Involvement of an Asn/Val cleavage site in the production of a soluble form of a human tumor necrosis factor (TNF) receptor. Site-directed mutagenesis of a putative cleavage site in the p55 TNF receptor chain.
Soluble forms of receptors for tumor necrosis factor (TNF) might be important for regulating the actions of TNF. Site-directed in vitro mutagenesis was employed to examine the processing of the p55 tumor necrosis factor receptor chain (TNF-R55) into soluble TNF-binding protein (TNF-R55-BP). An Asn/Val sequence close to the transmembrane region in TNF-R55 was indicated as a putative cleavage site for proteolytic processing. By mutagenesis, Asn and Val were replaced with Gly and Ala, respectively. Expression in Chinese hamster ovary (CHO) cells resulted in identical binding of ligand to mutated receptors as compared to receptors not subjected to mutagenesis. Turnover rates of receptors as judged by disappearance of TNF binding capacity after inhibition of de novo protein synthesis and downregulation in response to incubation with phorbol esters were also identical between wild-type and mutated receptors. However, mutations of the cleavage site resulted in a decreased spontaneous and phorbol ester-induced release of soluble receptor (TNF-R55-BP) which was almost abolished when both Asn and Val were mutated. Our results clearly demonstrate the importance of an Asn/Val sequence for proteolytic processing of the TNF-R55 into soluble TNF-R55-BP and indicate that phorbol ester-induced downregulation of the TNF-R55 may be dissociated from proteolytic cleavage of the receptor.
Gullberg
U
U
Department of Medicine, University of Lund, Sweden.
Lantz
M
M
Lindvall
L
L
Olsson
I
I
Himmler
A
A
D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D016466:CHO Cells; D006224:Cricetinae; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011956:Receptors, Cell Surface / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D018124:Receptors, Tumor Necrosis Factor; D011994:Recombinant Proteins / Q000096:biosynthesis; D012995:Solubility
D011956:Receptors, Cell Surface; D018124:Receptors, Tumor Necrosis Factor; D011994:Recombinant Proteins