Regulation of membrane metalloproteolytic cleavage of L-selectin (CD62L) by the epidermal growth factor domain

The adhesion molecule L-selectin is cleaved rapidly from the surface of act ivated leukocytes by tumor necrosis factor-a converting enzyme, a cell surf ace metalloprotease, and also undergoes slower constitutive shedding in una ctivated cells. The structural features that render it susceptible to shedd ing are poorly understood. We therefore analyzed the shedding of a series o f mutant and chimeric L-selectin molecules. Although murine L-selectin is c leaved at a specific location in the juxtamembrane region 11 amino acids di stal to the cell membrane, this cleavage has little sequence specificity. H owever, proline substitution at the P2 ' or P3 ' position or deletion of th e epidermal growth factor (EGF) domain completely blocks the rapid phorbol ester-induced cleavage, but does not affect the slower basal proteolytic sh edding. Insertion of the 15-residue membrane-proximal region (MPR) of L-sel ectin into the heterologous protein B7.2 results in a molecule that undergo es constitutive proteolytic turnover. In contrast, insertion of both the EG F domain and the MPR confers susceptibility to both slow constitutive shedd ing and the rapid proteolytic cleavage induced by phorbol 12-myristate 13-a cetate. These results demonstrate that constitutive and induced L-selectin cleavage are separable processes and that the rapid phorbol ester-induced s hedding requires the presence of the EGF domain, a sequence that is remote from the cleavage site.