Data_Sheet_1_Epitope-tagged and phosphomimetic mouse models for investigating natriuretic peptide-stimulated receptor guanylyl cyclases.docx

The natriuretic peptide receptors NPR1 and NPR2, also known as guanylyl cyclase A and guanylyl cyclase B, have critical functions in many signaling pathways, but much remains unknown about their localization and function in vivo. To facilitate studies of these proteins, we developed genetically modified mouse lines in which endogenous NPR1 and NPR2 were tagged with the HA epitope. To investigate the role of phosphorylation in regulating NPR1 and NPR2 guanylyl cyclase activity, we developed mouse lines in which regulatory serines and threonines were substituted with glutamates, to mimic the negative charge of the phosphorylated forms (NPR1-8E and NPR2-7E). Here we describe the generation and applications of these mice. We show that the HA-NPR1 and HA-NPR2 mice can be used to characterize the relative expression levels of these proteins in different tissues. We describe studies using the NPR2-7E mice that indicate that dephosphorylation of NPR2 transduces signaling pathways in ovary and bone, and studies using the NPR1-8E mice that indicate that the phosphorylation state of NPR1 is a regulator of heart, testis, and adrenal function.

钠尿肽受体NPR1和NPR2，亦称鸟苷酸环化酶A和鸟苷酸环化酶B，在众多信号通路中扮演着至关重要的角色，然而关于它们在体内的定位和功能仍有许多未知。为了促进对这些蛋白的研究，我们开发了一系列基因修饰小鼠品系，其中内源性的NPR1和NPR2被标记了HA表位。为了探究磷酸化在调节NPR1和NPR2鸟苷酸环化酶活性中的作用，我们创建了小鼠品系，其中调控的丝氨酸和苏氨酸被替换为谷氨酸，以模拟磷酸化形式的负电荷（NPR1-8E和NPR2-7E）。在此，我们描述了这些小鼠的生成及其应用。我们发现，HA-NPR1和HA-NPR2小鼠可用于表征这些蛋白在不同组织中的相对表达水平。我们描述了利用NPR2-7E小鼠的研究，这些研究表明NPR2的去磷酸化可以传导卵巢和骨的信号通路，以及利用NPR1-8E小鼠的研究，这些研究表明NPR1的磷酸化状态是心脏、睾丸和肾上腺功能调节的因子。