Bivalent binding of an anti-CD3 antibody to Jurkat cells induces association of the T cell receptor complex with the cytoskeleton.
Ligand binding and cross-linking of TCR/CD3 complex leads to T cell stimulation in immunologic responses. As a prelude to investigating the dynamic interactions of these receptors, we have characterized binding of the mAb OKT3 specific for CD3 on Jurkat cells. The association of both OKT3 and its Fab' fragment is rapid at 4 degrees C, and dissociation of Fab' is also rapid, but dissociation of OKT3 is slow, indicating bivalent binding in this case. Dissociation of OKT3 is substantially accelerated at 37 degrees C if internalization is prevented. From the concentration dependence, the binding of OKT3 at 4 degrees C appears to be very tight whereas binding of the Fab' fragment is weaker and biphasic. Scatchard analysis of the Fab' equilibrium binding data indicates two binding sites with KD values of 5.1 x 10(-9) M and 2.7 x 10(-8) M. The very tight binding of the bivalent antibody may be caused by inter- or intramolecular cross-linking between these sites. If Jurkat cells are warmed to 37 degrees C, there is an energy-dependent increase by about one-third of sites bound by OKT3 or its Fab' fragment over that seen at 4 degrees C. This increase may be related to a receptor recycling process because internalization of a similar number of the bound ligands occurs at similar rates. Other experiments have revealed that OKT3, but not its Fab' fragment, causes the receptor complex to become associated with the detergent-insoluble cytoskeleton, and there are also insoluble intracellular OKT3-binding sites. These cross-linking-induced receptor-cytoskeletal interactions are sensitive to moderate changes in salt concentration that should allow their molecular basis to be investigated.
Marano
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N
Department of Chemistry, Cornell University, Ithaca, NY 14853.
Holowka
D
D
Baird
B
B
D000911:Antibodies, Monoclonal / Q000502:physiology; D000945:Antigens, Differentiation, T-Lymphocyte / Q000276:immunology / Q000378:metabolism*; D001666:Binding Sites, Antibody; D002460:Cell Line; D003599:Cytoskeleton / Q000276:immunology / Q000378:metabolism* / Q000502:physiology; D004705:Endocytosis; D006801:Humans; D007700:Kinetics; D015458:Leukemia, T-Cell / Q000276:immunology / Q000378:metabolism*; D011948:Receptors, Antigen, T-Cell / Q000096:biosynthesis* / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology / Q000378:metabolism* / Q000502:physiology; D013696:Temperature
D000911:Antibodies, Monoclonal; D000945:Antigens, Differentiation, T-Lymphocyte; D011948:Receptors, Antigen, T-Cell