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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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125I–IL-4–binding to the IL-4R allotypes expressed as cell  surface molecules. Ligand binding analysis was performed with human TF-1  cells expressing C57BL/6 or BALB/c IL-4R. (A) Equilibrium binding  studies. Cells were incubated with various concentrations of 125I–IL-4 for  90 min at 4°C and assayed for binding as described in Materials and Methods. Data are corrected for nonspecific binding and are presented as saturation binding curve or Scatchard plot (inset). (B) Dissociation of IL-4  from the transmembrane receptors. Transfected TF-1 cells were incubated  in the presence of 1.2 nM 125I–IL-4 for 90 min at 4°C, washed twice, resuspended in medium, and incubated at 4°C. Aliquots of 5 × 105 cells were  taken at the indicated time points and the amount of IL-4 still bound to  the cell surface was determined. The calculated Koff values for this experiment are indicated.
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Figure 7: 125I–IL-4–binding to the IL-4R allotypes expressed as cell surface molecules. Ligand binding analysis was performed with human TF-1 cells expressing C57BL/6 or BALB/c IL-4R. (A) Equilibrium binding studies. Cells were incubated with various concentrations of 125I–IL-4 for 90 min at 4°C and assayed for binding as described in Materials and Methods. Data are corrected for nonspecific binding and are presented as saturation binding curve or Scatchard plot (inset). (B) Dissociation of IL-4 from the transmembrane receptors. Transfected TF-1 cells were incubated in the presence of 1.2 nM 125I–IL-4 for 90 min at 4°C, washed twice, resuspended in medium, and incubated at 4°C. Aliquots of 5 × 105 cells were taken at the indicated time points and the amount of IL-4 still bound to the cell surface was determined. The calculated Koff values for this experiment are indicated.

Mentions: Since the experiments described thus far were performed exclusively with soluble and most probably monomeric IL-4R molecules, we set out to analyze the ligand-binding characteristics of IL-4R complexes expressed at the cell surface. Equilibrium binding analysis was performed with transfected human TF-1 cells expressing functionally active C57BL/6 or BALB/c allotypic IL-4Rs. The resulting saturation binding curves and Scatchard plots revealed Kd values of 370 and 410 pM for the C57BL/6 and BALB/c IL-4Rs, respectively, which were expressed in comparable numbers (17,000–22,000 molecules/cell) on the transfected TF-1 cells (Fig. 7 A). Importantly, the kinetic measurements revealed a markedly enhanced dissociation rate for the BALB/c IL-4R (Fig. 7 B), while the association rates were similar (200–260 × 106 M−1 min−1). In several experiments, the calculated Koff values were in the range of 7–16-fold faster for the BALB/c IL-4R than for the C57BL/6 IL-4R.


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

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

125I–IL-4–binding to the IL-4R allotypes expressed as cell  surface molecules. Ligand binding analysis was performed with human TF-1  cells expressing C57BL/6 or BALB/c IL-4R. (A) Equilibrium binding  studies. Cells were incubated with various concentrations of 125I–IL-4 for  90 min at 4°C and assayed for binding as described in Materials and Methods. Data are corrected for nonspecific binding and are presented as saturation binding curve or Scatchard plot (inset). (B) Dissociation of IL-4  from the transmembrane receptors. Transfected TF-1 cells were incubated  in the presence of 1.2 nM 125I–IL-4 for 90 min at 4°C, washed twice, resuspended in medium, and incubated at 4°C. Aliquots of 5 × 105 cells were  taken at the indicated time points and the amount of IL-4 still bound to  the cell surface was determined. The calculated Koff values for this experiment are indicated.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199121&req=5

Figure 7: 125I–IL-4–binding to the IL-4R allotypes expressed as cell surface molecules. Ligand binding analysis was performed with human TF-1 cells expressing C57BL/6 or BALB/c IL-4R. (A) Equilibrium binding studies. Cells were incubated with various concentrations of 125I–IL-4 for 90 min at 4°C and assayed for binding as described in Materials and Methods. Data are corrected for nonspecific binding and are presented as saturation binding curve or Scatchard plot (inset). (B) Dissociation of IL-4 from the transmembrane receptors. Transfected TF-1 cells were incubated in the presence of 1.2 nM 125I–IL-4 for 90 min at 4°C, washed twice, resuspended in medium, and incubated at 4°C. Aliquots of 5 × 105 cells were taken at the indicated time points and the amount of IL-4 still bound to the cell surface was determined. The calculated Koff values for this experiment are indicated.
Mentions: Since the experiments described thus far were performed exclusively with soluble and most probably monomeric IL-4R molecules, we set out to analyze the ligand-binding characteristics of IL-4R complexes expressed at the cell surface. Equilibrium binding analysis was performed with transfected human TF-1 cells expressing functionally active C57BL/6 or BALB/c allotypic IL-4Rs. The resulting saturation binding curves and Scatchard plots revealed Kd values of 370 and 410 pM for the C57BL/6 and BALB/c IL-4Rs, respectively, which were expressed in comparable numbers (17,000–22,000 molecules/cell) on the transfected TF-1 cells (Fig. 7 A). Importantly, the kinetic measurements revealed a markedly enhanced dissociation rate for the BALB/c IL-4R (Fig. 7 B), while the association rates were similar (200–260 × 106 M−1 min−1). In several experiments, the calculated Koff values were in the range of 7–16-fold faster for the BALB/c IL-4R than for the C57BL/6 IL-4R.

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