Identification of multiple binding sites for atrial natriuretic factor by affinity cross-linking in cultured endothelial cells.
Identification of multiple binding sites for atrial natriuretic factor by affinity cross-linking in cultured endothelial cells.
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DOI：
10.1016/s0021-9258(18)67292-8
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发表时间：
1986-09
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影响因子：
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通讯作者：
D. Leitman;J. Andresen;T. Kuno;Y. Kamisaki;J. Chang;F. Murad
中科院分区：
文献类型：
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作者：
D. Leitman;J. Andresen;T. Kuno;Y. Kamisaki;J. Chang;F. Murad
在之前的研究中，我们发现心房肽I在增加培养内皮细胞环GMP方面的作用远弱于心房利钠因子（ANF-(8-33)） （EC50 = 0.3 nM） （EC50大于500 nM）。在这项研究中，我们使用交联试剂二琥珀酰亚基来研究活性的差异是否由于多个ANF受体的存在。当交联后内皮细胞上98%的ANF结合位点被酪氨酸-心房肽I占据时，在循环GMP积累方面，ANF-(8-33)的浓度-反应曲线没有差异。相比之下，当96%的结合位点被交联的ANF-占据时（8-33），读取ANF-后观察到最大循环GMP反应降低了60%（8-33）。这些结果表明，ANF-(8-33)结合到atriopeptin I不能有效结合的另一个位点。125I-ANF与完整内皮细胞的亲和交联导致Mr约66,000和约130,000两个位点的标记。大约94%的125I-ANF结合位点的Mr值约为66,000。酪氨酸-atriopeptin I （KI = 0.9 nM）和ANF-(8-33) （KI = 0.09 nM）均抑制该位点的标记。虽然0.1微米酪氨酸-心房肽（AP I）抑制66,000道尔顿位点标记的程度与ANF-(8-33)几乎相同，但与ANF-(8-33)相比，它只产生了4倍的环状GMP增加。这些结果表明66000道尔顿位点不耦合于鸟苷酸环化酶和环GMP的形成。酪氨酸- ap I （KI大于10 nM）对130,000道尔顿位点的竞争明显弱于ANF-(8-33) （KI = 0.075 nM）。由于酪氨酸- ap I（大于100 nM）和ANF-(8-33) （0.4 nM）的环GMP刺激的EC50更接近130,000道顿蛋白的KI值，因此该位点可能介导了环GMP的显著刺激。我们的研究结果表明，内皮细胞含有两个ANF-的结合位点（8-33），并且表明只有较少的位点（Mr约为130,000）是与鸟苷酸环化酶激活偶联的受体。
In a previous study, we found that atriopeptin I was much weaker (EC50 greater than 500 nM) than atrial natriuretic factor (ANF-(8-33)) (EC50 = 0.3 nM) at increasing cyclic GMP in cultured endothelial cells. In this study, we used the cross-linking reagent disuccinimidyl suberate to investigate whether the differences in activity were due to the presence of multiple ANF receptors. When 98% of the ANF-binding sites on endothelial cells were occupied by tyrosine-atriopeptin I after cross-linking, there was no difference in the concentration-response curve to ANF-(8-33) with regard to cyclic GMP accumulation. In contrast, when 96% of the binding sites were occupied by cross-linked ANF-(8-33), a 60% decrease in the maximal cyclic GMP response was observed after the readdition of ANF-(8-33). These results suggest that ANF-(8-33) is binding to an additional site that atriopeptin I does not effectively bind. Affinity cross-linking of 125I-ANF to intact endothelial cells resulted in the labeling of two sites of Mr approximately 66,000 and approximately 130,000. Approximately 94% of the 125I-ANF binding sites had an Mr approximately 66,000. Labeling of this site was inhibited by both tyrosine-atriopeptin I (KI = 0.9 nM) and ANF-(8-33) (KI = 0.09 nM). Although 0.1 microM tyrosine-atriopeptin (AP I) inhibited labeling of the 66,000-dalton site to nearly the same degree as ANF-(8-33), it produced only a 4-fold increase in cyclic GMP compared to a 400-fold increase with ANF-(8-33). These results suggest that the 66,000-dalton site is not coupled to guanylate cyclase and cyclic GMP formation. Tyrosine-AP I (KI greater than 10 nM) was much weaker at competing for the 130,000-dalton site than ANF-(8-33) (KI = 0.075 nM). Because the EC50 for cyclic GMP stimulation for tyrosine-AP I (greater than 100 nM) and ANF-(8-33) (0.4 nM) is closer to the KI values for the 130,000-dalton protein, this site probably mediates the marked stimulation of cyclic GMP. Our results demonstrate that endothelial cells contain two binding sites for ANF-(8-33) and suggest that only the less abundant site (Mr approximately 130,000) is the receptor coupled to the activation of guanylate cyclase.