Subcellular trafficking of guanylyl cyclase/natriuretic peptide receptor-A with concurrent generation of intracellular cGMP

Abstract

Atrial natriuretic peptide (ANP) activates guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA), which lowers blood pressure and blood volume. The objective of the present study was to visualize internalization and trafficking of enhanced GFP (eGFP)-tagged NPRA (eGFP-NPRA) in human embryonic kidney-293 (HEK-293) cells, using immunofluorescence (IF) and co-immunoprecipitation (co-IP) of eGFP-NPRA. Treatment of cells with ANP initiated rapid internalization and co-localization of the receptor with early endosome antigen-1 (EEA-1), which was highest at 5 min and gradually decreased within 30 min. Similarly, co-localization of the receptor was observed with lysosome-associated membrane protein-1 (LAMP-1); however, after treatment with lysosomotropic agents, intracellular accumulation of the receptor gradually increased within 30 min. Co-IP assays confirmed that the localization of internalized receptors occurred with subcellular organelles during the endocytosis of NPRA. Rab 11, which was used as a recycling endosome (Re) marker, indicated that ∼20% of receptors recycled back to the plasma membrane. ANP-treated cells showed a marked increase in the IF of cGMP, whereas receptor was still trafficking into the intracellular compartments. Thus, after ligand binding, NPRA is rapidly internalized and trafficked from the cell surface into endosomes, Res and lysosomes, with concurrent generation of intracellular cGMP.

Keywords: endosomes; guanylyl cyclase/natriuretic peptide receptor-A; immunofluorescence; lysosomes; receptor internalization.

Figure 1. Identification and localization of eGFP–NPRA fusion protein and kinetics of ANP-induced eGFP–NPRA and NPRA internalization in stably expressing recombinant HEK-293 cells

(A) Western blot analysis indicating the 162-kDa eGFP–NPRA fusion protein. (B) Western blot analyses of eGFP–NPRA and NPRA in stably expressing HEK-293 cells detected by using anti-NPRA antibody. (C) Schematic representation of eGFP–NPRA fusion protein. (D) The localization of NPRA through anti-NPRA antibody staining (Texas Red-conjugated secondary antibodies); (i) NPRA untransfected cells (overlay of anti-NPRA antibody staining and DAPI (blue) (ii) NPRA transfected cells (iii) overlay of anti-NPRA antibody staining and DAPI. (E) (i) localization of eGFP–NPRA (green fluorescence) (ii) localization of NPRA through anti-NPRA antibody staining; and (iii) overlay of anti-NPRA antibody staining, eGFP–NPRA and DAPI. (F) Series of single confocal plane images were taken from cells fixed with 4.0% formaldehyde to visualize the internalization of NPRA after stimulation by 100 nM ANP. These images of mid-focal planes are from five independent experiments. For the kinetic analyses of receptors, eGFP–NPRA and NPRA stably expressing cells, were pre-treated in the absence or presence of 200 μM chloroquine at 37°C for 1 h. Both control (G) and chloroquine-pre-treated (H) cells were allowed to bind ¹²⁵I-ANP at 4°C for 60 min. Cells were washed four times with ice-cold assay medium (2 ml per wash) to remove unbound ligand, then warmed to 37°C. At the indicated time intervals, cell-surface-associated (●, ○), internalized (■, □) and released (▲, ∆) ¹²⁵I-ANP radioactivity levels were determined in acid eluates, cell extracts and culture medium. (I and J) The composition of degraded ¹²⁵I-ANP in culture medium was analysed by determining the trichloroacetic acid-soluble degraded ¹²⁵I-ANP in supernatant. Each data point represents the mean ± S.E.M. of six separate experiments in triplicate dishes.

Figure 2. Internalization and co-localization of eGFP–NPRA with EEA-1 in HEK-293 cells

(A) In untreated cells, all receptors were localized in the plasma membrane. Cells were treated with 100 nM ANP for 5, 10, 15 or 30 min. Co-localization of eGFP–NPRA and EEA-1 marker was observed after 5 and 10 min of treatment with ANP, after which it gradually decreased from 15 to 30 min. The images represent mid-focal planes and five independent experiments. (B) Quantification of the percent of co-localization of eGFP–NPRA with EEA-1. Bars represent the mean ± S.E.M. ***P<0.001 relative to untreated cells. Scale bar=50 μm.

Figure 3. Co-localization of internalized eGFP–NPRA with LAMP-1 in HEK-293 cells

(A) In untreated cells, all receptors were localized in the plasma membrane. Cells were treated with 100 nM ANP for 5, 10, 15 or 30 min. Co-localization of eGFP–NPRA with LAMP-1 marker gradually increased from 5 to 30 min after treatment. The images represent mid-focal planes and are typical of 4–5 independent experiments. (B) Quantification of the percent co-localization of eGFP–NPRA with LAMP-1. Bars represent the mean ± S.E.M. ***P<0.001 relative to untreated cells. Scale bar=50 μm.

Figure 4. Co-localization of eGFP-NPRA with Rab11 in HEK-293 cells

(A) In untreated cells, all receptors were localized in the plasma membrane. Cells were treated with 100 nM ANP for 5, 10, 15 or 30 min. Co-localization of eGFP–NPRA and Rab 11 marker was observed after 10 and 30 min of treatment with ANP. Sites of co-localization for receptors and Rab11 are depicted as yellow foci. The images represent mid-focal planes and are typical of four independent experiments. (B) Quantification of the percent of co-localization of eGFP–NPRA with Rab11. Bars represent the mean ± S.E.M. ***P<0.001 relative to untreated cells. Scale bar=50 μm.

Figure 5. Lysosomal degradation of receptors in the presence or absence of lysosomotropic agents

(A) Analysis of eGFP–NPRA fusion protein degradation and effect of lysosomotropic agents. Cells were treated with 100 nM ANP for 5, 10, 15 or 30 min. Inhibition of eGFP–NPRA degradation was determined by Western blot after treatment with chloroquine (200 μM) and ammonium chloride (10 mM, both lysosomotropic agents). (B) Densitometric western blot quantification of (a) NPRA; (b) NPRA + ChL; (c) NPRA + NH₄Cl relative to untreated cells. (C) Co-IF analysis of the co-localization of cGMP with early endosomes in intact cells. (i) Untreated cells stained with DyLight™405 anti-rabbit antibody without prior incubation with the first rabbit antiserum. (ii–vi) Cells were treated with ANP for 1, 5, 10, 15 or 30 min, which show the co-localization (pink) of cGMP (blue) with EEA-1 (red). Co-localization of cGMP with EEA-1 clearly showed the concurrent generation of signal whereas receptor was trafficking. (D) Bars represent the densitometric analysis of the cGMP fluorescence intensities. (E) Stimulation of intracellular accumulation of cGMP in cells treated with 100 nM ANP. Cells were treated for 5, 10, 15 or 30 min at 37°C in the presence of IBMX. For each time point, untreated cells were used as controls. cGMP was quantified by ELISA. The images shown are typical of 4–5 independent experiments. Bars represent mean ± S.E.M. ***P<0.001 relative to untreated cells. Scale bar=50 μm.

Figure 6. Subcellular fractionation and co-IP of NPRA with pan-cadherin, EEA-1, LAMP-1 and Rab 11 in HEK-293 cells

To determine the association of NPRA with pan-cadherin, EEA-1, LAMP-1 and Rab 11, cells were stimulated with 100 nM ANP for different times. To confirm that lysate contained similar amounts of pan-cadherin, early endosome, lysosome and Res, equal amounts of proteins were immunoblotted with antibodies against each of the entities after subcellular fractionations. (A) Immunoblot of NPRA after IP of pan-cadherin, showed decreased association with increasing time. (B) Densitometric western blot quantification of NPRA association with pan-cadherin relative to untreated cells. (C) Co-IP of NPRA with EEA-1 showed maximum association at 5 min, after which it gradually decreased. (D) Densitometric western blot quantification of NPRA with EEA-1 relative to untreated cells. (E) Association of NPRA with lysosome gradually increased after 5 min, reaching its maximum at 30 min. (F) Densitometric western blot quantification of NPRA with LAMP-1 relative to untreated cells. (G) Strong association of receptor and Res was observed at 10 min, after which it gradually decreased. (H) Densitometric western blot quantification of NPRA with Rab 11 relative to untreated cells. The quantification of western blot results are presented in arbitrary units (AU). Bars represent the mean ± S.E.M. of five independent experiments.*P<0.05; **P<0.01; ***P<0.001 relative to untreated cells.

Figure 7. Schematic representation of intracellular trafficking of NPRA in HEK-293 cells

The scheme depicts the pathways of internalization, trafficking, recycling and degradation of ligand–receptor complexes. After binding of ligand (yellow), the receptor (blue) is activated and intracellular cGMP (green) is produced. The ligand–receptor complex enters the cell via coated pits. The ligand-bound receptor complex is trafficked intracellularly through endosomes and lysosomes. A small population of receptor recycles back to the plasma membrane through Res along with the concurrent generation of intracellular cGMP. Sorting of bound ANP–NPRA complexes (shown in a disrupted triangle in yellow for ligand and rectangles of blue and red colour for NPRA) occurs by endosomal-dissociation metabolic and lysosomal degradative pathways. Note that the multi-vesicular body formation probably places the receptor in the lumen of the lysosomes.