PDF(Pubmed)
Abstract：
The colony-stimulating factor 3 receptor (CSF3R) controls the growth of neutrophils, the most abundant type of white blood cell. In healthy neutrophils, signaling is dependent on CSF3R binding to its ligand, CSF3. A single amino acid mutation in CSF3R, T618I, instead allows for constitutive, ligand-independent cell growth and leads to a rare type of cancer called chronic neutrophilic leukemia. However, the disease mechanism is not well understood. Here, we investigated why this threonine to isoleucine substitution is the predominant mutation in chronic neutrophilic leukemia and how it leads to uncontrolled neutrophil growth. Using protein domain mapping, we demonstrated that the single CSF3R domain containing residue 618 is sufficient for ligand-independent activity. We then applied an unbiased mutational screening strategy focused on this domain and found that activating mutations are enriched at sites normally occupied by asparagine, threonine, and serine residues-the three amino acids which are commonly glycosylated. We confirmed glycosylation at multiple CSF3R residues by mass spectrometry, including the presence of GalNAc and Gal-GalNAc glycans at WT threonine 618. Using the same approach applied to other cell surface receptors, we identified an activating mutation, S489F, in the interleukin-31 receptor alpha chain. Combined, these results suggest a role for glycosylated hotspot residues in regulating receptor signaling, mutation of which can lead to ligand-independent, uncontrolled activity and human disease.
摘要：
集落刺激因子3受体（CSF3R）控制中性粒细胞的生长，最丰富的白细胞类型。在健康的中性粒细胞中，信号传导依赖于CSF3R与其配体的结合，CSF3.CSF3R单个氨基酸突变,T618I,相反，允许构成，不依赖配体的细胞生长，并导致一种罕见的癌症，称为慢性中性粒细胞白血病。然而,疾病的机制还不清楚。这里,我们调查了为什么这种苏氨酸到异亮氨酸的替代是慢性中性粒细胞白血病的主要突变,以及它是如何导致中性粒细胞生长失控的.使用蛋白质结构域作图，我们证明了含有残基618的单个CSF3R结构域对于配体非依赖性活性是足够的。然后，我们应用了针对该结构域的无偏突变筛选策略，发现激活突变在通常由天冬酰胺占据的位点富集，苏氨酸,和丝氨酸残基-通常被糖基化的三个氨基酸。我们通过质谱证实了多个CSF3R残基的糖基化，包括在WT苏氨酸618处存在GalNAc和Gal-GalNAc聚糖。使用相同的方法应用于其他细胞表面受体，我们发现了一个激活突变,S489F,在白细胞介素-31受体α链中。合并,这些结果表明糖基化热点残基在调节受体信号传导中的作用，其突变可导致不依赖配体，不受控制的活动和人类疾病。
