STRUCTURAL MAPPING OF FC-RECEPTOR BOUND IMMUNOGLOBULIN-E - PROXIMITY TO THE MEMBRANE-SURFACE OF THE ANTIBODY COMBINING SITE AND ANOTHER SITE IN THE FAB SEGMENTS

Resonance energy-transfer methods have been used to investigate the structure of immunoglobulin E (IgE) bound to its high-affinity receptor on plasma membrane vesicles derived from rat basophilic leukemia cells. The structural mapping of receptor-bound IgE was initiated in an earlier study [Holowka, D., and Baird, B. (1983) Biochemistry 22, 3475], and it is based on measuring the minimal distance from IgE sites that are selectively labeled with donor probes to a plane of amphipathic acceptors at the membrane surface. This paper describes the use of monoclonal IgE specific for 5-(dimethylamino)naphthalene-1-sulfonyl (DNS) to place a donor probe, DNS-L-Lys, in the antibody combining sites. The distance from these sites to the membrane surface was determined to be greater than 100 .ANG. with two different amphipathic acceptor probes. Another site in the Fab segments of monoclonal IgE (anti-dinitrophenyl) could be labeled selectively with N-[4-[7-(diethylamino)-4-methylcoumarin-3-yl]phenyl]maleimide (CPM) in the absence of reducing agents [CPM(-)], and the reaction could not be blocked by prereaction with N-ethylmaleimide. The pattern of CPM (-)-labeled proteolytic fragments and the lack of fluorescence quenching by (trinitrophenyl)lysine in the antibody combining sites suggested the CPM(-)-labeled site to be in the C.epsilon.1 domain of IgE. The distance between this site on receptor-bound IgE and the membrane surface was determined to be 75-87 .ANG. with two different amphipathic acceptors. When IgE was labeled with CPM at the inter heavy chain disulfides in C.epsilon.2 in the presence of reducing agents [CPM(+)] and employed in further energy-transfer experiments, we observed an apparent interaction between this probe and one particular amphipathic acceptor but only when the IgE was bound to receptor. This suggests a conformational change in IgE accompanies receptor binding. The results from the experiments reported here support our previous findings and provide additional information about the structural orientation of receptor-bound IgE.