title: Intracellular angiotensin II fusion protein alters AT1 receptor fusion protein distribution and activates CREB.
abstract: In recently published studies, we show that angiotensin II (AII) generated from an engineered rat angiotensinogen cDNA, and maintained intracellularly, is growth stimulatory for a rat hepatoma cell line. In the present study, we report that co-expression of AII fused to cyan fluorescent protein (ECFP/AII) and angiotensin type I receptor fused to yellow fluorescent protein (AT1R/EYFP) enhances proliferation of COS-7 and CHO-K1 cells by 59% and 64%, respectively, compared to cells expressing the corresponding independent proteins (P < 0.001 for both). This effect is inhibited by losartan, suggesting (as in our previous published studies) that losartan is internalized by the cells, via receptor-mediated endocytosis, and thus inhibits intracellular receptor-ligand interaction. The growth effect is independent of anti-AII antibodies suggesting that it does not reflect AII secretion into the culture media; AII is also undetectable in the media. Expression of AT1R/EYFP with ECFP/AIIC (control scrambled sequence AII fused to ECFP) has no effect upon cell proliferation. ECFP/AII also alters the cellular localization of AT1R/EYFP. ECFP/AII is concentrated in the nucleus, but shows diffuse cytoplasmic fluorescence as well. AT1R/EYFP, expressed independently, is visible in the endoplasmic reticulum and Golgi apparatus of COS-7 and CHO-K1 cells as early as 24-h post-transfection. At 72 h, it is visibly associated with the plasma membrane. By 144 h, 85% of the cells show detectable circumferential fluorescence. In contrast, in cells that express AT1R/EYFP and ECFP/AII, both proteins accumulate in the nucleus and only 13% of the cells show visible plasma membrane-associated yellow fluorescence at 144 h (P < 0.001). Furthermore, co-expression of ECFP/AII with AT1R/EYFP stimulates cAMP response element-binding protein (CREB) activity in CHO-K1 and COS-7 cells. Exogenous AII similarly significantly increases CREB activation in AT1R/EYFP-stably transfected CHO-K1 and COS-7 cells.
authors.affiliation: Division of Research, Ochsner Clinic Foundation, New Orleans, LA 70121, USA. jcook@ochsner.org
authors.forename: Julia L
authors.initials: JL
authors.lastname: Cook
authors.forename: Richard
authors.initials: R
authors.lastname: Re
authors.forename: Jawed
authors.initials: J
authors.lastname: Alam
authors.forename: Michael
authors.initials: M
authors.lastname: Hart
authors.forename: Zhuo
authors.initials: Z
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D000804:Angiotensin II / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics; D001483:Base Sequence; D016466:CHO Cells; D019556:COS Cells; D002455:Cell Division; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003412:Cricetulus; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D005260:Female; D049452:Green Fluorescent Proteins; D042541:Intracellular Space / Q000378:metabolism*; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D044140:Receptor, Angiotensin, Type 1 / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: D001426:Bacterial Proteins; D017362:Cyclic AMP Response Element-Binding Protein; D008164:Luminescent Proteins; D044140:Receptor, Angiotensin, Type 1; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D000804:Angiotensin II; D049452:Green Fluorescent Proteins