Intracellular angiotensin II fusion protein alters AT1 receptor fusion protein distribution and activates CREB.
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.
Cook
Julia L
JL
Division of Research, Ochsner Clinic Foundation, New Orleans, LA 70121, USA. jcook@ochsner.org
Re
Richard
R
Alam
Jawed
J
Hart
Michael
M
Zhang
Zhuo
Z
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
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