Potter L R, Hunter T
Molecular Biology and Virology Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037, USA.
Mol Biol Cell. 1999 Jun;10(6):1811-20. doi: 10.1091/mbc.10.6.1811.
Dephosphorylation of the natriuretic peptide receptor-A (NPR-A) is hypothesized to mediate its desensitization in response to atrial natriuretic peptide (ANP) binding. Recently, we identified six phosphorylation sites within the kinase homology domain of NPR-A and determined that the conversion of these residues to alanine abolished the ability of the receptor to be phosphorylated or to be activated by ANP and ATP. In an attempt to generate a form of NPR-A that mimics a fully phosphorylated receptor but that is resistant to dephosphorylation, we engineered a receptor variant (NPR-A-6E) containing glutamate substitutions at all six phosphorylation sites. Consistent with the known ability of negatively charged glutamate residues to substitute functionally, in some cases, for phosphorylated residues, we found that NPR-A-6E was activated 10-fold by ANP and ATP. As determined by guanylyl cyclase assays, the hormone-stimulated activity of the wild-type receptor declined over time in membrane preparations in vitro, and this loss was blocked by the serine/threonine protein phosphatase inhibitor microcystin. In contrast, the activity of NPR-A-6E was more linear with time and was unaffected by microcystin. The nonhydrolyzable ATP analogue adenosine 5'-(beta,gamma-imino)-triphosphate was half as effective as ATP in stimulating the wild-type receptor but was equally as potent in stimulating NPR-A-6E, suggesting that ATP is required to keep the wild-type but not 6E variant phosphorylated. Finally, the desensitization of NPR-A-6E in whole cells was markedly blunted compared with that of the wild-type receptor, consistent with its inability to shed the negative charge from its kinase homology domain via dephosphorylation. These data provide the first direct test of the requirement for dephosphorylation in guanylyl cyclase desensitization and they indicate that it is an essential component of this process.
利钠肽受体-A（NPR-A）的去磷酸化被认为介导了其对心房利钠肽（ANP）结合的脱敏作用。最近，我们在NPR-A的激酶同源结构域内鉴定出六个磷酸化位点，并确定将这些残基转化为丙氨酸消除了受体被ANP和ATP磷酸化或激活的能力。为了生成一种模拟完全磷酸化受体但对去磷酸化具有抗性的NPR-A形式，我们设计了一种受体变体（NPR-A-6E），在所有六个磷酸化位点都含有谷氨酸替代物。与带负电荷的谷氨酸残基在某些情况下能够在功能上替代磷酸化残基的已知能力一致，我们发现NPR-A-6E被ANP和ATP激活了10倍。通过鸟苷酸环化酶测定确定，在体外膜制剂中，野生型受体的激素刺激活性随时间下降，这种损失被丝氨酸/苏氨酸蛋白磷酸酶抑制剂微囊藻毒素阻断。相比之下，NPR-A-6E的活性随时间更呈线性，且不受微囊藻毒素影响。不可水解的ATP类似物腺苷5'-（β，γ-亚氨基）-三磷酸在刺激野生型受体方面的效力只有ATP的一半，但在刺激NPR-A-6E方面同样有效，这表明ATP是维持野生型而非6E变体磷酸化所必需的。最后，与野生型受体相比，NPR-A-6E在全细胞中的脱敏作用明显减弱，这与其无法通过去磷酸化从其激酶同源结构域去除负电荷一致。这些数据首次直接测试了鸟苷酸环化酶脱敏中去磷酸化的必要性，并表明它是该过程的一个重要组成部分。
