Evidence of an important and direct role for protein kinase C in agonist-induced phosphorylation leading to desensitization of the angiotensin AT(1A) receptor
Evidence of an important and direct role for protein kinase C in agonist-induced phosphorylation leading to desensitization of the angiotensin AT(1A) receptor
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DOI：
10.1038/sj.bjp.0701522
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发表时间：
1997-12-01
影响因子：
7.3
通讯作者：
Ball, SG
中科院分区：
文献类型：
--
作者：
Balmforth, AJ;Shepherd, FH;Ball, SG
1 蛋白激酶 C (PKC) 在血管紧张素 AT(1) 受体快速激动剂诱导脱敏机制中的作用仍未解决。一个主要问题是以充分纯化的形式分离这些受体，以便研究它们的磷酸化状态。2 将可裂解的 (His)(6) 亲和标签引入重组 AT(1A) 受体的 N 末端，并在人胚胎肾细胞中稳定表达。该亲和标签可以快速分离、纯化和测定 AT(1A) 受体的磷酸化状态。使用这些细胞，我们在相同条件下确定了 PKC 在激动剂诱导的受体磷酸化和脱敏中的作用。3 在低浓度和高浓度的血管紧张素 II (AII) 下均观察到激动剂诱导的 AT(1A) 受体磷酸化。将细胞与 Ro-31-8220（一种 PKC 特异性抑制剂）预孵育表明，在低浓度 AII (1 nM) 下，PKC 似乎是参与受体磷酸化的主要激酶。相比之下，在高浓度 AII (100 nM) 下，尽管观察到 PKC 介导的受体磷酸化，但这被第二种激酶所掩盖。4 在初步脱敏研究中，我们观察到，在低浓度 AII 下，与 Ro-31-8220 预孵育会减弱脱敏作用，而在高浓度 AII (100 nM) 下，它对观察到的脱敏水平影响很小或没有影响。5 这些数据直接证明了 PKC 诱导的受体磷酸化与低浓度 AII 脱敏之间的关联。由于 AII 的循环浓度在皮摩尔范围内，我们认为 PKC 是 AT(1) 受体脱敏的生理相关介质。
1 The role of protein kinase C (PKC) in the mechanism underlying rapid agonist-induced desensitization of angiotensin AT(1) receptors remains unresolved. A major problem has been to isolate these receptors in a sufficiently purified form to allow study of their phosphorylation state.2 A cleavable (His)(6) affinity tag was introduced into the N-terminus of the recombinant AT(1A) receptor and stably expressed in human embryonic kidney cells. This affinity tag allowed rapid isolation, purification and determination of the phosphorylation state of the AT(1A) receptor. Using these cells, we determined the role of PKC in both agonist-induced receptor phosphorylation and desensitization under identical conditions.3 Agonist-induced phosphorylation of the AT(1A) receptor was observed at both low and high concentrations of angiotensin II (AII). Preincubation of cells with Ro-31-8220 (a PKC specific inhibitor) revealed that at low concentrations of AII (1 nM), PKC appeared to be the main kinase involved in receptor phosphorylation. In contrast, at high concentrations of AII (100 nM), although PKC-mediated phosphorylation of the receptor was observed, this was overshadowed by a second kinase.4 In preliminary desensitization studies we observed that at a low concentration of AII, preincubation with Ro-31-8220 attenuated desensitization, whilst at high concentrations of AII (100 nM) it had little or no effect on the level of desensitization observed.5 These data directly demonstrate an association between PKC-induced receptor phosphorylation and desensitization at low concentrations of AII. Since circulating concentrations of AII are in the picomolar range, we propose that PKC is the physiologically relevant mediator of AT(1) receptor desensitization.