Antibody binding to a conformation-dependent epitope induces L-selectin association with the detergent-resistant cytoskeleton.
L-Selectin mediates leukocyte rolling on endothelium and immobilized leukocytes. Its regulation has been the subject of much study, and the conformation of the molecule may play an important role in its function. Here we report that a conformational change in L-selectin, induced by an anti-lectin domain mAb (LAM1-116) and recognized by another mAb directed to a conserved epitope on L-selectin (EL-246), predisposed L-selectin to cytoskeletal association. This effect was due to direct binding of the mAb, not to overt signaling events, and was specific to LAM1-116. Nineteen other anti-L-selectin mAbs directed against the lectin, epidermal growth factor, or short consensus repeat domains lacked this activity. The induced conformational change occurred at 37 degrees C, at 4 degrees C, in the presence of sodium azide and tyrosine kinase inhibitors herbimycin A and genistein, and with soluble detergent-extracted L-selectin. In the presence of LAM1-116, EL-246 induced cytoskeletal association of L-selectin in the absence of Ab cross-linking as visualized by L-selectin staining after low dose detergent treatment of the cells. We propose that the conformational change described herein regulates L-selectin-mediated events by exposing a high avidity binding site that, when engaged, triggers association of L-selectin with the cytoskeleton, which may lead to stronger tethers with physiological ligands.
Leid
J G
JG
Veterinary Molecular Biology, Montana State University, Bozeman, MT 59717, USA.
Steeber
D A
DA
Tedder
T F
TF
Jutila
M A
MA
D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis / Q000378:metabolism* / Q000494:pharmacology; D016227:Benzoquinones; D001666:Binding Sites, Antibody / Q000187:drug effects; D002417:Cattle; D002458:Cell Fractionation; D002460:Cell Line; D003488:Cyanogen Bromide; D003593:Cytoplasm / Q000235:genetics / Q000276:immunology; D003599:Cytoskeleton / Q000187:drug effects / Q000235:genetics / Q000276:immunology*; D003902:Detergents / Q000494:pharmacology*; D019040:E-Selectin / Q000235:genetics / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D000939:Epitopes / Q000096:biosynthesis / Q000276:immunology / Q000378:metabolism*; D019833:Genistein / Q000494:pharmacology; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000494:pharmacology; D019041:L-Selectin / Q000032:analysis / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D047029:Lactams, Macrocyclic; D007962:Leukocytes / Q000737:chemistry / Q000187:drug effects / Q000201:enzymology / Q000276:immunology; D051379:Mice; D008863:Microspheres; D017830:Octoxynol; D019007:P-Selectin / Q000235:genetics / Q000378:metabolism; D011092:Polyethylene Glycols / Q000494:pharmacology; D011233:Precipitin Tests; D011487:Protein Conformation / Q000187:drug effects; D017434:Protein Structure, Tertiary / Q000235:genetics; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D011809:Quinones / Q000494:pharmacology; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D017828:Rifabutin / Q000031:analogs & derivatives; D012756:Sheep; D012995:Solubility; D013194:Staining and Labeling; D014162:Transfection; D015854:Up-Regulation / Q000187:drug effects / Q000276:immunology
D000911:Antibodies, Monoclonal; D016227:Benzoquinones; D003902:Detergents; D019040:E-Selectin; D004791:Enzyme Inhibitors; D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; D047029:Lactams, Macrocyclic; D019007:P-Selectin; D011809:Quinones; D011994:Recombinant Proteins; D019041:L-Selectin; D017828:Rifabutin; D011092:Polyethylene Glycols; C020754:herbimycin; D017830:Octoxynol; C010615:Nonidet P-40; D019833:Genistein; D011505:Protein-Tyrosine Kinases; D003488:Cyanogen Bromide