based signal in the cytoplasmic tail of the class II beta chain is critical for this Ii-independent presentation. Elimination or mutation of this signal reduces the rate of re-entry of mature surface class II molecules into the endocytic pathway. Antigen presentation controlled by this signal does not require newly synthesized class II molecules and appears to involve determinants requiring only limited proteolysis for exposure, whereas the opposite is true for li-dependent determinants. This demonstrates that related leucine-based trafficking signals in li and class II control the functional presentation of protein determinants with distinct processing requirements, suggesting that the peptide binding sites of newly synthesized versus mature class II molecules are made available for antigen binding in distinct endocytic compartments under the control of these homologous cytoplasmic signals. This permits capture of protein fragments produced optimally under distinct conditions of pH and proteolytic activity.

Show MeSH
Related in: MedlinePlus