ADAM-17-independent shedding of L-selectin

Abstract

L-selectin is expressed by leukocytes and facilitates their adhesion under flow along the walls of blood vessels. As do a variety of membrane proteins, L-selectin undergoes ectodomain shedding. Using approaches that monitor full-length L-selectin in short-term assays, it has been determined that L-selectin shedding is defective in tumor necrosis factor alpha-converting enzyme (ADAM-17)-deficient cells. In this study, we examined the steady-state levels of L-selectin on ADAM-17-deficient cells using a monoclonal antibody to the cytoplasmic region of L-selectin, which allows for the detection of total L-selectin (full-length and the membrane-associated cleavage fragment). We demonstrate that ADAM-17-deficient cells generate a 6-kDa transmembrane fragment of L-selectin. Although inducible L-selectin shedding by phorbol 12-myristate 13-acetate stimulation was not observed by these cells in short-term assays, basal turnover did occur, resulting in the production of soluble L-selectin, as determined by enzyme-linked immunosorbent assay. L-selectin turnover was greatly increased upon ADAM-17 reconstitution. Truncating the juxtamembrane region of L-selectin blocked ADAM-17-independent shedding as did a hydroxymate metalloprotease inhibitor. Together, these findings demonstrate that a metalloprotease activity separate from ADAM-17 can use the cleavage domain of L-selectin. We speculate that separate proteolytic mechanisms of L-selectin shedding may regulate distinct antiadhesive mechanisms, such as inducible shedding for the rapid dissociation of cell-cell interactions and constitutive shedding for the homeostatic maintenance of high serum levels of soluble L-selectin, a potential adhesion buffer.

Cytoskeletal interactions regulate inducible L-selectin clustering

“…This methodology was shown previously to be insensitive to topographic redistribution of L-selectin (72). We also engineered a deletion in the cleavage region of ⌬16 Lselectin, which we reported previously blocks L-selectin shedding (52,82). Despite this cleavage resistance, we found that the adhesion defect by ⌬16 L-selectin was not altered (Fig.…”

“…Flow cytometry, immunoprecipitation, SDS-PAGE, and immunoblotting were performed as previously described (1,52,74,82,84). For flow cytometry, antibody-labeled cells were analyzed (10,000 cells/sample) or sorted on a FACSCalibur instrument (Becton-Dickinson Immunocytometry Systems, San Jose, CA).…”

Adam17-dependent shedding limits early neutrophil influx but does not alter early monocyte recruitment to inflammatory sites

“…Within tissues, neutrophil degranulation is a rich source of serine proteases, and their release may significantly contribute to the loss of L-selectin once they are within the peritoneum. Because some Adam17-independent cleavage of L-selectin can be inhibited by metalloproteinase inhibitors, 35 it is possible that there may be a shift in the enzymes controlling L-selectin cleavage as the inflammatory response evolves and/or at different steps in the emigration process. Recently, Adam8 mobilization from the neutrophil granules to the membrane was demonstrated after endothelial adhesion, and Adam8 increased L-selectin shedding.…”

Evidence for a Role of ADAM17 (TACE) in the Regulation of Platelet Glycoprotein V

“…For such an assay system, antibodies against the ϳ13-kDa C-terminal portion of the GPV ectodomain will be required that are currently being generated in our laboratory. ADAM 17 is a well known sheddase that cleaves a variety of receptors, including L-selectin, VCAM-1 (14,33,34), and, as shown very recently, platelet GPIb␣ (35). As in the regulation of these molecules, the release of GPV from the platelet surface can be stimulated by the phorbol ester PMA through activation of ADAM17, a pathway that seems to be conserved across multiple cell types.…”