MOLECULAR DISTRIBUTION OF HUMAN ADRENERGIC RECEPTORS

人肾上腺素能受体的分子分布

• 批准号: 6537047
• 负责人: Debra Anne Schwinn
• 金额: $ 38.43万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-09-01 至 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6537047
• 关键词: adrenergic receptor; affinity labeling; cell line; chimeric proteins; deoxyribonuclease I; gel mobility shift assay; gene deletion mutation; genetic promoter element; genetic regulatory element; hormone regulation /control mechanism; human tissue; in situ hybridization; laboratory rat; liver cells; nucleic acid sequence; phosphorylation; receptor expression; receptor sensitivity; second messengers; species difference; transcription factor; transfection

The overall long term objective of this research is to determine mechanisms underlying regulation of human adrenergic receptors (ARs). Within this context, this competitive renewal seeks to investigate two specific aspects of AR regulation using the alpha1aAR as a model--1) mechanisms underlying species specific tissue distribution of AR subtypes, and 2) mechanisms underlying agonist-induced regulation of the human alpha1aAR. During the last grant period we demonstrated AR subtype specific and species-dependent tissue expression. Although 5'- regulatory sequences have been shown to bind tissue specific transcription factors, this information is limited to detailed analysis of only a few tissues. Since comparative information regarding human and rat tissue distribution of alpha1AR subtypes was obtained during the last grant period, and other studies in our laboratory (not directly related to this grant) resulted in cloning and characterization of 6.2kb 5'UTR of the human alpha1aAR gene, we are in a unique position to test the hypothesis that species specific tissue expression (rat versus human) of ARs results from differences in 5'-regulatory gene sequences. This will be accomplished by cloning the rat alpha1aAR 5'UTR and comparing rat versus human reporter construct expression in tissues of interest (rat neonatal myocytes, hepatoma cell lines, SK-N-MC cells); DNase I assays using nuclei isolated from fresh human and rat tissues of interest (heart, mammary artery, liver) will also be utilized for these studies. During the previous grant period we also examined the influence of disease and age on human cardiovascular AR subtype expression. Hypertension, congestive heart failure, and aging all increase circulating catecholamine levels, hence a natural progression from our previous studies is to examine the response of ARs to agonist exposure. The alpha1aAR is unique in that agonist exposure in a rat neonatal myocyte model results in upregulation and continued signaling, while alpha1b and alpha1dARs undergo concurrent downregulation and desensitization; this pathway has been shown to be important in alpha1AR-mediated myocardial hypertrophy and raises the question of whether alpha1aARs are capable of undergoing desensitization. In preliminary studies we find that rapid (less than 5 min) acute partial desensitization of human alpha1aARs occurs (signaling remains 50 percent of naive receptor after 30 min of agonist exposure). Therefore we plan to test the hypothesis that alpha1aAR acute partial desensitization occurs via mechanisms distinct from other ARs, enabling persistent signaling in the presence of agonist. These studies will utilize a hemagglutinin (HA)-tagged human alpha1aAR and COOH-terminal deletion mutant to characterize agonist-dependent (norepinephrine [NE]) and agonist-independent (phorbol ester [PMA]) pathways, as well as study the role of receptor phosphorylation (using immunoprecipitation of HA tagged receptors) and sequestration (whole cell binding assays). Mutation of putative phosphorylation sites and chimeric alpha1a/alpha1bARs will then be used to further define mechanisms underlying this process. Extensive training in molecular pharmacology of ARs, experience in protein biochemistry of desensitization, experience in cloning 5'UTRs and characterizing the human alpha1aAR gene, as well as availability of several human alpha1aAR reporter constructs already in the laboratory, places us in a unique position to elucidate mechanisms underlying regulation of ARs using the alpha1aAR as a model. Results from these studies should facilitate therapeutic interventions (e.g. enhanced human tissue targeting for gene therapy) as well as understanding of mechanisms underlying cardiovascular diseases.

这项研究的总体长期目标是确定 人类肾上腺素受体（AR）的调节机制。 在此背景下，本次竞争性更新旨在调查两个 使用 alpha1aAR 作为模型的 AR 调节的具体方面--1) AR 物种特异性组织分布的机制 亚型，以及 2) 激动剂诱导调节的潜在机制 人类α1aAR。 在上一次资助期间，我们展示了 AR 亚型特异性和物种依赖性组织表达。 虽然5'- 调节序列已被证明可以结合组织特异性 转录因子，此信息仅限于详细分析 仅由少数组织组成。 由于有关人类的比较信息 并获得α1AR亚型的大鼠组织分布 最后的资助期，以及我们实验室的其他研究（不直接 与本次资助相关）导致 6.2kb 的克隆和表征 人类 alpha1aAR 基因的 5'UTR，我们处于测试的独特位置 物种特异性组织表达（大鼠与 人类）的 AR 是由 5' 调节基因序列的差异引起的。 这将通过克隆大鼠 alpha1aAR 5'UTR 和 比较大鼠和人类报告构建体在组织中的表达 兴趣（大鼠新生肌细胞、肝癌细胞系、SK-N-MC细胞）； 使用从新鲜人类和大鼠组织中分离的细胞核进行 DNase I 测定 感兴趣的（心脏、乳动脉、肝脏）也将用于 这些研究。 在上一次资助期间，我们还审查了 疾病和年龄对人类心血管AR亚型的影响 表达。 高血压、充血性心力衰竭和衰老 增加循环儿茶酚胺水平，因此是自然进展 我们之前的研究是为了检查 ARs 对激动剂的反应 接触。 α1aAR 在大鼠中的激动剂暴露中是独特的 新生儿肌细胞模型导致上调和持续信号传导， 而 alpha1b 和 alpha1dAR 同时发生下调，并且 脱敏；该途径已被证明在以下方面很重要 α1AR 介导的心肌肥大并提出了以下问题： α1aAR是否能够进行脱敏。 在 初步研究我们发现快速（少于 5 分钟）的急性部分 人类 α1aAR 发生脱敏（信号传导仍保持 50%） 暴露于激动剂 30 分钟后的幼稚受体）。 因此我们计划 检验α1aAR急性部分脱敏的假设 通过与其他 AR 不同的机制发生，从而实现持久 在激动剂存在的情况下发出信号。 这些研究将利用 血凝素 (HA) 标记的人 alpha1aAR 和 COOH 末端缺失 突变体具有激动剂依赖性（去甲肾上腺素 [NE]）和 激动剂独立（佛波酯 [PMA]）途径，以及研究 受体磷酸化的作用（使用HA标记的免疫沉淀 受体）和隔离（全细胞结合测定）。突变 然后假定的磷酸化位点和嵌合 alpha1a/alpha1bAR 用于进一步定义该过程背后的机制。 广泛的 AR分子药理学培训，蛋白质方面的经验 脱敏生物化学、克隆5'UTR的经验和 表征人类 alpha1aAR 基因，以及可用性 实验室中已经构建了几种人类 alpha1aAR 报告基因， 使我们处于一个独特的位置来阐明潜在的机制 使用 alpha1aAR 作为模型来调节 AR。 这些结果 研究应促进治疗干预（例如增强人类 基因治疗的组织靶向）以及对 心血管疾病的潜在机制。