Binding of cytoplasmic proteins to the CD19 intracellular domain is high affinity, competitive, and multimeric
Binding of cytoplasmic proteins to the CD19 intracellular domain is high affinity, competitive, and multimeric
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DOI：
10.4049/jimmunol.172.12.7556
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发表时间：
2004-06-15
影响因子：
4.4
通讯作者：
Carter, RH
中科院分区：
文献类型：
--
作者：
Brooks, SR;Kirkham, PM;Carter, RH
CD 19是B1和边缘区B细胞发育、Ab应答和B细胞记忆所必需的。CD 19免疫沉淀物含有胞质蛋白复合物，包括林恩、Vav、磷脂酶C γ 2（PLC γ 2）、Grb 2和磷脂酰肌醇3-激酶的p85亚基。其中哪些直接与CD 19结合以及相互作用的强度尚不清楚。这些问题对于理解CD 19的信号功能非常重要，CD 19对正常B细胞生理学至关重要。使用纯化的重组蛋白，我们现在表明，这些信号蛋白中的每一个包含至少一个Src同源2（SH 2）结构域，直接与磷酸化的CD 19胞质结构域相互作用。通过表面等离子体共振（Biacore）分析，Vav、p85和Grb 2的SH 2结构域与CD 19的结合亲和力各自在纳摩尔范围内。林恩和PLC γ 2的结合不符合1：1建模。然而，结合数据分析（林恩）和竞争实验（PLC γ 2）表明，这些结合具有相当的亲和力。竞争实验表明，其结合依赖于相同的CD 19酪氨酸的SH 2结构域竞争结合，但这些SH 2结构域不阻碍不同的SH 2结构域与其他CD 19酪氨酸的结合。我们的结论是，结合到CD 19胞质结构域是多聚体，高亲和力，和竞争性。相互作用的高亲和力也表明，酪氨酸在体内是非必需的，但功能。初步的结构模型表明，CD 19形成了一个信号复合物，其中含有多个细胞质蛋白，这些蛋白彼此非常接近，并与质膜紧密相连。
CD19 is required for the development of B1 and marginal zone B cells, for Ab responses, and for B cell memory. CD19 immunoprecipitates contain a complex of cytoplasmic proteins, including Lyn, Vav, phospholipase Cgamma2 (PLCgamma2), Grb2, and the p85 subunit of phosphatidylinositol 3-kinase. Which of these bind directly to CD19 and the strengths of the interactions are unknown. These issues are important in understanding the signaling functions of CD19, which are crucial for normal B cell physiology. Using purified, recombinant proteins, we now show that each of these signaling proteins contains at least one Src homology 2 (SH2) domain that interacts directly with the phosphorylated CD19 cytoplasmic domain. The affinities of binding of the SH2 domains of Vav, p85, and Grb2 to CD19 are each in the nanomolar range by surface plasmon resonance (Biacore) analysis. Binding of Lyn and PLCgamma2 do not fit 1:1 modeling. However, analyses of binding data (Lyn) and competition experiments (PLCgamma2) suggest that these bind with comparable affinity. Competition experiments demonstrate that SH2 domains whose binding is dependent on the same CD19 tyrosine(s) compete for binding, but these SH2 domains do not impede binding of different SH2 domains to other CD19 tyrosines. We conclude that binding to the CD19 cytoplasmic domain is multimeric, high affinity, and competitive. The high affinity of the interactions also suggests that tyrosines that were nonessential in vivo are nevertheless functional. A preliminary structural model suggests that CD19 forms a signaling complex containing multiple cytoplasmic proteins in close proximity to each other and to the plasma membrane.