de Koning J P, Soede-Bobok A A, Schelen A M, Smith L, van Leeuwen D, Santini V, Burgering B M, Bos J L, Lowenberg B, Touw I P
Institute of Hematology, Erasmus University and Dr. Daniel den Hoed Cancer Center, Rotterdam, The Netherlands.
Blood. 1998 Mar 15;91(6):1924-33.
The membrane-distal region of the cytoplasmic domain of human granulocyte colony-stimulating factor receptor (G-CSF-R) contains four conserved tyrosine residues: Y704, Y729, Y744, and Y764. Three of these (Y729, Y744, and Y764) are located in the C-terminal part of G-CSF-R, previously shown to be essential for induction of neutrophilic differentiation. To determine the role of the tyrosines in G-CSF-mediated responses, we constructed tyrosine-to-phenylalanine (Y-to-F) substitution mutants and expressed these in a differentiation competent subclone of 32D cells that lacks endogenous G-CSF-R. We show that all tyrosines can be substituted essentially without affecting the differentiation signaling properties of G-CSF-R. However, substitution of one specific tyrosine, ie, Y764, markedly influenced proliferation signaling as well as the timing of differentiation. 32D cells expressing wild-type (WT) G-CSF-R (or mutants Y704F, Y729F, or Y744F) proliferated in G-CSF-containing cultures until day 8 and then developed into mature neutrophils. In contrast, 32D/Y764F cells arrested in the G1 phase of the cell cycle within 24 hours and showed complete neutrophilic differentiation after 3 days of culture. This resulted in an average 30-fold reduction of neutrophil production as compared with the 32D/WT controls. Importantly, G-CSF-mediated activation of Shc, p21Ras and the induction of c-myc were severely reduced by substitution of Y764. These findings indicate that Y764 of G-CSF-R is crucial for maintaining the proliferation/differentiation balance during G-CSF-driven neutrophil development and suggest a role for multiple signaling mechanisms in maintaining this balance.
人粒细胞集落刺激因子受体（G-CSF-R）胞质结构域的膜远端区域含有四个保守的酪氨酸残基：Y704、Y729、Y744和Y764。其中三个（Y729、Y744和Y764）位于G-CSF-R的C末端，先前已证明其对诱导嗜中性粒细胞分化至关重要。为了确定酪氨酸在G-CSF介导的反应中的作用，我们构建了酪氨酸到苯丙氨酸（Y-to-F）替代突变体，并在缺乏内源性G-CSF-R的32D细胞的分化能力亚克隆中表达这些突变体。我们发现所有酪氨酸都可以被替代，基本上不影响G-CSF-R的分化信号特性。然而，一个特定酪氨酸即Y764的替代显著影响增殖信号以及分化的时间。表达野生型（WT）G-CSF-R（或突变体Y704F、Y729F或Y744F）的32D细胞在含G-CSF的培养物中增殖至第8天，然后发育成成熟的嗜中性粒细胞。相比之下，32D/Y764F细胞在24小时内停滞在细胞周期的G1期，并在培养3天后表现出完全的嗜中性粒细胞分化。与32D/WT对照相比，这导致嗜中性粒细胞产生平均减少30倍。重要的是，Y764的替代严重降低了G-CSF介导的Shc、p21Ras激活以及c-myc的诱导。这些发现表明，G-CSF-R的Y764对于在G-CSF驱动的嗜中性粒细胞发育过程中维持增殖/分化平衡至关重要，并提示多种信号机制在维持这种平衡中发挥作用。
