Laboratory of Structural Biology and Cell Signaling, Institute of Microbiology, The Czech Academy of Sciences, Vídeňská 1083, 14220 Prague 4, Czech Republic.
Department of Biophysical Chemistry, J. Heyrovsky Institute of Physical Chemistry, The Czech Academy of Sciences, Dolejškova 2155/3, 18223 Prague 8, Czech Republic.
Int J Mol Sci. 2019 Apr 16;20(8):1884. doi: 10.3390/ijms20081884.
Mouse activating Nkrp1 proteins are commonly described as type II transmembrane receptors with disulfide-linked homodimeric structure. Their function and the manner in which Nkrp1 proteins of mouse strain (C57BL/6) oligomerize are still poorly understood. To assess the oligomerization state of Nkrp1 proteins, mouse activating EGFP-Nkrp1s were expressed in mammalian lymphoid cells and their oligomerization evaluated by Förster resonance energy transfer (FRET). Alternatively, Nkrp1s oligomers were detected by Western blotting to specify the ratio between monomeric and dimeric forms. We also performed structural characterization of recombinant ectodomains of activating Nkrp1 receptors. Nkrp1 isoforms c1, c2 and f were expressed prevalently as homodimers, whereas the Nkrp1a displays larger proportion of monomers on the cell surface. Cysteine-to-serine mutants revealed the importance of all stalk cysteines for protein dimerization in living cells with a major influence of cysteine at position 74 in two Nkrp1 protein isoforms. Our results represent a new insight into the oligomerization of Nkrp1 receptors on lymphoid cells, which will help to determine their function.
鼠源激活型 Nkrp1 蛋白通常被描述为具有二硫键连接的同源二聚体结构的 II 型跨膜受体。其功能以及鼠源（C57BL/6）Nkrp1 蛋白的寡聚化方式仍知之甚少。为了评估 Nkrp1 蛋白的寡聚化状态，在哺乳动物淋巴样细胞中表达了鼠源激活型 EGFP-Nkrp1s，并通过Förster 共振能量转移（FRET）评估其寡聚化。或者，通过 Western blot 检测 Nkrp1 蛋白寡聚体以确定单体和二聚体形式之间的比例。我们还对激活型 Nkrp1 受体的重组胞外结构域进行了结构表征。Nkrp1 同种型 c1、c2 和 f 主要以同源二聚体的形式表达，而 Nkrp1a 在细胞表面上表现出更大比例的单体。半胱氨酸到丝氨酸突变体揭示了所有突环半胱氨酸对活细胞中蛋白二聚化的重要性，其中两个 Nkrp1 蛋白同种型中第 74 位半胱氨酸的影响较大。我们的研究结果为淋巴样细胞上 Nkrp1 受体的寡聚化提供了新的见解，这将有助于确定它们的功能。
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