Pullen S S, Miller H G, Everdeen D S, Dang T T, Crute J J, Kehry M R
Department of Biology, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, Connecticut 06877-0368, USA.
Biochemistry. 1998 Aug 25;37(34):11836-45. doi: 10.1021/bi981067q.
CD40 is a TNF receptor superfamily member that provides activation signals in antigen-presenting cells such as B cells, macrophages, and dendritic cells. Multimerization of CD40 by its ligand initiates signaling by recruiting TNF receptor-associated factors (TRAFs) to the CD40 cytoplasmic domain. Recombinant human TRAF proteins overexpressed in insect cells were biochemically characterized and used to finely map TRAF binding regions in the human CD40 cytoplasmic domain. TRAF1, TRAF2, TRAF3, and TRAF6, but not TRAF4 or TRAF5, bound directly to the CD40 cytoplasmic domain. CD40 interactions with TRAF2 and TRAF3 were stronger than the interactions with TRAF1 and TRAF6. Full-length TRAF3 and TRAF5 formed hetero-oligomers, presumably through their predicted isoleucine zippers. TRAF3-TRAF5 hetero-oligomers interacted with CD40, indicating that TRAF5 can be indirectly recruited to the CD40 cytoplasmic domain. Overlapping peptides synthesized on cellulose membranes were used to map each TRAF interaction region. TRAF1, TRAF2, and TRAF3 interacted with the same region. The recognition site for TRAF6 was a nonoverlapping membrane proximal region. Using peptides with progressive deletions, a minimal TRAF1, TRAF2, and TRAF3 binding region was mapped to the PVQET sequence in the CD40 cytoplasmic domain. The minimal region for TRAF6 binding was the sequence QEPQEINF. These studies demonstrate that the CD40 cytoplasmic domain contains two nonoverlapping TRAF binding regions and suggest that TRAF1, TRAF2, and TRAF3 could bind competitively to one site. Relative affinities and competition of individual and hetero-oligomeric TRAF proteins for CD40 binding sites may contribute to receptor specificity and cell-type selectivity in CD40-dependent signaling.
CD40是肿瘤坏死因子受体超家族成员，可在抗原呈递细胞（如B细胞、巨噬细胞和树突状细胞）中提供激活信号。CD40与其配体多聚化通过将肿瘤坏死因子受体相关因子（TRAFs）募集到CD40胞质结构域来启动信号传导。在昆虫细胞中过表达的重组人TRAF蛋白经生化特性鉴定，并用于精细定位人CD40胞质结构域中的TRAF结合区域。TRAF1、TRAF2、TRAF3和TRAF6可直接与CD40胞质结构域结合，而TRAF4或TRAF5则不能。CD40与TRAF2和TRAF3的相互作用强于与TRAF1和TRAF6的相互作用。全长TRAF3和TRAF5形成异源寡聚体，推测是通过其预测的异亮氨酸拉链。TRAF3-TRAF5异源寡聚体与CD40相互作用，表明TRAF5可间接募集到CD40胞质结构域。在纤维素膜上合成的重叠肽用于定位每个TRAF相互作用区域。TRAF1、TRAF2和TRAF3与同一区域相互作用。TRAF6的识别位点是一个不重叠的膜近端区域。使用具有逐步缺失的肽，将TRAF1、TRAF2和TRAF3的最小结合区域定位到CD40胞质结构域中的PVQET序列。TRAF6结合的最小区域是序列QEPQEINF。这些研究表明，CD40胞质结构域包含两个不重叠的TRAF结合区域，并提示TRAF1、TRAF2和TRAF3可能竞争性结合到一个位点。单个和异源寡聚体TRAF蛋白对CD40结合位点的相对亲和力和竞争可能有助于CD40依赖性信号传导中的受体特异性和细胞类型选择性。
