REGULATION OF RECEPTOR COUPLED ADENYLYLCYCLASE

受体偶联腺苷酸环化酶的调节

• 批准号: 3760245
• 负责人: P H FISHMAN
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3760245
• 关键词: adenylate cyclase; beta adrenergic agent; beta adrenergic receptor; cyclic AMP; human tissue; phosphorylation; protein kinase A; protein structure function; receptor binding; receptor coupling; receptor expression; receptor sensitivity; tissue /cell culture; western blottings

The goal of this project is to identify molecular mechanisms involved in the regulation of beta-adrenergic receptor (beta-AR)-coupled adenylylcyclase. We previously reported that human beta1AR and beta2AR are regulated differently by agonists whether the receptors are endogenously expressed in human cell lines or stably expressed in transfected hamster cell lines. When exposed to agonist, the cells expressing beta2AR exhibit a rapid, typical pattern of desensitization of agonist-stimulated adenylylcyclase. Both maximum stimulation (V/max) is reduced and dose response (K/act) is shifted to lower sensitivity. By contrast, agonist-treated cells expressing beta1AR display a shift in K/act, but little or no reduction in V/max. It has been shown that desensitization of the beta2AR is mediated by protein kinase A (PKA) via phosphorylation of the third intracellular loop of the receptor and by the beta-adrenergic receptor kinase (betaARK) via phosphorylation of the C-terminus. By using specific kinase inhibitors, we have found that the reduction in V/max was mediated by betaARK. This included the modest reduction observed for beta1AR. The shift in K/act was mediated by PKA. This was further confirmed by exposing the cells to a cAMP derivative. With cells expressing either subtype, there was a shift in K/act, but no reduction in V/max. These results indicated that human beta1AR was resistant to betaARK-mediated desensitization. By contrast, hamster cells expressing rat beta1AR displayed a pattern of agonist-mediated desensitization similar to that of cells expressing human beta2AR. Whereas human beta1AR and beta2AR are only 54% homologous, the human and rat beta1AR are 91% homologous. Thus, only a small region of the receptor may be involved in betaARK-mediated desensitization.

该项目的目标是确定参与的分子机制， β-肾上腺素能受体（β-AR）-偶联调节 腺苷酸环化酶我们以前报道过，人类β 1 AR和β 2 AR 受激动剂的调节不同， 在人细胞系中内源表达或在人细胞系中稳定表达。 转染的仓鼠细胞系。 当暴露于激动剂时，细胞 表达β 2 AR的人表现出快速的典型脱敏模式， 激动剂刺激的腺苷酸环化酶 两种最大刺激（V/max） 降低，剂量响应（K/act）向较低灵敏度移动。 相比之下，激动剂处理的表达β 1 AR的细胞显示出在细胞周期中的变化。 K/act，但V/max很少或没有降低。 已经显示 β 2 AR的脱敏是由蛋白激酶A（PKA）介导的， 受体的第三胞内环的磷酸化， β-肾上腺素能受体激酶（betaARK）通过磷酸化 C端通过使用特异性激酶抑制剂，我们发现， V/max的降低由β ARK介导。这包括温和的 观察到β 1 AR减少。 PKA介导K/act的变化。 通过将细胞暴露于cAMP衍生物进一步证实了这一点。 对于表达任一亚型的细胞，K/act发生了变化，但没有变化。 降低V/max。 这些结果表明，人β 1 AR 对β-ARK介导的脱敏有抵抗力。 相比之下，仓鼠 表达大鼠β 1 AR的细胞显示激动剂介导的 与表达人β 2AR的细胞类似的脱敏。 尽管人类β 1 AR和β 2 AR只有54%同源，但人类和 大鼠β 1 AR同源性为91%。 因此，只有一个小区域的 受体可能参与β-ARK介导的脱敏。