Jh. Stoddart et al., PROTEASE-RESISTANT L-SELECTIN MUTANTS - DOWN-MODULATION BY CROSS-LINKING BUT NOT CELLULAR ACTIVATION, The Journal of immunology, 157(12), 1996, pp. 5653-5659
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.