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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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Murine sIL-4R constructs designated for the expression in  293-EBNA cells. Boxes represent the coding regions of the sIL-4Rs derived from C57BL/6 (black) or from BALB/c (white) mice. The amino acids differing between the C57BL/6 and BALB/c sequences are indicated.  The names of the constructs include the substituted amino acids in the  C57BL/6 sIL-4R and their position in the mature peptide. See Materials  and Methods for details of the cloning strategies.
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Figure 3: Murine sIL-4R constructs designated for the expression in 293-EBNA cells. Boxes represent the coding regions of the sIL-4Rs derived from C57BL/6 (black) or from BALB/c (white) mice. The amino acids differing between the C57BL/6 and BALB/c sequences are indicated. The names of the constructs include the substituted amino acids in the C57BL/6 sIL-4R and their position in the mature peptide. See Materials and Methods for details of the cloning strategies.

Mentions: A panel of mAbs obtained either by immunization of BALB/c mice with the recombinant C57BL/6 IL-4R or from similarly immunized rats was analyzed with regard to their IL-4R binding characteristics. mAbs were coupled to microtiter plates and the IL-4R ELISAs were developed with polyclonal rabbit Abs. While all of the murine mAbs were allotype specific, recognizing the C57BL/6-type IL-4R, but not the IL-4R of BALB/c, AKR/J, and SJL/J mice, the rat mAbs did not distinguish between IL-4Rs obtained from the different inbred strains of mice (Fig. 2). Thus, these binding data obtained with cell culture supernatants as well as sera (data not shown) confirmed the allotypic differences deduced from the cDNA sequences. To map the allotype-specific binding epitopes of the different murine mAbs, we cloned C57BL/6, BALB/c, and C57BL/6-BALB/c-hybrid sIL-4Rs and expressed them in 293-EBNA cells. The two hybrid receptors, C57BL/6-C34R-T49I and C57BL/6-M168T, were constructed using the internal restriction site EcoRI as indicated in Fig. 3. The PCR mismatch technique was used to introduce a single point mutation in the C57BL/6 sIL-4R cDNA leading to the C57BL/6-T49I sIL-4R.


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

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

Murine sIL-4R constructs designated for the expression in  293-EBNA cells. Boxes represent the coding regions of the sIL-4Rs derived from C57BL/6 (black) or from BALB/c (white) mice. The amino acids differing between the C57BL/6 and BALB/c sequences are indicated.  The names of the constructs include the substituted amino acids in the  C57BL/6 sIL-4R and their position in the mature peptide. See Materials  and Methods for details of the cloning strategies.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Murine sIL-4R constructs designated for the expression in 293-EBNA cells. Boxes represent the coding regions of the sIL-4Rs derived from C57BL/6 (black) or from BALB/c (white) mice. The amino acids differing between the C57BL/6 and BALB/c sequences are indicated. The names of the constructs include the substituted amino acids in the C57BL/6 sIL-4R and their position in the mature peptide. See Materials and Methods for details of the cloning strategies.
Mentions: A panel of mAbs obtained either by immunization of BALB/c mice with the recombinant C57BL/6 IL-4R or from similarly immunized rats was analyzed with regard to their IL-4R binding characteristics. mAbs were coupled to microtiter plates and the IL-4R ELISAs were developed with polyclonal rabbit Abs. While all of the murine mAbs were allotype specific, recognizing the C57BL/6-type IL-4R, but not the IL-4R of BALB/c, AKR/J, and SJL/J mice, the rat mAbs did not distinguish between IL-4Rs obtained from the different inbred strains of mice (Fig. 2). Thus, these binding data obtained with cell culture supernatants as well as sera (data not shown) confirmed the allotypic differences deduced from the cDNA sequences. To map the allotype-specific binding epitopes of the different murine mAbs, we cloned C57BL/6, BALB/c, and C57BL/6-BALB/c-hybrid sIL-4Rs and expressed them in 293-EBNA cells. The two hybrid receptors, C57BL/6-C34R-T49I and C57BL/6-M168T, were constructed using the internal restriction site EcoRI as indicated in Fig. 3. The PCR mismatch technique was used to introduce a single point mutation in the C57BL/6 sIL-4R cDNA leading to the C57BL/6-T49I sIL-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