B cell antibody responses are triggered by the binding of antigen to the clonally distributed B cell antigen receptors (BCRs). Over the last several years a great deal has been learned about the biochemistry of the complex signal cascades triggered by BCR antigen engagement. However, what remains relatively poorly understood are the molecular events that trigger the initiation of signaling. We understand that the BCR, like other members of the multichain immune recognition receptor family, is composed of a ligand binding chain, for the BCR a membrane form of Ig (mIg) the most common forms of which, mIgM and mIgD, have short cytoplasmic domains with no capacity to interact directly with the components of the signaling cascades. To do so the mIg associates with two additional chains, Ig alpha and Ig beta, that contain within their cytoplasmic domains immunoregulatory tyrosine activation motifs (ITAMs). The BCR has no inherent kinase activity but following antigen binding one of the first events observed is the phosphorylation of the BCR ITAMs by the Src-family kinase, Lyn. This project represents an approach to gain an understanding of the changes that occur in the BCR following antigen binding that allow Lyn to discriminate antigen-bound BCRs from the unbound BCRs. Based on the crystal structure of antigens bound to Fab of antibodies there is at present no evidence for an antigen-induced structural change in the BCR that could propagate the information that the BCR has bound antigen from the BCR ectodomains, across the membrane, to the cytoplasmic domains. In the absence of such an allosteric effect of antigen binding one is left with antigen-induced clustering of the BCR as the trigger for the initiation of signaling. Thus, the goal of this project is to gain an understanding of how antigen induces BCRs to cluster and how BCR clustering leads to the initiation of signaling. The critical events that trigger signaling are likely to occur within seconds of antigen binding to the BCR and to be highly dynamic, involving many weak protein-protein and protein-lipid interactions. In general, the biochemical approaches that have been used so effectively to describe the BCR signaling pathways are inadequate to capture events that occur as rapidly and as transiently as those predicted to initiate BCR signaling. In addition, antigen binding to the B cell involves a dramatic spatial change in the BCRs resulting in their patching and capping and the formation of an immune synapse. All this potentially important spatial information is lost with the addition of detergents to cells for biochemical analyses. The use of detergents is particularly problematic in the study of the early membrane changes that accompany antigen binding by BCRs. Consequently we have taken advantage of new live cell imaging technologies that allow analyses of the BCR and components of the BCR signaling pathway with the temporal and spatial resolution necessary to view the earliest events in B cell activation at the single molecule level without the complications that the addition of detergents introduce. Over the last year we have applied total internal reflection fluorescence microscopy (TIRFM) in conjunction with single particle tracking (SPT) and fluorescence resonance energy transfer (FRET) to better resolve the early events in B cell activation both spatially and temporally. We approached a fundamental question in B cell biology namely what advantage is conferred on B cell in activation by high affinity, isotype switched BCRs. Previously, we showed that in B cells responding to antigen presented on a planar lipid bilayer, simulating an antigen presenting cell, BCRs form signaling active microclusters by a mechanism that does not require physical crosslinking of BCRs by multivalent antigens. We observed that in response to either monovalent or multivalent antigens the BCRs accumulate and form microclusters at the initial points of contact of the B cell membrane with the bilayer. Clustering did not depend on signaling active BCRs revealing an intrinsic tendency of the BCRs to cluster. The microclusters grew by trapping mobile BCRs and the larger clusters were actively organized into an immune synapse. The kinetics of these events and signaling were identical for monovalent and multivalent antigens. We determined that the membrane-proximal ecto-domain of the BCR mIg was both necessary and sufficient for BCR oligomerization and signaling. BCRs that contained a mIg in which Cmu4 was deleted failed to cluster and to signal when engaging monovalent antigen. Conversely, Cmu4 expressed alone on the B cell surface spontaneously clustered and activated B cells. These findings lead us to propose a novel mechanism by which BCRs form signaling active microclusters that we termed the conformation induced oligomerization model for the initiation of BCR signaling. According to our model BCR in the resting state are not in an oligomerization receptive conformation such that random bumping has no repercussion. The binding of antigen on an opposing membrane exerts a force on the BCR to bring it into an oligomerization receptive form so that when two antigen-bound BCRs bump they oligomerize. Over the last year we demonstrated that high affinity BCRs, as compared to low affinity BCR form immobile, signaling active BCR clusters more efficiently providing evidence that affinity discrimination is a BCR intrinsic function. We also determined that as compared to mIgM-containing BCRs, mIgG BCRs more efficiently form signaling active BCR oligomers by a mechanism that depends on the cytoplasmic domain of the mIgG. These results are important in providing a molecular understanding of the functional advantage of high affinity, isotype-switched BCRs. In order to understand the molecular mechanisms by which antigen binding to the BCRs induces change in the BCRs that allows oligomerization it will ultimately be necessary to obtain the structure of the BCR in the membrane. As a step toward obtaining such a structure in collaboration with Dr. Sun in LIG we have determined the structure of a dimer of the Ig(beta) ectodomain. Based on this dimer structure and a consideration of the conserved residues of Ig(alpha) we modeled an Ig(alpha)Ig(beta) heterodimer. Based on this model and the known structure of Fc(alpha) we carried out a mutational study that identified a potential Ig(alpha)Ig(beta)-IgM interaction face. Remarkably, this face includes regions from all three chains and suggest that conformational changes in Cmu4 induced by BCR antigen binding could induce conformational changes in Ig(alpha-beta) that could be translated across the membrane to initiate signaling.
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