REGULATION OF HORMONE-RESPONSIVE ADENYLATE CYCLASE

激素反应性腺苷酸环化酶的调节

• 批准号: 3846196
• 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/3846196
• 关键词: CHO cells; adenosine triphosphate; adenylate cyclase; alkylation; beta adrenergic receptor; clone cells; dopamine receptor; enzyme inhibitors; enzyme mechanism; heparin; hormone regulation /control mechanism; human tissue; isoproterenol; neoplastic cell culture for noncancer research; protein kinase A; protein structure; receptor binding; receptor coupling; receptor expression; receptor sensitivity; transfection

Exposing cells to beta-adrenergic agonists results in multiple effects on the beta-adrenergic receptor-coupled adenylyl cyclase. The receptors rapidly become desensitized and less efficient at stimulating adenylyl cyclase; then the receptors become sequestered and inaccessible to hydrophilic ligands; and finally, the receptors are down-regulated as evidenced by a loss of antagonist binding. We previously found that the endogenous beta1-adrenergic receptors expressed by human neurotumor SK-N- MC cells are resistent to desensitization by the beta-adrenergic receptor kinase. The latter has been shown to desensitize the human beta2- adrenergic receptor. We also observed that the beta1 receptors are resistant to down-regulation but undergo sequestration. In order to explore possible differences in the regulation of the two receptor subtypes. we transfected BHK and CHW hamster cells with an expression vector into which we inserted a cDNA encoding one or the other receptor subtypes, and isolated stable receptor-expressing transformants. In addition, we obtained transfected beta1- and beta2- CHO hamster cells and beta2-L mouse cells from other laboratories. The different cell lines were exposed to the agonist isoproterenol for different times and assayed for sequestration, down-regulation and desensitization. In all the transfected cell lines tested, beta2 receptors underwent sequestration faster and more extensively than beta1 receptors. Down-regulation was much more complicated and appeared to be affected by cell type. Although we have only been able to thoroughly assess three cell lines for desensitization, the results are encouraging. When cells were exposed to agonist for 30 min, the two cells lines expressing beta1 receptors exhibited no significant reduction in maximum stimulation of adenylyl cyclase by agonist. By contrast, the one beta2 receptor-expressing cell line tested so far underwent a 43% desensitization. Although these results are incomplete, they suggest differences in agonist-mediated regulation of human beta1- and beta2-adrenergic receptors, particularly sequestration and desensitization. These differences may relate to structural differences in the two receptors, especially their C-termini.

细胞暴露于β-肾上腺素能激动剂会产生多种效应 关于β-肾上腺素能受体偶联的腺酰环化酶。受体 迅速变得不敏感，刺激腺苷的效率降低 环化酶；然后受体变得隔离，无法与 亲水性配体；最后，受体被下调为 证据是失去了拮抗剂的结合。我们之前发现， 人神经瘤SK-N-受体表达的内源性β1-肾上腺素能受体 MC细胞抵抗β-肾上腺素能受体的脱敏作用 激活剂。后者已被证明可以使人的β2脱敏。 肾上腺素能受体。我们还观察到Beta1受体是 抵制下调监管，但经历自动减支。为了 探索这两种受体在调节上的可能差异 子类型。我们用BHK和CHW仓鼠细胞进行了表达 我们在载体中插入了编码一种或另一种受体的cDNA 亚型，以及分离的稳定的受体表达转化子。在……里面 此外，我们还获得了转基因的Beta1和Beta2-CHO仓鼠细胞和 Beta2-L小鼠细胞来自其他实验室。不同的细胞系 分别与激动剂异丙肾上腺素作用不同时间后测定 用于隔离、下调调控和脱敏。在所有的 测试了转染性细胞系，β2受体发生了隔离 比Beta1受体更快、更广泛。下调监管是 要复杂得多，而且似乎受到细胞类型的影响。虽然 我们只能彻底评估三种细胞系 脱敏，结果令人鼓舞。当细胞暴露在 激动剂作用30min后，两株细胞表达β1受体 对腺苷酸的最大刺激没有明显的降低 激动剂的环化酶。相比之下，表达Beta2受体的细胞 到目前为止，测试的品系经历了43%的脱敏。尽管这些 结果是不完整的，它们表明了激动剂介导的差异 人β1和β2肾上腺素能受体的调节 隔离和脱敏。这些差异可能与 两种受体的结构差异，特别是它们的C-末端。