Phosphate (Pi)-regulated heterodimerization of the high-affinity sodium-dependent Pi transporters PiT1/Slc20a1 and PiT2/Slc20a2 underlies extracellular Pi sensing independently of Pi uptake
Phosphate (Pi)-regulated heterodimerization of the high-affinity sodium-dependent Pi transporters PiT1/Slc20a1 and PiT2/Slc20a2 underlies extracellular Pi sensing independently of Pi uptake
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DOI：
10.1074/jbc.m117.807339
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发表时间：
2018-02-09
影响因子：
4.8
通讯作者：
Beck, Laurent
中科院分区：
文献类型：
--
作者：
Bon, Nina;Couasnay, Greig;Beck, Laurent
细胞外磷酸盐（P-i）可以作为信号分子直接改变基因表达和细胞生理学。细胞或生物体检测细胞外 P-i 水平变化的能力意味着存在向身体或单个细胞发出信号的 P-i 传感机制。然而，与原核生物、酵母和植物不同，哺乳动物中参与 P-i 传感的分子参与者仍然未知。在这项研究中，我们研究了高亲和力、钠依赖性 P-i 转运蛋白 PiT1 和 PiT2 在介导骨骼细胞中 P-i 信号传导中的作用。我们发现 PiT1 或 PiT2 的缺失减弱了 P-i 依赖性 ERK1/2 介导的磷酸化以及随后的矿化抑制剂基质 Gla 蛋白和骨桥蛋白的基因上调。这一结果表明两个 PiT 对于 P-i 信号传导都是必需的。此外，ERK1/2 磷酸化可以通过过度表达 P-i 转运缺陷的 PiT 突变体来挽救。使用交联和生物发光共振能量转移方法，我们发现 PiT1 和 PiT2 形成高丰度同二聚体和 P-i 调节的低丰度异二聚体。有趣的是，在缺乏钠依赖性 P-i 转运活性的情况下，PiT1-PiT2 异二聚化仍然受到细胞外 P-i 水平的调节。值得注意的是，当两个假定的 P-i 结合残基 Ser-128（PiT1 中）和 Ser-113（PiT2 中）被丙氨酸取代时，PiT1-PiT2 异二聚化不再受细胞外 P-i 调节。这些观察结果表明，P-i 结合而不是 P-i 摄取可能是通过 PiT 蛋白介导 P-i 信号传导的关键因素。综上所述，这些结果表明 P-i 调节的 PiT1-PiT2 异二聚化介导独立于 P-i 摄取的 P-i 传感。
Extracellular phosphate (P-i) can act as a signaling molecule that directly alters gene expression and cellular physiology. The ability of cells or organisms to detect changes in extracellular P-i levels implies the existence of a P-i-sensing mechanism that signals to the body or individual cell. However, unlike in prokaryotes, yeasts, and plants, the molecular players involved in P-i sensing in mammals remain unknown. In this study, we investigated the involvement of the high-affinity, sodium-dependent P-i transporters PiT1 and PiT2 in mediating P-i signaling in skeletal cells. We found that deletion of PiT1 or PiT2 blunted the P-i-dependent ERK1/2-mediated phosphorylation and subsequent gene up-regulation of the mineralization inhibitors matrix Gla protein and osteopontin. This result suggested that both PiTs are necessary for P-i signaling. Moreover, the ERK1/2 phosphorylation could be rescued by overexpressing P-i transport-deficient PiT mutants. Using cross-linking and bioluminescence resonance energy transfer approaches, we found that PiT1 and PiT2 form high-abundance homodimers and P-i-regulated low-abundance heterodimers. Interestingly, in the absence of sodium-dependent P-i transport activity, the PiT1-PiT2 heterodimerization was still regulated by extracellular P-i levels. Of note, when two putative P-i-binding residues, Ser-128 (in PiT1) and Ser-113 (in PiT2), were substituted with alanine, the PiT1-PiT2 heterodimerization was no longer regulated by extracellular P-i. These observations suggested that P-i binding rather than P-i uptake may be the key factor in mediating P-i signaling through the PiT proteins. Taken together, these results demonstrate that P-i-regulated PiT1-PiT2 heterodimerization mediates P-i sensing independently of P-i uptake.