MOLECULAR DISTRIBUTION OF HUMAN ADRENERGIC RECEPTORS

这项研究的总体长期目标是确定 人类肾上腺素能受体(ARs)的调节机制。 在这一背景下，这一竞争性更新试图调查两个 以Alpha1aAR为模型的AR监管的具体方面--1) 变应性鼻炎物种特异性组织分布的机制 亚型，以及2)激动剂诱导的调节机制。 人类字母1aAR。在上一次授权期间，我们演示了AR 亚型特异性和物种依赖性的组织表达。尽管5英尺- 已有研究表明，调节序列可与组织特异性结合 转录因子，这些信息仅限于详细分析 只有几张纸巾。由于关于人类的比较信息 并获得大鼠α1AR亚型的组织分布。 上一次资助期间，以及在我们实验室进行的其他研究(非直接 导致了6.2kb的克隆和鉴定 人类α1aAR基因的5‘非编码区，我们处于一个独特的位置来检测 物种特异性组织表达(大鼠与 人类)是由5‘-调节基因序列的差异引起的。 这将通过克隆大鼠α1aAR 5‘UTR和 大鼠与人报告基因在大鼠脑内组织中表达的比较 感兴趣(新生大鼠心肌细胞、肝癌细胞系、SK-N-MC细胞)； 用从人和大鼠新鲜组织中分离的细胞核进行DNA酶I分析 感兴趣的(心脏、乳动脉、肝脏)也将用于 这些研究。在上一个授权期内，我们也研究了 疾病和年龄对人体心血管AR亚型的影响 表情。高血压、充血性心力衰竭和老龄化 增加循环中的儿茶酚胺水平，因此是一种自然进展 从我们以前的研究中得到的结论是检查ars对激动剂的反应。 曝光。在大鼠暴露于激动剂中，α1aAR是独一无二 新生儿心肌细胞模型导致上调和持续的信号传递， 而alpha1b和alpha1dar同时经历下调和 脱敏；这一途径已被证明在 α1AR介导的心肌肥厚并提出以下问题 Alpha1aAR是否能够进行脱敏。在……里面 初步研究发现，快速(不到5分钟)急性部分 人类α1aAR发生脱敏(信令保持50% 激动剂暴露30分钟后的幼稚受体)。因此，我们计划 来检验Alpha1aAR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α1aAR和COOH末端缺失 突变体以表征激动剂依赖(去甲肾上腺素[NE])和 非激动剂(佛波酯[PMA])途径，以及研究 受体磷酸化的作用(使用标记的HA的免疫沉淀法 受体)和隔离(全细胞结合试验)。基因突变 假定的磷酸化位点和嵌合的alpha1a/alpha1bARs 用来进一步定义这一进程背后的机制。广泛性 Ars分子药理学培训，蛋白质方面的经验 脱敏的生物化学、克隆5‘UTRs和 人类α1aAR基因的特征，以及 几个人类Alpha1aAR记者已经在实验室里构建了， 使我们处于独特的位置来阐明潜在的机制 以α1aAR为模型对AR进行调控。来自这些的结果 研究应促进治疗性干预(例如，增强人类 组织靶向用于基因治疗)以及对 心血管疾病的潜在机制。

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.