The endocytic inhibitors MDC and CPZ and dynamin

The endocytic inhibitors MDC and CPZ and dynamin GTPase blocker (dynamin mutant) have been shown to significantly decrease the intracellular accumulation of cGMP. Moreover, earlier reports suggested that G-protein-coupled receptor (GPCRs) continue to signal by generating cAMP throughout internalization processes with their bound ligands [124]. Signaling from inside the cell is persistent and appears to trigger specific downstream effects. We have recently demonstrated that intracellular accumulation of cGMP significantly decreases after treatment with endocytic inhibitors MDC and CPZ and transfection with dynamin mutant GTPase blocker [69]. Diminution in the intracellular accumulation of cGMP indicates that these same endocytic inhibitors and dynamin GTPase blocker (dynamin mutant) have a concerted regulatory role in NPRA trafficking and signaling process.
Post-endocytic sorting of NPRA Recently we visualized the internalization and subcellular trafficking of enhanced GFP (eGFP)-tagged NPRA (eGFP–NPRA) in intact recombinant HEK-293 TAME and primary MMCs, using immunofluorescence and co-IP analyses of eGFP–NPRA [26,42]. These studies showed that treatment of cells with ANP initiated rapid internalization and co-localization of the receptor with early endosome antigen-1 (EEA-1), which was increased in a time-dependent manner. The highest visualization, which occurred at 5 min, gradually decreased within 30 min. Similarly, co-localization of the receptor was time-dependently accomplished with lysosome-associated membrane protein-1 (LAMP-1) in MMCs [26]. These immunofluorescence and co-IP studies demonstrated that after trafficking in the endosomes, eGFP-NPRA routed intracellularly within the late endosomes and/or lysosomes. The accumulation of eGFP–NPRA in intracellular compartments was observed after treatment of MMCs with lysosomotropic agents such as chloroquine and NH4Cl2. In ANP-treated cells, eGFP–NPRA was time-dependently co-localized with LAMP-1; however, after treatment with lysosomotropic agents, intracellular accumulation of the receptor gradually increased within 30 min. It is known that both chloroquine and NH4Cl2 inhibit lysosomal degradation of intracellular trafficking of the various ligand-receptor complexes [34,35,40,125]. Moreover, lysosomotropic agents did not completely block the receptor degradation of ANP/NPRA complexes in lysosomes, suggesting that the trafficking of intact eGFP–NPRA also occurred through a lysosome-independent pathway. In our studies, co-IP assays confirmed that the localization of internalized NPRA occurred within subcellular organelles during endocytosis of eGFP-NPRA [42]. The endocytic recycling marker Rab 11, which was used as a recycling endosome, showed that ∼20 % of receptors recycled back to the plasma membrane [26,42]. Our results support the notion that ANP-treated cells exhibit a marked increase in the IF of cGMP, whereas receptor still trafficked into intracellular compartments. This suggests that after ligand binding, NPRA is rapidly internalized and trafficked from the cell surface into endosomes, including recycling endosomes, late endosomes, and lysosomes, with concurrent generation of intracellular second-messenger cGMP. On the other hand, subcellular trafficking of NPRA indicated that IF of the mutated receptor with EEA-1, LAMP-1, and Rab 11 markers was decreased by almost 50%-58% in early endosomes, 45%-50% in lysosomes, and 35%-45% in recycling endosomes, respectively, compared with WT NPRA in MMCs and HEK-293 cells [26,42]. To support the findings of immunofluorescence-based internalization kinetics and cell trafficking of WT and mutated eGFP-NPRA in intact MMCs, we have used the 125I-ANP binding assay to examine cell-surface and intracellular ligand-receptor complexes [26]. These studies demonstrated that 125I-ANP binds to cell-surface eGFP-NPRA, enters through the process of receptor-mediated endocytosis, and then is delivered to intracellular compartments until it reaches a steady-state level, which occurs after 15-30 min. The kinetic rates of internalization, degradation, and release of 125I-ANP were markedly decreased in the mutant receptor, suggesting that the FQQI motif is important for the internalization and subcellular trafficking itinerary of eGFP-NPRA in intact cells [26]. Thus, it seems that homeostatic regulation of native NPRA and the cell sensitivity of ANP depends on a dynamic equilibrium and reuse of ligand-receptor complexes from the cell surface to intracellular compartments.