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MOLECULAR DISTRIBUTION OF HUMAN ADRENERGIC RECEPTORS

MOLECULAR DISTRIBUTION OF HUMAN ADRENERGIC RECEPTORS
人肾上腺素受体的分子分布
批准号:
6183596
负责人:
Debra Anne Schwinn
金额:
$27.33万
依托单位:
依托单位国家:
美国
项目类别:
财政年份:
1993
资助国家:
美国
项目状态:
已结题
起止时间:
1993-09-01 至 2004-03-31

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中文摘要
翻译
本研究的总体长期目标是确定 人肾上腺素能受体(AR)的潜在调节机制。 在这种情况下,这种竞争性的更新旨在调查两个 使用α 1aAR作为模型的AR调节的特定方面--1) AR种属特异性组织分布的机制 亚型,和2)激动剂诱导的调节的潜在机制, 人α 1aAR。 在上一个赠款期间,我们展示了AR 亚型特异性和物种依赖性组织表达。 虽然5 '- 调节序列已经显示出与组织特异性结合, 转录因子,这些信息仅限于详细的分析 只有几个组织。 因为关于人类的比较信息 并在实验过程中获得了α 1 AR亚型的大鼠组织分布。 上一个资助期,以及我们实验室的其他研究(不直接 相关的补助金)导致克隆和表征6.2kb 5 'UTR的人alpha 1aAR基因,我们在一个独特的位置,以测试 种属特异性组织表达(大鼠与 人)的AR的差异是由5 '调控基因序列的差异引起的。 这将通过克隆大鼠α 1aAR 5 'UTR和 比较大鼠与人报告基因构建体在组织中的表达, 感兴趣的(大鼠新生肌细胞、肝癌细胞系、SK-N-MC细胞); 使用从新鲜人和大鼠组织中分离的细胞核的DNA酶I测定 感兴趣的(心脏、乳腺动脉、肝脏)也将用于 这些研究。 在上一个资助期内,我们亦研究了 疾病和年龄对人心血管AR亚型影响 表情 高血压充血性心力衰竭和衰老 增加循环中的儿茶酚胺水平,因此是一种自然的进展 我们之前的研究是检查AR对激动剂的反应 exposure. alpha 1aAR在大鼠中的激动剂暴露是独特的, 新生肌细胞模型导致上调和持续的信号传导, 而alpha 1b和alpha 1dAR同时下调, 脱敏;这一途径已被证明在 α 1AR介导的心肌肥大,并提出了一个问题, alpha 1aAR是否能够经历脱敏。 在 初步研究我们发现,快速(小于5分钟)急性部分 人类α 1aAR发生脱敏(信号传导保持50% 在激动剂暴露30分钟后的初始受体)。 因此,我们计划 为了检验α 1aAR急性部分脱敏 通过与其他AR不同的机制发生, 在激动剂存在下的信号传导。 这些研究将利用 血凝素(HA)标记的人α 1aAR和COOH末端缺失 突变体表征激动剂依赖性(去甲肾上腺素[NE]), 激动剂非依赖性(佛波酯[PMA])途径,以及研究 受体磷酸化的作用(使用HA标记的 受体)和螯合(全细胞结合测定)。突变 假定的磷酸化位点和嵌合的α 1a/α 1bAR将 用于进一步界定这一进程的机制。 广泛 AR分子药理学培训,蛋白质 脱敏的生物化学,克隆5 'UTR的经验, 表征人α 1aAR基因,以及 实验室中已经存在的几种人α 1aAR报告基因构建体, 使我们处于一个独特的位置, 使用alpha 1aAR作为模型调节AR。 从这些 研究应促进治疗干预(例如,增强人类 组织靶向基因治疗)以及理解 心血管疾病的潜在机制。
英文摘要
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.
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Anesthesiology & Perioperative Medicine Research Training
  • 批准号:
    7876973
  • 项目类别:
  • 资助金额:
    $20.48万
  • 财政年份:
    2009
  • 负责人:
    Debra Anne Schwinn
  • 依托单位:
Anesthesiology & Perioperative Medicine Research Training
  • 批准号:
    7560479
  • 项目类别:
  • 资助金额:
    $13.6万
  • 财政年份:
    2009
  • 负责人:
    Debra Anne Schwinn
  • 依托单位:
Anesthesiology & Perioperative Medicine Research Training
  • 批准号:
    8094372
  • 项目类别:
  • 资助金额:
    $21.02万
  • 财政年份:
    2009
  • 负责人:
    Debra Anne Schwinn
  • 依托单位:
PHARMACOGENETICS OF CX(1A)-ADRENOCEPTORS IN HYPERTENSION
  • 批准号:
    7198453
  • 项目类别:
  • 资助金额:
    $3.48万
  • 财政年份:
    2005
  • 负责人:
    Debra Anne Schwinn
  • 依托单位:
海外基金