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1 Genetics Graduate Program, University of Iowa, Iowa City, IA, USA
2 Department of Internal Medicine, University of Iowa, Iowa City, IA, USA
3 Genetics Graduate Program, University of Iowa, Iowa City, IA, USA; Department of Internal Medicine, University of Iowa, Iowa City, IA, USA; Department of Physiology and Biophysics, University of Iowa, Iowa City, IA, USA
* To whom correspondence should be addressed. E-mail: curt-sigmund{at}uiowa.edu.
In the brain, angiotensinogen (AGT) is primarily expressed in astrocytes; and brain Ang-II derived from locally produced AGT has been shown to influence blood pressure. In order to better understand the molecular basis of AGT expression in the brain, we identified a human astrocytoma cell line, CCF-STTG1 (CCF) that expresses endogenous AGT mRNA and produces AGT protein. Studies examining CCF cell AGT after N- and O-glycosidase suggests that AGT may not be post-translationally modified by glycosylation in these cells as it is in plasma. Small amounts of AGT (5% of HepG2) were detected in the culture media suggesting a low rate of AGT secretion. Immunocytochemical examination of AGT in CCF cells revealed mainly nuclear localization. Although this was has not been previously reported, it is consistent with nuclear localization of other serpin family members. To examine this further, we generated a fusion protein consisting of green fluorescent protein (GFP) and human AGT and examined subcellular localization by confocal microscopy after confirming expression of the fusion protein by Western blot. In CCF cells, a control GFP construct lacking AGT was mainly localized in the cytoplasm, whereas the GFP-AGT fusion protein was primarily localized in the nucleus. To map the location of a potential nuclear localization signal, overlapping 500 bp fragments of hAGT cDNA were fused in frame downstream of GFP. Whereas, four of the fusion proteins exhibited either perinuclear or cytoplasmic localization, one fusion protein encoding the C-terminus of AGT was localized in the nucleus. Importantly, nuclear localization of human AGT was confirmed in primary cultures of glial cells isolated from transgenic mice expressing the human AGT under the control of its own endogenous promoter. Our results suggest that AGT may have a novel intracellular role in the brain apart from its predicted endocrine function.
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