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

We constructed an expression vector for a fusion protein [ANG II type 1a re ceptor-green fluorescent protein (AT(1a)R-GFP)] consisting of enhanced GFP attached to the COOH terminus of the rat AT(1a)R. Chinese hamster ovary (CH O) cells transfected with AT(1a)R-GFP demonstrated specific, high-affinity I-125-labeled ANG II binding (IC50 21 nM). ANG II exposure stimulated sodiu m-proton exchange and cytoplasmic calcium release to a similar extent in ce lls transfected with AT(1a)R or AT(1a)R-GFP; these responses were desensiti zed by prior exposure to ANG II and were sensitive to the AT(1)R blocker lo sartan. ANG II-driven internalization of AT(1a)R-GFP in transfected CHO cel ls was demonstrated both by radioligand binding and by laser scanning confo cal 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 AT(1a)R-GFP exhibits similar pharmacol ogical behavior to that of the native AT(1a)R. Our observations also suppor t previous evidence for the presence of AT(1a)R in the nucleus and suggest that the density of AT(1a)R in the nucleus may be regulated by exposure to its ligand.