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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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Inhibition of IL-4–induced proliferation by different sIL-4R  variants. The Th L1/1 (A) or HT-2 (B) were incubated with 2 ng/ml recombinant murine IL-4 and serial diluted concentrations of C57BL/6,  BALB/c, or C57BL/6-T49I sIL-4Rs, respectively. After 48 h [3H]-thymidine was added and 16 h later the cells were harvested and radioactivity  was measured in a β counter. Sigmoidal dose–response curves were calculated for each set of data.
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Figure 6: Inhibition of IL-4–induced proliferation by different sIL-4R variants. The Th L1/1 (A) or HT-2 (B) were incubated with 2 ng/ml recombinant murine IL-4 and serial diluted concentrations of C57BL/6, BALB/c, or C57BL/6-T49I sIL-4Rs, respectively. After 48 h [3H]-thymidine was added and 16 h later the cells were harvested and radioactivity was measured in a β counter. Sigmoidal dose–response curves were calculated for each set of data.

Mentions: Since the C57BL/6 and BALB/c sIL-4R display different biochemical properties, we addressed the question of whether the allotypic differences also result in functional changes. Therefore, the sIL-4R proteins were analyzed for their capacity to inhibit IL-4–induced proliferation of the Th2 clone L1/1 and HT-2 cells. The dose–response curves clearly revealed a diminished IL-4–neutralizing effect of the BALB/c sIL-4R when compared to the C57BL/6 sIL-4R, irrespective of the cell type used (Fig. 6 A and B). The calculated ED50 values obtained from seven experiments performed with different sIL-4R preparations averaged 640 ± 158 pM for the C57BL/6 and 1,650 ± 193 pM for the BALB/c sIL-4R. Interestingly, the C57BL/6-T49I sIL-4R construct displayed a dose–response curve (Fig. 6 A and B) and an ED50 value (1,370 ± 243 pM) very similar to that of the BALB/c sIL-4R. This indicates that the substitution of Thr49 and the resulting loss of N-glycosylation at this part of the IL-4R causes the allotypic differences of IL-4 neutralization.


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

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

Inhibition of IL-4–induced proliferation by different sIL-4R  variants. The Th L1/1 (A) or HT-2 (B) were incubated with 2 ng/ml recombinant murine IL-4 and serial diluted concentrations of C57BL/6,  BALB/c, or C57BL/6-T49I sIL-4Rs, respectively. After 48 h [3H]-thymidine was added and 16 h later the cells were harvested and radioactivity  was measured in a β counter. Sigmoidal dose–response curves were calculated for each set of data.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 6: Inhibition of IL-4–induced proliferation by different sIL-4R variants. The Th L1/1 (A) or HT-2 (B) were incubated with 2 ng/ml recombinant murine IL-4 and serial diluted concentrations of C57BL/6, BALB/c, or C57BL/6-T49I sIL-4Rs, respectively. After 48 h [3H]-thymidine was added and 16 h later the cells were harvested and radioactivity was measured in a β counter. Sigmoidal dose–response curves were calculated for each set of data.
Mentions: Since the C57BL/6 and BALB/c sIL-4R display different biochemical properties, we addressed the question of whether the allotypic differences also result in functional changes. Therefore, the sIL-4R proteins were analyzed for their capacity to inhibit IL-4–induced proliferation of the Th2 clone L1/1 and HT-2 cells. The dose–response curves clearly revealed a diminished IL-4–neutralizing effect of the BALB/c sIL-4R when compared to the C57BL/6 sIL-4R, irrespective of the cell type used (Fig. 6 A and B). The calculated ED50 values obtained from seven experiments performed with different sIL-4R preparations averaged 640 ± 158 pM for the C57BL/6 and 1,650 ± 193 pM for the BALB/c sIL-4R. Interestingly, the C57BL/6-T49I sIL-4R construct displayed a dose–response curve (Fig. 6 A and B) and an ED50 value (1,370 ± 243 pM) very similar to that of the BALB/c sIL-4R. This indicates that the substitution of Thr49 and the resulting loss of N-glycosylation at this part of the IL-4R causes the allotypic differences of IL-4 neutralization.

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