Pullen S S, Labadia M E, Ingraham R H, McWhirter S M, Everdeen D S, Alber T, Crute J J, Kehry M R
Department of Biology, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, Connecticut 06877-0368, USA.
Biochemistry. 1999 Aug 3;38(31):10168-77. doi: 10.1021/bi9909905.
Signaling by some TNF receptor family members, including CD40, is mediated by TNF receptor-associated factors (TRAFs) that interact with receptor cytoplasmic domains following ligand-induced receptor oligomerization. Here we have defined the oligomeric structure of recombinant TRAF domains that directly interact with CD40 and quantitated the affinities of TRAF2 and TRAF3 for CD40. Biochemical and biophysical analyses demonstrated that TRAF domains of TRAF1, TRAF2, TRAF3, and TRAF6 formed homo-trimers in solution. N-terminal deletions of TRAF2 and TRAF3 defined minimal amino acid sequences necessary for trimer formation and indicated that the coiled coil TRAF-N region is required for trimerization. Consistent with the idea that TRAF trimerization is required for high-affinity interactions with CD40, monomeric TRAF-C domains bound to CD40 significantly weaker than trimeric TRAFs. In surface plasmon resonance studies, a hierarchy of affinity of trimeric TRAFs for trimeric CD40 was found to be TRAF2 > TRAF3 >> TRAF1 and TRAF6. CD40 trimerization was demonstrated to be sufficient for optimal NF-kappaB and p38 mitogen activated protein kinase activation through wild-type CD40. In contrast, a higher degree of CD40 multimerization was necessary for maximal signaling in a cell line expressing a mutated CD40 (T254A) that signaled only through TRAF6. The affinities of TRAF proteins for oligomerized receptors as well as different requirements for degree of receptor multimerization appear to contribute to the selectivity of TRAF recruitment to receptor cytoplasmic domains.
包括CD40在内的一些肿瘤坏死因子受体（TNF）家族成员的信号传导是由TNF受体相关因子（TRAFs）介导的，这些因子在配体诱导的受体寡聚化后与受体胞质结构域相互作用。在此，我们确定了与CD40直接相互作用的重组TRAF结构域的寡聚结构，并定量了TRAF2和TRAF3对CD40的亲和力。生化和生物物理分析表明，TRAF1、TRAF2、TRAF3和TRAF6的TRAF结构域在溶液中形成同源三聚体。TRAF2和TRAF3的N端缺失确定了三聚体形成所需的最小氨基酸序列，并表明卷曲螺旋TRAF-N区域是三聚化所必需的。与TRAF三聚化是与CD40高亲和力相互作用所必需的观点一致，与CD40结合的单体TRAF-C结构域的亲和力明显低于三聚体TRAFs。在表面等离子体共振研究中，发现三聚体TRAFs对三聚体CD40的亲和力等级为TRAF2 > TRAF3 >> TRAF1和TRAF6。已证明CD40三聚化足以通过野生型CD40实现最佳的核因子κB（NF-κB）和p38丝裂原活化蛋白激酶激活。相比之下，在表达仅通过TRAF6信号传导的突变型CD40（T254A）的细胞系中，最大信号传导需要更高程度的CD40多聚化。TRAF蛋白对寡聚化受体的亲和力以及对受体多聚化程度的不同要求似乎有助于TRAF募集到受体胞质结构域的选择性。
