Related Experiment Videos
Protease-resistant L-selectin mutants. Down-modulation by cross-linking but not cellular activation
J H Stoddart1, R R Jasuja, M A Sikorski
1Division of Hematology-Oncology and Tupper Research Institute, Department of Medicine, New England Medical Center, Tufts University School of Medicine, Boston, MA 02111, USA.
Journal of Immunology (Baltimore, Md. : 1950)
|December 15, 1996
Summary
The adhesion molecule L-selectin (CD62L) shedding is mediated by proteases that tolerate minor cleavage site alterations. L-selectin down-modulation occurs via multiple mechanisms, including protein kinase C-independent pathways.
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- L-selectin (CD62L) is a crucial adhesion molecule on leukocytes.
- L-selectin shedding from the plasma membrane is triggered by proteolytic cleavage during leukocyte activation.
- Down-modulation of L-selectin also occurs upon cross-linking, potentially via a similar proteolytic mechanism.
Purpose of the Study:
- To investigate the mechanism of L-selectin down-modulation.
- To characterize the role of the cleavage site in L-selectin shedding and down-regulation.
- To differentiate between distinct pathways of L-selectin surface modulation.
Main Methods:
- Generation and transfection of L-selectin mutants with altered cleavage sites into COS cells.
- Analysis of L-selectin shedding into culture supernatant.
- Expression of wild-type and mutant L-selectin in L1-2 pre-B cells.
- Assessment of PMA- and antibody-induced L-selectin down-modulation.
- Inhibition studies using staurosporine.
Main Results:
- Mutants with minor substitutions at the cleavage site were efficiently shed.
- Deletion or replacement of a nine-amino acid stretch at the cleavage site rendered L-selectin cell-surface retained and protease-resistant.
- The L-selectin deletion mutant (321del.9) resisted PMA-induced down-regulation but not cross-linking-induced loss.
- PMA-induced down-modulation was inhibited by staurosporine, while cross-linking-induced down-modulation was not.
Conclusions:
- L-selectin proteases can accommodate minor structural changes at the cleavage site.
- L-selectin down-modulation can be mediated by distinct mechanisms.
- Cross-linking-induced L-selectin down-modulation is independent of protein kinase C.