ALPHA1-AR LOCALIZATION IN CARDIAC MYOCYTES

这个子项目是许多利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 本项目研究了α 1-肾上腺素能受体（α 1-AR）在成人心肌细胞中的定位，我们以前证明，它只表达α 1A和α 1B亚型。先前的报道表明，α 1-AR在心肌细胞的小窝中表达，尽管由于缺乏α 1-AR抗体，该证据是从小窝中α 1-AR信号伴侣的存在推断的。因此，为了确定α 1-AR在肌细胞中的定位，我们采用了另一种方法来检查WT肌细胞中内源性α 1-AR的定位。我们使用了α 1-AR拮抗剂哌唑嗪的荧光类似物BODIPY-哌唑嗪，它以相同的亲和力结合所有α 1-AR亚型，仅在与受体结合时发出荧光。共聚焦显微镜显示，内源性α 1-AR在培养的成年小鼠心肌细胞的核周（而不是在膜上）。为了进一步确定α 1亚型的定位，我们开发了表达α 1荧光融合蛋白与绿色荧光蛋白（GFP）的腺病毒构建体，以在缺乏内源性α 1-AR的培养的α 1 ABKO心肌细胞中重建α 1-AR。我们最初的实验表明，α 1A-和α 1B-GFP定位于α 1ABKO心肌细胞的核膜，与核膜蛋白LAP 2共定位证实。总之，我们证明了α 1-AR在心肌细胞的核膜上表达。

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.