Mutation of Proline 211 Reduces Shedding of the Human p75 TNF Receptor

We have identified a residue necessary for the cleavage of human p75 TNF-R from the cell surface by deletion and mutagenesis analysis of the membrane-proximal domain between amino acids 147 and 221. Deletion analysis of this area showed that residues between amino acids 207 and 216 are required for shedding. Site-directed mutagenesis of proline 211 to glycine reduced PMA-induced shedding of human p75 TNF-R from COS-7 or Raw 264.7 cells. Mutation of glycine 210 to aspartic acid did not affect receptor shedding. Mutation of serine 212 to leucine did not affect the PMA-induced shedding from the surface of COS-7 cells, but reduced the efficiency of shedding from the surface of Raw 264.7 macrophages by PMA or LPS. Deletion of putative elastase cleavage sites at amino acids 151 to 153, 162 to 163, and 177 to 178 (also a putative metalloprotease site similar to the cleavage site of TNF-α) or mutation of a serine-threonine-serine motif resembling a similar motif at the site of L-selectin cleavage at amino acids 202 to 204 did not reduce shedding of p75 TNF-R after treatment of cells with PMA. This work shows that a single amino acid mutation at proline 211 of human p75 TNF-R can prevent shedding from the cell surface, and that deletion of other previously proposed putative cleavage sites of the human p75 TNF-R does not prevent its shedding.