Identification of a putative nuclear localization sequence within ANG II AT(1A) receptor associated with nuclear activation.

Angiotensin II (ANG II) type 1 (AT(1)) receptors, similar to other G protein-coupled receptors, undergo desensitization and internalization, and potentially nuclear localization, subsequent to agonist interaction. Evidence suggests that the carboxy-terminal tail may be involved in receptor nuclear localization. In the present study, we examined the carboxy-terminal tail of the receptor for specific regions responsible for the nuclear translocation phenomenon and resultant nuclear activation. Human embryonic kidney cells stably expressing either a wild-type AT(1A) receptor-green fluorescent protein (AT(1A)R/GFP) construct or a site-directed mutation of a putative nuclear localization sequence (NLS) [K307Q]AT(1A)R/GFP (KQ/AT(1A)R/GFP), were examined for differences in receptor nuclear trafficking and nuclear activation. Receptor expression, intracellular signaling, and ANG II-induced internalization of the wild-type/GFP construct and of the KQ/AT(1A)R/GFP mutant was similar. Laser scanning confocal microscopy showed that in cells expressing the AT(1A)R/GFP, trafficking of the receptor to the nuclear area and colocalization with lamin B occurred within 30 min of ANG II (100 nM) stimulation, whereas the KQ/AT(1A)R/GFP mutant failed to demonstrate nuclear localization. Immunoblotting of nuclear lysates with an anti-GFP antibody confirmed these observations. Nuclear localization of the wild-type receptor correlated with increase transcription for both EGR-1 and PTGS-2 genes while the nuclear-deficient KQ/AT(1A)R/GFP mutant demonstrated increases for only the EGR-1 gene. These results suggest that a NLS (KKFKKY; aa307-312) is located within the cytoplasmic tail of the AT(1A) receptor and that nuclear localization of the receptor corresponds with specific activation of transcription for the COX-2 gene PTGS-2.