Cross-linking of IgE-Receptor Complexes by Rigid Bivalent Antigens >200 A in Length Triggers Cellular Degranulation

We have examined the effect of crosslinking IgE-receptor complexes with variable receptor-receptor distances on the transmembrane signaling that leads to degranulation of rat basophilic leukemia cells. Linear polymers of the biotin-binding protein avidin were generated with bis biotin-l,12-diamidododecane, and a dinitrophenyl-biotin conjugate was bound at each end of the polymers to form a series of rigid bivalent haptens of well-defined length. The polymers were fractionated by size with nondenaturing PAGE, electroeluted, and tested for their ability to stimulate degranulation of rat basophilic leukemia cells sensitized with anti-DNP IgE. We found that hexamers of avidin (of length >/240.4,) were as effective in triggering degranulation as dimers (of length ~80 .~), while the monomeric avidin antigen (of length '~40 .~) elicited a poorer degranulation response from the cells. The mechanism by which aggregation of cell surface receptors can initiate signal transduction is discussed in light of these results. T H E aggregation of cell surface receptors in response to biological stimuli is an important mechanism for initiating signal transduction across cell membranes. In particular, the aggregation of cell surface molecules plays an important role in the immune response; for example, T cellindependent antigens activate B cells by cross-linking surface Ig (Monroe and Cambier, 1983; Dintzis et al., 1983). Perhaps the most widely studied example of this process is the cross-linking of IgE-receptor complexes on mast cells, basophils, and rat basophilic leukemia (RBL) ~ cells, a tumor cell line of mast cell lineage (Seldin et al., 1985). These cells all have cell surface receptors that bind monomeric IgE with high affinity and secretory granules that contain histamine, serotonin, and other mediators of the allergic response. The binding of IgE to its receptor causes no detectable perturbation of the cells, but in response to cross-linking of the IgE receptors by antigens or other means, a complex series of biochemical events is initiated, culminating in the degranulation of these cells (Metzger et al., 1986). Since the earliest postulation of the importance of crosslinking (Ovary, 1961), the aggregation of IgE receptors on the cell surface has been well-established as the initial requirement for signal transduction leading to degranulation (Metzger, 1983). However, it is still not known what aspects of the cross-linking event are critical for generation of a transmem1. Abbreviations used in this paper: bis-biotin, bis-biotin-l,12-diamidododecane; BS 3, bis-sulfosuccinimidyl suberate; DNP, 2,4-dinitrophenyl; DNP-Ct2-biotin, 1-DNP-12-biotin amidododecane; (DNP-Cwbiotin)-avidin, DNP-C~,-biotin bound to avidin after dialysis to remove unbound or loosely bound hapten: RBL, rat basophilic leukemia. brane signal. For example, cross-linking could induce contact between receptors, perhaps forming an ion channel or causing a mutual conformational change in the receptors. Alternatively, the simple tethering of receptors to each other, with the orientational constraints and potential for multivalent interactions with other cellular components inherent in this tethering, could initiate signal transduction. In this study, we have attempted to address the question of whether cross-linked receptors must be brought into contact to trigger degranulation by using a series of rigid bivalent haptens of defined length to aggregate the IgE-receptor complexes. For these experiments, we used linear polymers of the tetravalent, biotin-binding protein, avidin. The polymers were prepared by the addition of bis-biotin-l,12-diamidododecane (bis-biotin) to avidin as described by Green and colleagues (1971, 1975). Dinitrophenyl (DNP) groups, bound to the free biotin binding sites at each end of the linear polymers as a DNP-biotin conjugate, served to cross-link antiDNP IgE-receptor complexes into linear chains as was shown for unpolymerized avidin antigens by Kane et al. (Kane, P., D. Holowka, and B. Baird, manuscript submitted for publication). The results described here indicate that directly cross-linked receptors may not need to be brought into contact to stimulate degranulation. Materials and Methods