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The soluble human IL-6 receptor. Mutational characterization of the proteolytic cleavage site
J Müllberg1, W Oberthür, F Lottspeich
1Department of Biochemistry, Rheinisch-Westfälische Technische Hochschule Aachen, Germany.
Abstract:
Like many proteins with a single transmembrane domain the IL-6R exists in a membrane-associated and soluble form. The soluble IL-6R is generated by limited proteolysis of the membranous receptor. This process, also called shedding, is drastically enhanced by PMA, an activator of protein kinase C. The soluble receptor protein was purified to homogeneity from supernatants of COS-7 cells transfected with a cDNA coding for the transmembrane IL-6R. The COOH-terminus of the shed receptor protein was analyzed by carboxypeptidase treatment and subsequent amino acid analysis. The established cleavage site Gln357/Asp358 was extensively altered by point mutations and small deletions to define the structural requirements for cleavage. Although point mutations around the cleavage site reduced shedding of the IL-6R up to fivefold, deletions of 5 or 10 amino acids almost completely abolished shedding. Deletion of the cytoplasmic domain of the receptor had no influence on shedding of the protein. It turned out that a potential N-glycosylation site close to the proteolytic cleavage site of the IL-6R is used. However this N-glycosylation does not affect the efficiency of the shedding process. Furthermore, we demonstrate for the first time that the human IL-6R is constitutively phosphorylated and that this phosphorylation can be stimulated by PMA but is not correlated with shedding of the receptor protein. The knowledge of the mechanism by which the soluble IL-6R is generated will help to identify the processing enzyme involved and to analyze its regulation.
Insights
Researchers investigated the shedding of the Interleukin-6 Receptor (IL-6R), finding that deletions near the cleavage site significantly inhibit this process. Phosphorylation of IL-6R is stimulated by PMA but not linked to shedding.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- The Interleukin-6 Receptor (IL-6R) exists in both membrane-bound and soluble forms.
- Soluble IL-6R is generated through limited proteolysis (shedding) of the membrane-bound receptor.
- This shedding process is notably enhanced by phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C.
Purpose of the Study:
- To elucidate the structural requirements governing the proteolytic cleavage site of the IL-6R.
- To investigate the role of N-glycosylation and phosphorylation in the shedding mechanism of IL-6R.
- To understand the regulation of IL-6R shedding, potentially identifying the processing enzyme.
Main Methods:
- Purification of soluble IL-6R from transfected COS-7 cell supernatants.
- Carboxypeptidase treatment and amino acid analysis to characterize the COOH-terminus.
- Site-directed mutagenesis (point mutations and deletions) at the Gln357/Asp358 cleavage site.
- Analysis of N-glycosylation and constitutive/stimulated phosphorylation of IL-6R.
Main Results:
- Point mutations near the cleavage site reduced IL-6R shedding by up to fivefold.
- Deletions of 5 or 10 amino acids at the cleavage site almost completely abolished shedding.
- Deletion of the cytoplasmic domain did not affect shedding efficiency.
- A potential N-glycosylation site near the cleavage site is utilized but does not influence shedding.
- Human IL-6R is constitutively phosphorylated, and this is stimulated by PMA, but not correlated with shedding.
Conclusions:
- Specific structural elements around the IL-6R cleavage site are critical for efficient shedding.
- N-glycosylation and cytoplasmic domain presence do not play a significant role in IL-6R shedding.
- IL-6R phosphorylation, while regulated by PMA, is independent of the shedding process.
- Further research into the identified cleavage site and regulatory mechanisms is warranted to identify the responsible enzyme.