REGULATION OF HORMONE-RESPONSIVE ADENYLATE CYCLASE

激素反应性腺苷酸环化酶的调节

• 批准号: 3968963
• 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/3968963
• 关键词: Leydig cells; adenylate cyclase; affinity chromatography; aging; beta adrenergic agent; chloroquine; chorionic gonadotropin; corpus striatum; cyclic AMP; drug interactions; glioma; hormone regulation /control mechanism; hypersensitivity desensitization; immunochemistry; isoproterenol; membrane activity; membrane fusion; membrane permeability; phosphorylation; testis neoplasms; tissue /cell culture

1. Exposure of mammalian cells to isoproterenol resulted in a rapid loss of agonist-stimulated adenylate cyclase activity. Desensitization was accompanied by a sequestration of Beta-adrenergic receptors into a lighter density membrane fraction inaccessible to hydrophilic antagonists. When the cells were washed free of agonist, resensitization and reappearance of the sequestered receptors occurred. Pretreatment of the cells with concanavalin A blocked sequestration but not desensitization. Using a membrane fusion technique to transfer the receptors to a foreign adenylate cyclase, we were able to show that a reduction in receptor function occurred during desensitization and in the absence of sequestration. Upon resensitization, receptor function was recovered. Thus, the key event in agonist-mediated desensitization is a reduction in receptor function. 2. Exposure of rat striatal membranes to N-ethylmaleimide (NEM) caused a loss of D-1 dopamine receptors. D-1 specific agonists or antagonists, respectively, fully and partially protected the receptors from inactivation by NEM. Upon transfer of the receptors to a foreign adenylate cyclase by membrane fusion, agonist- but not antagonist-protected D-1 receptors remained functional as measured by agonist stimulation of the cyclase. We have now succeeded in solubilizing the D-1 receptor and are using the selective protection by agonist as a means to purify the receptor. 3. Deglycosylated human chorionic gonadotropin (DG-hCG) is a potent antagonist of hCG. When DG-hCG is bound to hCG-receptors on MLTC-1 cells, addition of anti-hCG reverses the antagonism and results in stimulation of adenylate cyclase. Rabbit antibodies raised to DG-hCG cross-reacted with hCG. When the antiserum was purified on an hCG-agarose affinity column, the nonabsorbed fraction only reacted with DG-hCG and did not reverse its antagonism. In contrast, the absorbed antibodies reacted preferentially with the Beta-subunit of hCG and DG-hCG, reversed the antagonism of DG-hCG and this reversal was blocked by prior incubation of the antibodies with the Beta- but not the Alpha-subunits. Thus, epitopes on the Beta-subunit may be important for determining whether the hormone is an agonist or antagonist.

1.将哺乳动物细胞暴露于异丙肾上腺素， 激动剂刺激的腺苷酸环化酶活性。 脱敏是 伴随着β-肾上腺素能受体的隔离进入打火机 亲水性拮抗剂不可及的密度膜部分。 当 洗涤细胞，使其不含激动剂，使其再敏化，并使其再出现。 被隔离的受体出现了。 用以下物质预处理细胞 伴刀豆球蛋白A阻断隔离但不阻断脱敏。 使用 膜融合技术将受体转移到外源腺苷酸 环化酶，我们能够证明受体功能的降低 发生在脱敏过程中，没有隔离。 后 复敏后，受体功能恢复。 因此， 激动剂介导的脱敏是受体功能的降低。 2. 大鼠纹状体膜暴露于N-乙基马来酰亚胺（NEM）， D-1多巴胺受体。 D-1特异性激动剂或拮抗剂， 分别完全和部分保护受体免于失活 的NEM。 当受体转移到外源腺苷酸环化酶时， 膜融合，激动剂保护而非拮抗剂保护的D-1受体 通过环化酶的激动剂刺激来测量保持功能。 我们 现在已经成功地溶解了D-1受体，并正在使用 通过激动剂的选择性保护作为纯化受体的手段。 3. 去糖基化人绒毛膜促性腺激素（DG-hCG）是一种有效的拮抗剂 的hCG。 当DG-hCG与MLTC-1细胞上的hCG-受体结合时，添加 抗-hCG逆转拮抗作用并导致腺苷酸的刺激 环化酶 抗DG-hCG的兔抗体与hCG发生交叉反应。 当 在hCG-琼脂糖亲和柱上纯化抗血清， 未吸收部分仅与DG-hCG反应，不逆转其 对抗 相反，被吸收的抗体优先反应， 与hCG β亚基和DG-hCG作用，逆转DG-hCG的拮抗作用 这种逆转被预先将抗体与 β亚基而不是α亚基 因此，β亚基上的表位 对于确定激素是否是激动剂或 拮抗剂