Department of Physiology 1, University of Tübingen, Gmelinstr. 5, D-72076 Tübingen, Germany.
Biochem Biophys Res Commun. 2013 Feb 8;431(2):186-91. doi: 10.1016/j.bbrc.2012.12.137. Epub 2013 Jan 9.
Na(+) coupled phosphate transporter NaPiIIa is the main carrier accomplishing phosphate transport across the apical cell membrane of proximal renal tubules and thus renal tubular phosphate reabsorption. The carrier is regulated by a wide variety of hormones and cellular signaling molecules. Hormones stimulating renal tubular phosphate transport and thus leading to hyperphosphatemia include growth hormone. Signaling of growth hormone involves activation of janus-activated kinase-2 JAK2, which has previously been shown to participate in the regulation of several Na(+) coupled transporters. Experiments exploring the effect of JAK2 on phosphate transport have, however, never been reported. The present study thus addressed the effect of JAK2 on NaPiIIa.
cRNA encoding NaPiIIa was injected into Xenopus oocytes with or without additional injection of cRNA encoding wild type JAK2, the gain of function mutant JAK2(V617F) or inactive JAK2(K882E). Phosphate-induced current (I(NaPi)) reflecting electrogenic phosphate transport was determined by two electrode voltage clamp. Moreover, NaPiIIa protein abundance in the cell membrane was determined by chemiluminescence.
No appreciable I(NaPi) was observed in water injected oocytes or in oocytes expressing JAK2 alone. In NaPiIIa expressing oocytes I(NaPi) was significantly increased by additional expression of JAK2 or JAK2(V617F), but not by coexpression of JAK2(K882E). In oocytes expressing both, NaPiIIa and JAK2, I(NaPi) was gradually decreased by JAK2 inhibitor AG490 (40 μM). Coexpression of NaPiIIa and JAK2 or JAK2(V617F), but not of JAK2(K882E) increased NaPiIIa protein abundance in the cell membrane. Disruption of carrier protein insertion with Brefeldin A (5 μM) was followed by a decline of I(NaPi) to a similar extent in Xenopus oocytes expressing NaPiIIa with JAK2 and in Xenopus oocytes expressing NaPiIIa alone, suggesting that JAK2 did not affect carrier stability in the cell membrane.
JAK2 contributes to the regulation of phosphate transporter NaPiIIa.
Na(+) 偶联磷酸盐转运体 NaPiIIa 是在近端肾小管的顶端细胞膜上完成磷酸盐转运并实现肾小管磷酸盐重吸收的主要载体。该载体受多种激素和细胞信号分子调节。刺激肾小管磷酸盐转运从而导致高磷酸盐血症的激素包括生长激素。生长激素的信号转导涉及到 Janus 激活激酶-2 (JAK2) 的激活，此前已证明 JAK2 参与了几种 Na(+) 偶联转运体的调节。然而，从未有研究报道 JAK2 对磷酸盐转运的影响。本研究因此探讨了 JAK2 对 NaPiIIa 的影响。
用编码 NaPiIIa 的 cRNA 注射非洲爪蟾卵母细胞，或在注射编码野生型 JAK2、功能获得性突变体 JAK2(V617F)或无活性 JAK2(K882E)的 cRNA 。通过双电极电压钳测定反映电致磷酸化转运的磷酸诱导电流 (I(NaPi))。此外，通过化学发光法测定质膜中 NaPiIIa 蛋白的丰度。
在水注射卵母细胞或单独表达 JAK2 的卵母细胞中，未观察到明显的 I(NaPi)。在表达 NaPiIIa 的卵母细胞中，I(NaPi) 显著增加了 JAK2 或 JAK2(V617F)的表达，但不增加 JAK2(K882E)的表达。在同时表达 NaPiIIa 和 JAK2 的卵母细胞中，JAK2 抑制剂 AG490（40 μM）逐渐降低 I(NaPi)。NaPiIIa 和 JAK2 或 JAK2(V617F)的共表达，但不是 JAK2(K882E)的共表达，增加了质膜中 NaPiIIa 蛋白的丰度。用布雷非德菌素 A（5 μM）破坏载体蛋白插入后，在同时表达 NaPiIIa 和 JAK2 的非洲爪蟾卵母细胞中和在单独表达 NaPiIIa 的非洲爪蟾卵母细胞中，I(NaPi) 均下降到相似程度，表明 JAK2 不影响质膜中载体的稳定性。
JAK2 有助于磷酸盐转运体 NaPiIIa 的调节。
