TRIO encodes a cytoskeletal regulatory protein with three catalytic domains—two guanine exchange factor (GEF) domains, GEF1 and GEF2, and a kinase domain—as well as several accessory domains that have not been extensively studied. Function-damaging variants in the TRIO gene are known to be enriched in individuals with neurodevelopmental disorders (NDDs). Disease variants in the GEF1 domain or the nine adjacent spectrin repeats (SRs) are enriched in NDDs, suggesting that dysregulated GEF1 activity is linked to these disorders. We provide evidence here that the Trio SRs interact intramolecularly with the GEF1 domain to inhibit its enzymatic activity. We demonstrate that SRs 6-9 decrease GEF1 catalytic activity both in vitro and in cells and show that NDD-associated variants in the SR8 and GEF1 domains relieve this autoinhibitory constraint. Our results from chemical cross-linking and bio-layer interferometry indicate that the SRs primarily contact the pleckstrin homology region of the GEF1 domain, reducing GEF1 binding to the small GTPase Rac1. Together, our findings reveal a key regulatory mechanism that is commonly disrupted in multiple NDDs and may offer a new target for therapeutic intervention for TRIO-associated NDDs.

TRIO编码的细胞骨架调节蛋白具有三个催化结构域——两个鸟嘌呤交换因子 (GEF) 结构域、GEF1 和 GEF2，以及一个激酶结构域——以及几个尚未被广泛研究的附属结构域。已知TRIO基因中的功能破坏变异在患有神经发育障碍 (NDD) 的个体中丰富。GEF1 结构域或九个相邻的血影蛋白重复序列​​ (SR) 中的疾病变异在 NDD 中富集，表明 GEF1 活性失调与这些疾病有关。我们在此提供证据表明 Trio SR 与 GEF1 结构域在分子内相互作用以抑制其酶活性。我们证明 SRs 6-9在体外降低 GEF1 催化活性并在细胞中显示 SR8 和 GEF1 域中与 NDD 相关的变体解除了这种自抑制约束。我们的化学交联和生物层干涉测量结果表明，SR 主要接触 GEF1 结构域的 pleckstrin 同源区域，从而减少 GEF1 与小 GTPase Rac1 的结合。总之，我们的研究结果揭示了一种关键的调节机制，这种机制通常在多种 NDD 中被破坏，并且可能为TRIO相关 NDD 的治疗干预提供新的目标。