The soluble human IL-6 receptor. Mutational characterization of the proteolytic cleavage site.
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.
Müllberg
J
J
Department of Biochemistry, Rheinisch-Westfälische Technische Hochschule Aachen, Germany.
Oberthür
W
W
Lottspeich
F
F
Mehl
E
E
Dittrich
E
E
Graeve
L
L
Heinrich
P C
PC
Rose-John
S
S
D000595:Amino Acid Sequence; D002478:Cells, Cultured; D004252:DNA Mutational Analysis; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017354:Point Mutation; D018123:Receptors, Interleukin / Q000737:chemistry / Q000378:metabolism*; D019947:Receptors, Interleukin-6; D012680:Sensitivity and Specificity
D018123:Receptors, Interleukin; D019947:Receptors, Interleukin-6