GRB2 is an adaptor protein required for facilitating cytoplasmic signaling complexes from a wide array of binding partners. GRB2 has been reported to exist in either a monomeric or dimeric state in crystal and solution. GRB2 dimers are formed by the exchange of protein segments between domains, otherwise known as “domain-swapping”. Swapping has been described between SH2 and C-terminal SH3 domains in the full-length structure of GRB2 (SH2/C–SH3 domain-swapped dimer), as well as between α-helixes in isolated GRB2 SH2 domains (SH2/SH2 domain-swapped dimer). Interestingly, SH2/SH2 domain-swapping has not been observed within the full-length protein, nor have the functional influences of this novel oligomeric conformation been explored. We herein generated a model of full-length GRB2 dimer with an SH2/SH2 domain-swapped conformation supported by in-line SEC–MALS–SAXS analyses. This conformation is consistent with the previously reported truncated GRB2 SH2/SH2 domain-swapped dimer but different from the previously reported, full-length SH2/C-terminal SH3 (C–SH3) domain-swapped dimer. Our model is also validated by several novel full-length GRB2 mutants that favor either a monomeric or a dimeric state through mutations within the SH2 domain that abrogate or promote SH2/SH2 domain-swapping. GRB2 knockdown and re-expression of selected monomeric and dimeric mutants in a T cell lymphoma cell line led to notable defects in clustering of the adaptor protein LAT and IL-2 release in response to TCR stimulation. These results mirrored similarly-impaired IL-2 release in GRB2-deficient cells. These studies show that a novel dimeric GRB2 conformation with domain-swapping between SH2 domains and monomer/dimer transitions are critical for GRB2 to facilitate early signaling complexes in human T cells.

GRB2 是一种接头蛋白，是促进多种结合配偶体形成细胞质信号复合物所需的。据报道，GRB2 在晶体和溶液中以单体或二聚体状态存在。 GRB2二聚体是通过域之间的蛋白质片段交换形成的，也称为“域交换”。 GRB2 全长结构中的 SH2 和 C 端 SH3 结构域之间的交换（SH2/C-SH3 结构域交换二聚体）以及孤立的 GRB2 SH2 结构域中的 α 螺旋之间的交换（SH2/SH2 结构域-交换二聚体）。有趣的是，在全长蛋白质中尚未观察到 SH2/SH2 结构域交换，也没有探索这种新型寡聚构象的功能影响。我们在此生成了全长 GRB2 二聚体模型，其具有 SH2/SH2 结构域交换构象，并得到在线 SEC-MALS-SAXS 分析的支持。该构象与之前报道的截短的GRB2 SH2/SH2结构域交换二聚体一致，但与之前报道的全长SH2/C末端SH3 (C–SH3)结构域交换二聚体不同。我们的模型还得到了几种新型全长 GRB2 突变体的验证，这些突变体通过 SH2 结构域内的突变促进单体或二聚体状态，从而消除或促进 SH2/SH2 结构域交换。 T 细胞淋巴瘤细胞系中 GRB2 敲低和选定单体和二聚体突变体的重新表达导致响应 TCR 刺激的接头蛋白 LAT 和 IL-2 释放的聚集出现显着缺陷。这些结果反映了 GRB2 缺陷细胞中 IL-2 释放的类似受损。 这些研究表明，具有 SH2 结构域之间的结构域交换和单体/二聚体转换的新型二聚体 GRB2 构象对于 GRB2 促进人类 T 细胞中的早期信号复合物至关重要。