Pre-BCR expression level regulates cellular functions

描述（由申请方提供）：本项目的长期目标是鉴定控制B细胞分化的分子、生物化学和细胞事件。我们的重点将是了解前B细胞受体（前BCR）的作用。preBCR控制多个分化事件，包括等位基因排斥、分化为pre-BII、克隆扩增和L链重排。待检验的假设是，这些功能对前BCR表达水平的敏感性不同，并且存在功能层次。我们的分析将集中在具有IG VH 12 H链转基因的小鼠上。VH 12前B细胞分化过程中的选择是极端的，提供了一个独特的机会，以进一步了解前BCR功能。选择有利于VH 12前B细胞，其具有10个氨基酸的VHCDR 3序列和在第四位置的Gly，命名为10/G4。10/G4前B细胞存活并分化至B细胞阶段，而大多数（>95%）非10/G4 VH 12 B细胞在前BI至前BII细胞转变时死亡。为了确定大多数非10/G4前B细胞丢失的基础，我们已经产生了具有编码8个氨基酸的CDR 3的VH 12重排并且没有Gly（8/GO）的非10/G4转基因（Tg）小鼠系。8/GO pre-BCR诱导等位基因排斥、向pre-BII的分化和L链重排，但不诱导前B细胞克隆扩增或向B细胞的分化。我们假设这种缺陷是由于低前BCR表达水平。提出了三个具体目标来检验这一假设，并确定一个层次的功能。首先，将测定离体8/GO前B细胞的细胞表面上和脂筏中8/GO前BCR表达的水平。此外，将比较8/GO和10/G4前BCR之间的信号转导和脂筏易位。在第二项研究中，将通过使用逆转录病毒骨髓感染和建立额外的非10/G4 Tg小鼠，操作前BCR水平，以确定前BCR功能的层次结构。在最终目的中，将确定8/GO Tg小鼠不能产生未成熟B细胞的基础是否是由于前BCR或BCR缺陷。

DESCRIPTION (provided by the applicant): The long-term goal of this project is to identify the molecular, biochemical, and cellular events that control B cell differentiation. Our focus will be on understanding the role of the pre-B cell receptor (pre-BCR). The preBCR controls multiple differentiative events including allelic exclusion, differentiation to pre-BII, clonal expansion, and L chain rearrangement. The hypothesis to be tested is that these functions are differentially sensitive to pre-BCR expression level and that there is a hierarchy of function. Our analysis will center on mice with Ig VH12 H chain transgenes. VH12 pre-B cell selection during differentiation is extreme, providing a unique opportunity to further our understanding of pre-BCR function. Selection favors VH12 pre-B cells with VHCDR3 sequences of 10 amino acids and a Gly in the fourth position, designated 10/G4. 10/G4 pre-B cells survive and differentiate to the B cell stage, whereas the majority (>95%) of non-10/G4 VH12 B cells die at the pre-BI to pre-BII cell transition. To determine the basis for the loss of most non- 10/G4 pre-B cells, we have generated a non-10/G4 transgenic (Tg) mouse line with a VH12 rearrangement encoding an 8 amino acid CDR3 and no Gly (8/GO). The 8/GO pre-BCR induces allelic exclusion, differentiation to pre-BII, and L chain rearrangement, but does not induce pre-B cell clonal expansion or differentiation to a B cell. We hypothesize this deficiency is due to low pre-BCR expression level. Three specific aims are proposed to test this hypothesis and to determine a hierarchy of function. In the first, the level of 8/GO pre-BCR expression on the cell surface and in lipid rafts of ex vivo 8/GO pre-B cells will be determined. In addition, signal transduction and lipid raft translocation will be compared between 8/GO and 10/G4 pre-BCRs. In the second the level of the pre-BCR will be manipulated to determine a hierarchy of pre-BCR function through the use of retroviral bone marrow infection and the establishment of additional non-10/G4 Tg mice. In the final aim whether the basis for the inability of 8/GO Tg mice to generate immature B cells is due to a pre-BCR or BCR deficiency will be determined.