Laboratory for GPCR Biology, Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA; Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
Laboratory for GPCR Biology, Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
J Biol Chem. 2022 May;298(5):101836. doi: 10.1016/j.jbc.2022.101836. Epub 2022 Mar 17.
Phosphate homeostasis, mediated by dietary intake, renal absorption, and bone deposition, is incompletely understood because of the uncharacterized roles of numerous implicated protein factors. Here, we identified a novel role for one such element, regulator of G protein signaling 14 (RGS14), suggested by genome-wide association studies to associate with dysregulated Pi levels. We show that human RGS14 possesses a carboxy-terminal PDZ ligand required for sodium phosphate cotransporter 2a (NPT2A) and sodium hydrogen exchanger regulatory factor-1 (NHERF1)-mediated renal Pi transport. In addition, we found using isotope uptake measurements combined with bioluminescence resonance energy transfer assays, siRNA knockdown, pull-down and overlay assays, and molecular modeling that secreted proteins parathyroid hormone (PTH) and fibroblast growth factor 23 inhibited Pi uptake by inducing dissociation of the NPT2A-NHERF1 complex. PTH failed to affect Pi transport in cells expressing RGS14, suggesting that it suppresses hormone-sensitive but not basal Pi uptake. Interestingly, RGS14 did not affect PTH-directed G protein activation or cAMP formation, implying a postreceptor site of action. Further pull-down experiments and direct binding assays indicated that NPT2A and RGS14 bind distinct PDZ domains on NHERF1. We showed that RGS14 expression in human renal proximal tubule epithelial cells blocked the effects of PTH and fibroblast growth factor 23 and stabilized the NPT2A-NHERF1 complex. In contrast, RGS14 genetic variants bearing mutations in the PDZ ligand disrupted RGS14 binding to NHERF1 and subsequent PTH-sensitive Pi transport. In conclusion, these findings identify RGS14 as a novel regulator of hormone-sensitive Pi transport. The results suggest that changes in RGS14 function or abundance may contribute to the hormone resistance and hyperphosphatemia observed in kidney diseases.
磷酸盐稳态由饮食摄入、肾脏吸收和骨骼沉积介导，但由于许多涉及的蛋白因子的作用尚未确定，因此尚未完全了解。在这里，我们通过全基因组关联研究发现了一种新的蛋白因子调节剂的作用，该蛋白因子调节剂是 G 蛋白信号转导 14（RGS14），该蛋白因子调节剂与磷酸盐水平失调有关。我们证明了人类 RGS14 具有羧基末端 PDZ 配体，该配体是钠磷酸盐共转运蛋白 2a（NPT2A）和钠氢交换调节因子 1（NHERF1）介导的肾脏磷酸盐转运所必需的。此外，我们通过同位素摄取测量、生物发光共振能量转移测定、siRNA 敲低、下拉和覆盖测定以及分子建模发现，分泌蛋白甲状旁腺激素（PTH）和成纤维细胞生长因子 23 通过诱导 NPT2A-NHERF1 复合物的解离来抑制磷酸盐摄取。PTH 未能影响表达 RGS14 的细胞中的磷酸盐转运，表明它抑制了激素敏感但不是基础磷酸盐摄取。有趣的是，RGS14 不影响 PTH 定向的 G 蛋白激活或 cAMP 形成，这意味着它在受体后发挥作用。进一步的下拉实验和直接结合测定表明，NPT2A 和 RGS14 结合 NHERF1 上不同的 PDZ 结构域。我们表明，人肾近端小管上皮细胞中 RGS14 的表达阻断了 PTH 和成纤维细胞生长因子 23 的作用，并稳定了 NPT2A-NHERF1 复合物。相比之下，携带 PDZ 配体突变的 RGS14 遗传变异破坏了 RGS14 与 NHERF1 的结合以及随后的 PTH 敏感的磷酸盐转运。总之，这些发现确定了 RGS14 是一种新的激素敏感磷酸盐转运调节剂。结果表明，RGS14 功能或丰度的变化可能导致肾脏疾病中观察到的激素抵抗和高磷酸盐血症。
