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 gener ated 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 homogen eity from supernatants of COS-7 cells transfected with a cDNA coding f or the transmembrane IL-6R. The COOH-terminus of the shed receptor pro tein was analyzed by carboxypeptidase treatment and subsequent amino a cid analysis. The established cleavage site Gln357/Asp358 was extensiv ely altered by point mutations and small deletions to define the struc tural requirements for cleavage. Although point mutations around the c leavage sire reduced shedding of the IL-6R up to fivefold, deletions o f 5 or 10 amino acids almost completely abolished shedding. Deletion o f the cytoplasmic domain of the receptor had no influence on shedding of the protein. It turned out that a potential N-glycosylation site cl ose to the proteolytic cleavage site of the IL-6R is used. However thi s N-glycosylation does not affect the efficiency of the shedding proce ss. Furthermore, we demonstrate for the first time that the human IL-6 R is constitutively phosphorylated and that this phosphorylation can b e stimulated by PMA but is not correlated with shedding of the recepto r protein. The knowledge of the mechanism by which the soluble IL-6R i s generated will help to identify the processing enzyme involved and t o analyze its regulation.