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Molecular characterization and functional analysis of murine interleukin 4 receptor allotypes.

Schulte T, Kurrle R, Röllinghoff M, Gessner A - J. Exp. Med. (1997)

Bottom Line: The murine interleukin 4 receptor (IL-4R) exists as a transmembrane protein transducing pleiotropic IL-4 functions, or as soluble (s)IL-4-binding molecule with potent immunoregulatory effects.In this study we identified and characterized a murine IL-4R allotype.The extracellular Thr49 to Ile substitution abrogates one N-glycosylation site in the naturally occurring BALB/c IL-4R as well as in the experimentally point mutated C57BL/6-T49I sIL-4R, and both molecules display a nearly threefold reduction in IL-4-neutralizing activity compared to the C57BL/6 sIL-4R.

View Article: PubMed Central - PubMed

Affiliation: Institute of Clinical Microbiology and Immunology, University of Erlangen-Nürnberg, 91054 Erlangen, Germany.

ABSTRACT
The murine interleukin 4 receptor (IL-4R) exists as a transmembrane protein transducing pleiotropic IL-4 functions, or as soluble (s)IL-4-binding molecule with potent immunoregulatory effects. In this study we identified and characterized a murine IL-4R allotype. Sequence analysis of the IL-4R cDNA of BALB/c mice revealed 18 base substitutions leading to three extracellular and five cytoplasmic amino acid changes when compared with the published IL-4R sequence of C57BL/6 mice. Analyses with allotype-specific mAbs revealed that AKR/J and SJL/J mice possess the newly identified BALB/c IL-4R allotype whereas the IL-4Rs of C3H, CBA, DBA-2, and FVB/N mice are identical to that of the C57BL/6 mouse. The extracellular Thr49 to Ile substitution abrogates one N-glycosylation site in the naturally occurring BALB/c IL-4R as well as in the experimentally point mutated C57BL/6-T49I sIL-4R, and both molecules display a nearly threefold reduction in IL-4-neutralizing activity compared to the C57BL/6 sIL-4R. In line with this, a significantly enhanced dissociation rate of IL-4 was detected for the BALB/c IL-4R allotype by surface plasmon resonance and in radioligand binding studies with IL-4R-transfected cell lines. These findings suggest that the altered ligand binding behavior of the newly described IL-4R allotype may influence the IL-4 responsiveness, thus contributing to the diverse phenotypes of inbred mouse strains in IL-4-dependent diseases.

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Flow cytometric analysis of IL-4R allotypes expressed on human TF-1 cells. Transfected TF-1 cells stably expressing the transmembrane IL-4R of C57BL/6 or BALB/c mice were stained with the rat  mAb M1 or the mouse mAb 999-461, respectively. Fluorescence intensity of the background staining with the FITC-conjugated secondary Abs  alone (white graphs) or the specific binding in the presence of the primary  mAbs (black graphs) was determined by use of a FACScan®.
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Figure 4: Flow cytometric analysis of IL-4R allotypes expressed on human TF-1 cells. Transfected TF-1 cells stably expressing the transmembrane IL-4R of C57BL/6 or BALB/c mice were stained with the rat mAb M1 or the mouse mAb 999-461, respectively. Fluorescence intensity of the background staining with the FITC-conjugated secondary Abs alone (white graphs) or the specific binding in the presence of the primary mAbs (black graphs) was determined by use of a FACScan®.

Mentions: To investigate whether the binding epitopes on the transmembrane IL-4R are accessible for the mouse mAbs, FACS® analyses of transfected TF-1 cells were performed. The transfectants expressed functionally active C57BL/6 or BALB/c transmembrane IL-4Rs, since these factor-dependent human cells were grown with species-specific murine IL-4 for several months (data not shown). All three Cys34-dependent mAbs, but only one of six Met168-dependent mAbs, bound to the C57BL/6 IL-4R at the cell surface, whereas the BALB/c IL-4R was not recognized (Table 2 and Fig. 4). The mAb M1 was capable of staining both IL-4R allotypes expressed on the cell surface (Fig. 4). These results show that the Cys34-containing epitope is easily accessible whereas the region around Met168 may be more hidden through steric interactions with the cell membrane, with the IL-4R itself, or with other cell surface molecules. Two of the seven rat mAbs were able to bind to the cell surface IL-4R, but in an allotype-independent manner as expected (Table 2 and data not shown).


Molecular characterization and functional analysis of murine interleukin 4 receptor allotypes.

Schulte T, Kurrle R, Röllinghoff M, Gessner A - J. Exp. Med. (1997)

Flow cytometric analysis of IL-4R allotypes expressed on human TF-1 cells. Transfected TF-1 cells stably expressing the transmembrane IL-4R of C57BL/6 or BALB/c mice were stained with the rat  mAb M1 or the mouse mAb 999-461, respectively. Fluorescence intensity of the background staining with the FITC-conjugated secondary Abs  alone (white graphs) or the specific binding in the presence of the primary  mAbs (black graphs) was determined by use of a FACScan®.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199121&req=5

Figure 4: Flow cytometric analysis of IL-4R allotypes expressed on human TF-1 cells. Transfected TF-1 cells stably expressing the transmembrane IL-4R of C57BL/6 or BALB/c mice were stained with the rat mAb M1 or the mouse mAb 999-461, respectively. Fluorescence intensity of the background staining with the FITC-conjugated secondary Abs alone (white graphs) or the specific binding in the presence of the primary mAbs (black graphs) was determined by use of a FACScan®.
Mentions: To investigate whether the binding epitopes on the transmembrane IL-4R are accessible for the mouse mAbs, FACS® analyses of transfected TF-1 cells were performed. The transfectants expressed functionally active C57BL/6 or BALB/c transmembrane IL-4Rs, since these factor-dependent human cells were grown with species-specific murine IL-4 for several months (data not shown). All three Cys34-dependent mAbs, but only one of six Met168-dependent mAbs, bound to the C57BL/6 IL-4R at the cell surface, whereas the BALB/c IL-4R was not recognized (Table 2 and Fig. 4). The mAb M1 was capable of staining both IL-4R allotypes expressed on the cell surface (Fig. 4). These results show that the Cys34-containing epitope is easily accessible whereas the region around Met168 may be more hidden through steric interactions with the cell membrane, with the IL-4R itself, or with other cell surface molecules. Two of the seven rat mAbs were able to bind to the cell surface IL-4R, but in an allotype-independent manner as expected (Table 2 and data not shown).

Bottom Line: The murine interleukin 4 receptor (IL-4R) exists as a transmembrane protein transducing pleiotropic IL-4 functions, or as soluble (s)IL-4-binding molecule with potent immunoregulatory effects.In this study we identified and characterized a murine IL-4R allotype.The extracellular Thr49 to Ile substitution abrogates one N-glycosylation site in the naturally occurring BALB/c IL-4R as well as in the experimentally point mutated C57BL/6-T49I sIL-4R, and both molecules display a nearly threefold reduction in IL-4-neutralizing activity compared to the C57BL/6 sIL-4R.

View Article: PubMed Central - PubMed

Affiliation: Institute of Clinical Microbiology and Immunology, University of Erlangen-Nürnberg, 91054 Erlangen, Germany.

ABSTRACT
The murine interleukin 4 receptor (IL-4R) exists as a transmembrane protein transducing pleiotropic IL-4 functions, or as soluble (s)IL-4-binding molecule with potent immunoregulatory effects. In this study we identified and characterized a murine IL-4R allotype. Sequence analysis of the IL-4R cDNA of BALB/c mice revealed 18 base substitutions leading to three extracellular and five cytoplasmic amino acid changes when compared with the published IL-4R sequence of C57BL/6 mice. Analyses with allotype-specific mAbs revealed that AKR/J and SJL/J mice possess the newly identified BALB/c IL-4R allotype whereas the IL-4Rs of C3H, CBA, DBA-2, and FVB/N mice are identical to that of the C57BL/6 mouse. The extracellular Thr49 to Ile substitution abrogates one N-glycosylation site in the naturally occurring BALB/c IL-4R as well as in the experimentally point mutated C57BL/6-T49I sIL-4R, and both molecules display a nearly threefold reduction in IL-4-neutralizing activity compared to the C57BL/6 sIL-4R. In line with this, a significantly enhanced dissociation rate of IL-4 was detected for the BALB/c IL-4R allotype by surface plasmon resonance and in radioligand binding studies with IL-4R-transfected cell lines. These findings suggest that the altered ligand binding behavior of the newly described IL-4R allotype may influence the IL-4 responsiveness, thus contributing to the diverse phenotypes of inbred mouse strains in IL-4-dependent diseases.

Show MeSH
Related in: MedlinePlus