Structural mapping of membrane-bound immunoglobulin E-receptor complexes: use of monoclonal anti-IgE antibodies to probe the conformation of receptor-bound IgE.
Previous resonance energy-transfer measurements have suggested that immunoglobulin E (IgE) may bend near the junction of its Fc and Fab segments in order to bind to its high-affinity receptor on rat basophilic leukemia cells. In order to test this possibility, two monoclonal antibodies were employed that bind specifically to rat IgE (IgER) when IgER is in solution and when it is bound to receptors on the plasma membrane. The F(ab')2 fragment of one monoclonal (B5) that is specific for the Fab region of IgER was labeled with donor probes and bound to IgER, and the quenching of the fluorescence of these donors due to simultaneous binding of the Fab' fragment of an anti-Fc monoclonal (A2) that was labeled with an acceptor probe at its interchain disulfide bond was measured. Significantly less energy transfer between these probes was observed when IgER was bound to its receptor on membrane vesicles than when it was free in solution, and this result is interpreted in light of other energy-transfer measurements using A2 and B5 that were preferentially labeled near their combining sites with donors and acceptors, respectively, as well as measurements of the distance of closest approach between these sites and the membrane surface. These results along with previous energy-transfer measurements and other biochemical information form the basis for a working model of the conformation and orientation of receptor-bound IgE. This study demonstrates the use of fluorescently labeled monoclonal antibodies as highly selective energy-transfer probes in assessing structures of macromolecular complexes on the plasma membrane.
D000818:Animals; D000911:Antibodies, Monoclonal; D001491:Basophils / Q000276:immunology; D002462:Cell Membrane / Q000276:immunology; D004735:Energy Transfer; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007073:Immunoglobulin E / Q000378:metabolism*; D007700:Kinetics; D007942:Leukemia, Experimental / Q000276:immunology; D008958:Models, Molecular; D011487:Protein Conformation; D051381:Rats; D011961:Receptors, Fc / Q000302:isolation & purification / Q000378:metabolism*; D017455:Receptors, IgE; D011971:Receptors, Immunologic / Q000302:isolation & purification / Q000378:metabolism*; D013861:Thiocyanates
D000911:Antibodies, Monoclonal; D005452:Fluoresceins; D005456:Fluorescent Dyes; D011961:Receptors, Fc; D017455:Receptors, IgE; D011971:Receptors, Immunologic; D013861:Thiocyanates; D007073:Immunoglobulin E; D016650:Fluorescein-5-isothiocyanate