VIB Center for Inflammation Research, VIB, Ghent, Belgium; Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium.
Department of Biochemistry, Stellenbosch University, Stellenbosch, South Africa.
J Biol Chem. 2022 Feb;298(2):101574. doi: 10.1016/j.jbc.2022.101574. Epub 2022 Jan 8.
The glucocorticoid (GC) receptor (GR) is essential for normal development and in the initiation of inflammation. Healthy GR mice with reduced dimerization propensity due to a point mutation (A465T) at the dimer interface of the GR DNA-binding domain (DBD) (here GR) have previously helped to define the functions of GR monomers and dimers. Since GR retains residual dimerization capacity, here we generated the dimer-nullifying double mutant GR mice, featuring an additional mutation (I634A) in the ligand-binding domain (LBD) of GR. These mice are perinatally lethal, as are GR mice (these mice have the I634A mutation but not the A465T mutation), displaying improper lung and skin formation. Using embryonic fibroblasts, high and low doses of dexamethasone (Dex), nuclear translocation assays, RNAseq, dimerization assays, and ligand-binding assays (and K values), we found that the lethal phenotype in these mice is due to insufficient ligand binding. These data suggest there is some correlation between GR dimerization potential and ligand affinity. We conclude that even a mutation as subtle as I634A, at a position not directly involved in ligand interactions sensu stricto, can still influence ligand binding and have a lethal outcome.
糖皮质激素（GC）受体（GR）对于正常发育和炎症的发生至关重要。由于 GR DNA 结合域（DBD）二聚体界面上的点突变（A465T），健康的 GR 小鼠（GR）的二聚化倾向降低，此前曾有助于定义 GR 单体和二聚体的功能。由于 GR 仍保留残余的二聚化能力，因此我们生成了二聚体缺失的双突变 GR 小鼠，其 GR 配体结合域（LBD）中还有一个额外的突变（I634A）。这些小鼠在围产期内具有致死性，就像 GR 小鼠一样（这些小鼠具有 I634A 突变但没有 A465T 突变），显示出肺部和皮肤形成不当。使用胚胎成纤维细胞、高剂量和低剂量地塞米松（Dex）、核易位测定、RNAseq、二聚化测定、配体结合测定（和 K 值），我们发现这些小鼠的致死表型是由于配体结合不足引起的。这些数据表明，GR 二聚化潜力与配体亲和力之间存在一定的相关性。我们得出结论，即使是像 I634A 这样微妙的突变，位于与配体相互作用无关的位置，也仍然可以影响配体结合并产生致死后果。
