ALPHA1-AR LOCALIZATION IN CARDIAC MYOCYTES

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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.