RESUMEN

We constructed an expression vector for a fusion protein [ANG II type 1a receptor-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 (CHO) cells transfected with AT(1a)R-GFP demonstrated specific, high-affinity (125)I-labeled ANG II binding (IC(50) 21 nM). ANG II exposure stimulated sodium-proton exchange and cytoplasmic calcium release to a similar extent in cells transfected with AT(1a)R or AT(1a)R-GFP; these responses were desensitized by prior exposure to ANG II and were sensitive to the AT(1)R blocker losartan. ANG II-driven internalization of AT(1a)R-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 AT(1a)R-GFP exhibits similar pharmacological behavior to that of the native AT(1a)R. Our observations also support 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.

Asunto(s)

Núcleo Celular/metabolismo , Indicadores y Reactivos/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Receptores de Angiotensina/genética , Receptores de Angiotensina/metabolismo , Angiotensina II/farmacología , Animales , Antihipertensivos/farmacología , Transporte Biológico/efectos de los fármacos , Transporte Biológico/genética , Células CHO , Calcio/metabolismo , Cricetinae , Colorantes Fluorescentes , Genes Reporteros , Proteínas Fluorescentes Verdes , Radioisótopos de Yodo , Losartán/farmacología , Microscopía Confocal , Compuestos de Quinolinio , Ensayo de Unión Radioligante , Ratas , Receptores de Angiotensina/agonistas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Tiazoles