PROTEASE-RESISTANT L-SELECTIN MUTANTS - DOWN-MODULATION BY CROSS-LINKING BUT NOT CELLULAR ACTIVATION

The adhesion molecule L-selectin (CD62L) is rapidly shed from the plas ma membrane during leukocyte activation as a result of proteolytic cle avage between Lys(321) and Ser(322) within the extracellular domain, L -selectin is also down-modulated from the surface in response to cross -linking, possibly through a similar mechanism. To further characteriz e the mechanism of downmodulation, several L-selectin mutants were gen erated and transfected into COS cells, Wild-type L-selectin as well as mutants with one or two amino acid substitutions at the cleavage site were nearly quantitatively shed into the culture supernatant. However , mutants in which a nine-amino acid stretch that included the proteas e-sensitive site was either deleted or replaced with a polyglycine spa cer or a comparable region of E-selectin were retained on the cell sur face and not detected in the supernatant, These results are consistent with other reports describing protease resistant L-selectin mutants. We also demonstrate that when expressed in L1-2 pre-B cells, the L-sel ectin nine-amino acid deletion mutant (321del.9), but not wild-type L- selectin, is resistant to down-regulation induced by PMA. However, bot h wild-type and mutant 321del.9 are completely lost from the cell surf ace in response to cross-linking with an L-selectin Ab. PMA-induced- b ut not L-selectin cross-linking-induced down-modulation was inhibited by staurosporine. These data are consistent with the idea that the L-s electin protease(s) can tolerate minor structural alterations at the c leavage site, and that L-selectin down-modulation can be induced by mo re than one mechanism, at least one of which (cross-linking) is protei n kinase C independent.