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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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Related in: MedlinePlus

Restriction analysis of sIL-4R cDNAs. The sIL-4R cDNAs  of the different mouse strains indicated were PCR-amplified, gel-purified, and digested with the enzymes Eco47III or HhaI. Resulting fragments were analyzed on a 1.5% agarose gel. The pUC Mix Marker (Fermentas AB, Vilnius, Lithuania) was used for the determination of the  fragment sizes.
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Figure 1: Restriction analysis of sIL-4R cDNAs. The sIL-4R cDNAs of the different mouse strains indicated were PCR-amplified, gel-purified, and digested with the enzymes Eco47III or HhaI. Resulting fragments were analyzed on a 1.5% agarose gel. The pUC Mix Marker (Fermentas AB, Vilnius, Lithuania) was used for the determination of the fragment sizes.

Mentions: The PCR-amplified sIL-4R cDNAs from different mouse strains were digested with the restriction enzymes Eco47III or HhaI, and the resulting fragments were size compared. Except for the PCR products from BALB/c and SCID mice, all tested DNA fragments were digested by Eco47III, yielding two fragments of 573 and 172 bp (Fig. 1). In the case of the BALB/c and SCID sIL-4R cDNAs, the T to C base substitution at position 78 led to the loss of the restriction site and to an undigestable DNA fragment of 745 bp. Digestion of PCR products from C57BL/6-type cDNAs with HhaI resulted in three fragments of 449, 170, and 126 bp, whereas the BALB/c and SCID cDNAs yielded four fragments of 359, 170, 126, and 90 bp due to an additional HhaI restriction site at position 168 (T to C substitution).


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

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

Restriction analysis of sIL-4R cDNAs. The sIL-4R cDNAs  of the different mouse strains indicated were PCR-amplified, gel-purified, and digested with the enzymes Eco47III or HhaI. Resulting fragments were analyzed on a 1.5% agarose gel. The pUC Mix Marker (Fermentas AB, Vilnius, Lithuania) was used for the determination of the  fragment sizes.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Restriction analysis of sIL-4R cDNAs. The sIL-4R cDNAs of the different mouse strains indicated were PCR-amplified, gel-purified, and digested with the enzymes Eco47III or HhaI. Resulting fragments were analyzed on a 1.5% agarose gel. The pUC Mix Marker (Fermentas AB, Vilnius, Lithuania) was used for the determination of the fragment sizes.
Mentions: The PCR-amplified sIL-4R cDNAs from different mouse strains were digested with the restriction enzymes Eco47III or HhaI, and the resulting fragments were size compared. Except for the PCR products from BALB/c and SCID mice, all tested DNA fragments were digested by Eco47III, yielding two fragments of 573 and 172 bp (Fig. 1). In the case of the BALB/c and SCID sIL-4R cDNAs, the T to C base substitution at position 78 led to the loss of the restriction site and to an undigestable DNA fragment of 745 bp. Digestion of PCR products from C57BL/6-type cDNAs with HhaI resulted in three fragments of 449, 170, and 126 bp, whereas the BALB/c and SCID cDNAs yielded four fragments of 359, 170, 126, and 90 bp due to an additional HhaI restriction site at position 168 (T to C substitution).

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