ALPHA1-AR LOCALIZATION IN CARDIAC MYOCYTES

ALPHA1-AR 在心肌细胞中的定位

• 批准号: 7610349
• 负责人: Timothy D O'Connell
• 金额: $ 0.23万
• 依托单位: UNIVERSITY OF SOUTH DAKOTA
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7610349
• 关键词: Adrenergic Receptor; Adult; Affinity; Antibodies; Binding; Cardiac Myocytes; Caveolae; Chimeric Proteins; Computer Retrieval of Information on Scientific Projects Database; Confocal Microscopy; Funding; Grant; Green Fluorescent Proteins; Institution; Localized; Membrane; Membrane Proteins; Mus; Muscle Cells; Nuclear Envelope; Prazosin; Reporting; Research; Research Personnel; Resources; Signal Transduction; Source; United States National Institutes of Health; analog; receptor; reconstitution; research study

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This project examined the localization of alpha1-adrenergic receptors (alpha1-AR) in adult cardiac myocytes, which we previously demonstrated express only the alpha1A- and alpha1B-subtypes. Previous reports suggest that alpha1-ARs are expressed in caveolae in cardiac myocytes, although due to the lack of alpha1-AR antibodies this evidence was infered from the pressence of alpha1-AR signaling partners in caveolae. Therefore, to define alpha1-AR localization in myocytes, we took an alternative approach to examine the localization of endogenous alpha1-ARs in WT myocytes. We used a fluorescent analog of the alpha1-AR antagonist prazosin, BODIPY-prazosin, which binds all alpha1-AR subtypes with equal affinity and fluoresces only when bound to receptor. Confocal microscopy revealed that endogenous alpha1-ARs were perinuclear (not on the membrane) in cultured adult mouse cardiac myocytes. To define further alpha1-subtype localization, we developed adenoviral constructs that express alpha1-fluorescent fusion proteins with green fluorescent protein (GFP) to reconstitute alpha1-ARs in cultured alpha1ABKO cardiac myocytes, which lack endogenous alpha1-ARs. Our initial experiments showed that both the alpha1A- and alpha1B-GFP localized to the nuclear membrane in alpha1ABKO myocytes, as verified by co-localization with the nuclear membrane protein LAP2. In summary, we demonstrated that alpha1-ARs are expressed on the nuclear membrane in cardiac myocytes.

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 本课题研究了α1肾上腺素能受体(Alpha1-AR)在成人心肌细胞中的定位，我们先前证明该受体仅表达Alpha1A和Alpha1B亚型。以前的报道表明，α1-AR在心肌细胞的小窝中表达，尽管由于缺乏α1-AR抗体，这一证据是从小窝中α1-AR信号伙伴的存在推断出来的。因此，为了确定α1-AR在心肌细胞中的定位，我们采用了另一种方法来检测内源性α1-AR在WT肌细胞中的定位。我们使用了α1-AR拮抗剂哌唑嗪的荧光类似物BODIPY-哌唑嗪，它以同样的亲和力结合所有α1-AR亚型，只有在与受体结合时才会发出荧光。共聚焦显微镜显示，在培养的成年小鼠心肌细胞中，内源性α1-Ars位于核周(不在膜上)。为了进一步确定Alpha1亚型的定位，我们开发了表达Alpha1荧光融合蛋白和绿色荧光蛋白(GFP)的腺病毒载体，以在缺乏内源性Alpha1-Ars的培养的Alpha1ABKO心肌细胞中重建Alpha1-Ars。我们的初步实验表明，Alpha1A-和Alpha1B-GFP都定位于Alpha1ABKO肌细胞的核膜，与核膜蛋白LAP2共同定位证实了这一点。综上所述，我们证明了α1-Ars在心肌细胞的核膜上表达。