A functional angiotensin II receptor-GFP fusion protein: evidence for agonist-dependent nuclear translocation

Abstract

We constructed an expression vector for a fusion protein [ANG II type 1a receptor-green fluorescent protein (AT1aR-GFP)] consisting of enhanced GFP attached to the COOH terminus of the rat AT1aR. Chinese hamster ovary (CHO) cells transfected with AT1aR-GFP demonstrated specific, high-affinity 125I-labeled ANG II binding (IC50 21 nM). ANG II exposure stimulated sodium-proton exchange and cytoplasmic calcium release to a similar extent in cells transfected with AT1aR or AT1aR-GFP; these responses were desensitized by prior exposure to ANG II and were sensitive to the AT1R blocker losartan. ANG II-driven internalization of AT1aR-GFP in transfected CHO cells was demonstrated both by radioligand binding and by laser scanning confocal microscopy. Colocalization of GFP fluorescence with that of the nuclear stain TOTO-3 in confocal images was increased more than twofold after 1 h of ANG II exposure. We conclude that AT1aR-GFP exhibits similar pharmacological behavior to that of the native AT1aR. Our observations also support previous evidence for the presence of AT1aR in the nucleus and suggest that the density of AT1aR in the nucleus may be regulated by exposure to its ligand.