Membrane Microdomains And B- Cell Signaling

B细胞抗体反应是由抗原与克隆分布的B细胞抗原受体（BCRs）结合而引发的。在过去的几年里，人们对BCR抗原结合引发的复杂信号级联的生物化学原理有了大量的了解。信号传导是由Src家族激酶的膜相关成员Lyn对BCR的磷酸化启动的。目前，B细胞活化中使抗原结合的BCR与Lyn接触的起始事件尚不清楚。现在看来，富含胆固醇和鞘脂的膜微域，称为脂筏，作为BCR信号传导的平台。脂筏可以从细胞中分离出来，基于它们在4摄氏度下在某些非离子洗涤剂中的相对不溶性。使用洗涤剂不溶性分离筏，我们了解到在静息细胞中，BCR被排除在浓缩Lyn的筏中，但在多价抗原结合时，BCR寡聚并与筏结合，在那里它被Lyn磷酸化并启动信号传导。BCR在筏中的易位不需要BCR信号传导的两个最早的事件，即Lyn对BCR的磷酸化或BCR与肌动蛋白细胞骨架的关联。然而，BCR不能发出信号或与肌动蛋白细胞骨架结合导致BCR与脂质筏只有微弱和短暂的联系。在筏中的信号开始之后，筏聚集，最终形成一个高度有组织的结构，称为免疫突触，BCR信号可以从中延长。从我们对BCR与筏的关系的研究中出现了一个令人兴奋的主题，其中BCR筏关联受多种因素的调节，这些因素控制着B细胞与抗原相遇的结果，包括B细胞的发育状态、辅受体的参与和病毒感染。确定这些因素如何影响BCR/筏关联应该从根本上增加我们对筏如何运作的理解。

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. Signaling is initiated by phosphorylation of the BCR by a membrane associated member of the Src family kinase, Lyn. At present, the initiating event in B cell activation that brings the antigen bound BCR into contact with Lyn is not known. It now appears that cholesterol- and sphingolipid-rich membrane microdomains, termed lipid rafts, serve as a platform for BCR signaling. Lipid rafts can be isolated from cells based on their relative insolubility in certain nonionic detergents at 4 degrees Celsius. Using detergent insolubility to isolate rafts we learned that in resting cells the BCR is excluded from rafts that concentrate Lyn but upon multivalent antigen binding, the BCR oligomerizes and associates with rafts where it is phosphorylated by Lyn and signaling is initiated. The translocation of the BCR into rafts does not require two of the earliest events in BCR signaling, namely the phosphorylation of the BCR by Lyn or association of the BCR with the actin cytoskeleton. However, the failure of the BCR to signal or to associate with the actin cytoskeleton results in only weak and transient association of the BCR with lipid rafts. The initiation of signaling in the rafts is followed by raft clustering and ultimately by the formation of a highly organized structure termed an immunological synapse from which BCR signaling may be prolonged. An exciting theme that emerged from our studies of the relationship of the BCR with rafts is one in which BCR raft association is regulated by a variety of factors that control the outcome of the B cell's encounter with antigen including the developmental state of the B cell, the engagement of coreceptors and viral infection. Determining how these factors influence BCR/raft association should add fundamentally to our understanding of how rafts function. Over the last year we have made progress in defining the mechanisms by which coreceptors function to regulate the association of the BCR with lipid rafts and as a consequence regulate signaling. The B cell coreceptors CD19/CD21 when coligated to the BCR through the binding of complement tagged antigens prolongs BCR residency in and signaling from rafts. We determined that the ability of the CD19/CD21 complex to function in rafts was dependent on a tetraspanin CD81 that is a component of the CD19/CD21 complex. Thus, in B cells from CD81-deficient mice and B cells expressing chimeric CD19 receptors that fail to associate with CD81, the CD19/CD21 complex when coligated to the BCR failed to stabilize the BCR in rafts. Many proteins that associate with lipid rafts do so by virtue of their acylation in particular by their palmitoylation, a reversible acylation event. We determined that CD81 becomes palmitoylated in the lipid rafts following crosslinking of the BCR and the CD19/CD21 complex. Palmitoylation appeared essential for the function of CD81 as blocking palmitoylation using the inhibitor 2-bromopalmitate blocked the ability of the CD19/CD21 complex to stabilize the BCR in lipid rafts when coligated to the BCR. Studies are in progress to determine the nature of the palmitoylating enzyme and to determine if raft stabilization through palmitoylation is a shared function of other members of the tetraspanin family. Progress was also made in determining how the FcgammaRIIB, a potent negative regulator of BCR signaling when coligated to the BCR, signals for apoptosis when crosslinked to itself. We learned that the FcgammaRIIB when crosslinked to itself becomes associated with lipid rafts and signals for apoptosis by a mechanism dependent on c-Abl but independent of both the phosphatase SHIP and the FcgammaRIIB?s ITIM motifs that are required for FcgammaRIIB?s inhibition of BCR signaling. Although the signaling pathways following homo-versus hetero-aggregation are distinct there appears to be a feedback mechanism by which once signaling is initiated in one pathway signaling is shut down in the opposing pathway. Studies are in progress to define the role of c-Abl and SHIP in this feedback mechanism. Progress has also been made in establishing a method to measure the association of the BCR with lipid rafts in intact cells in the absence of detergent. The use of detergents to analyze rafts is associated with a number of potential artifacts including the possibility that the detergent treatment creates the insoluble membranes. To measure association between the BCR and lipid rafts in intact cells, B cell lines have been generated that express a chimeric BCR Ig alpha chain containing in its cytoplasmic domain a cyan fluorescent protein (CFP) and a chimeric, raft-resident Lyn kinase that contains in its cytoplasmic domain yellow fluorescent protein (YFP). Association of the BCR and Lyn results in fluorescent resonance energy transfer (FRET) between CFP and YFP that is detected using a META 510 spectral confocal microscope. Using this system we have determined that following BCR oligomerization BCR comes into close proximity with the raft-targeted Lyn and shows a significant increase in FRET. These results verify those obtained using detergent extraction to define rafts. This system should allow a more detailed view of the interactions of the BCR with lipid rafts and its regulation. Lastly, a collaboration has been established with Dr. Mark Shlomchik's laboratory at Yale University to determine the antigen affinity and valency requirements for the induction of the association of the BCR with lipid rafts. B cells from mice that express transgenes for BCR with either high, medium or low affinity for the small chemical haptin, nitrophenol (NP) are stimulated with NP-modified proteins containing increasing numbers of NP groups to determine a binding affinity and valency threshold for the induction of BCR raft association. These studies should provide important details concerning the initiating event in antigen-dependent B cell activation.