Antibody tracking demonstrates cell type-specific and ligand-independent internalization of guanylyl cyclase a and natriuretic peptide receptor C

Abstract

Atrial natriuretic peptide (ANP) binds guanylyl cyclase-A (GC-A) and natriuretic peptide receptor-C (NPR-C). Internalization of GC-A and NPR-C is poorly understood, in part, because previous studies used (125)I-ANP binding to track these receptors, which are expressed in the same cell. Here, we evaluated GC-A and NPR-C internalization using traditional and novel approaches. Although HeLa cells endogenously express GC-A, (125)I-ANP binding and cross-linking studies only detected NPR-C, raising the possibility that past studies ascribed NPR-C-mediated processes to GC-A. To specifically measure internalization of a single receptor, we developed an (125)I-IgG-binding assay that tracks extracellular FLAG-tagged versions of GC-A and NPR-C independently of each other and ligand for the first time. FLAG-GC-A bound ANP identically with wild-type GC-A and was internalized slowly (0.5%/min), whereas FLAG-NPR-C was internalized rapidly (2.5%/min) in HeLa cells. In 293 cells, (125)I-ANP and (125)I-IgG uptake curves were superimposable because these cells only express a single ANP receptor. Basal internalization of both receptors was 8-fold higher in 293 compared with HeLa cells and ANP did not increase internalization of FLAG-GC-A. For FLAG-NPR-C, neither ANP, BNP, nor CNP increased its internalization in either cell line. Prolonged ANP exposure concomitantly reduced surface and total GC-A levels, consistent with rapid exchange of extracellular and intracellular receptor pools. We conclude that ligand binding does not stimulate natriuretic peptide receptor internalization and that cellular environment determines the rate of this process. We further deduce that NPR-C is internalized faster than GC-A and that increased internalization is not required for GC-A down-regulation.

Fig. 1.

A, ¹²⁵I-ANP uptake in tTA-HeLa cells transiently transfected with GFP or FLAG-GC-A. Cells were labeled with subsaturating concentrations of ¹²⁵I-ANP at 4°C. Aliquots of labeled cells were incubated at 37°C for the times indicated before acid washing and counting. Values represent average ± the range of the determinations, where n = 2. The graph is representative of multiple experiments. B, FLAG-GC-A and wild-type GC-A bind and are activated by ANP similarly. The 293 cells were transiently transfected with wild-type GC-A or FLAG-GC-A and incubated with increasing concentrations of ANP for 1 min. Cellular cGMP concentrations were measured and plotted as a function of peptide concentration. The data points represent the mean ± S.E.M. assayed in triplicate. C, FLAG-GC-A has similar affinity for ANP as wild-type GC-A. Transiently transfected 293 cells were incubated for 1 h at 4°C with ¹²⁵I-ANP in the presence or absence of increasing concentrations of unlabeled ligand. Specifically bound ¹²⁵I-ANP was plotted as a function of competing peptide concentration. The data points represent the mean ± S.E.M. assayed in triplicate.

Fig. 2.

HeLa cells endogenously express high and low levels of NPR-C and GC-A, respectively. HeLa and tTA-HeLa cells were incubated with ¹²⁵I-ANP in the absence or presence of 1 μM ANP or CNP for 2 h at 4°C before cross-linking ¹²⁵I-ANP to the receptors with disuccinimidyl suberate. Membrane fractions were separated by SDS-PAGE, and ¹²⁵I-ANP-receptor complexes were visualized by autoradiography.

Fig. 3.

The ¹²⁵I-IgG uptake assay specifically measures FLAG-GC-A internalization. A, tTA-Hela cells were transiently transfected with GFP or FLAG-GCA. The cells were dispensed into tubes and incubated with 0.05 (1× primary) or 0.1 μl (20× primary) of anti-FLAG-M2 antibody. Excess antibody was removed before addition of 5 (1×), 50 (10× secondary), or 100 μl of ¹²⁵I-IgG. Cellular radioactivity was measured directly or after acid-stripping to remove surface ¹²⁵I-sIgG. Values represent the range of determinations, where n = 2. The graph is representative of more than three experiments. B, 293 cells were transiently transfected with 10 (1×), 1, or 5 μg of FLAG-GC-A plasmid DNA. Internalization assays were performed 48 h later. An equal number of cells from each transfection were separated by SDS-PAGE, blotted to an Immobilon membrane, and GC-A expression was detected by Western blot using an anti-GC-A antibody (inset). Values represent average ± S.E.M., where n = 6.

Fig. 4.

GC-A is slowly internalized in HeLa cells. A, tTA-HeLa cells transiently transfected with FLAG-GC-A were labeled with ¹²⁵I-ANP or anti-FLAG-M2 antibody followed by anti-mouse ¹²⁵I-IgG at 4°C. Values represent average ± S.E.M. where n = 4 (B) ANP increases GC-A uptake at longer but not shorter periods of time. Cells transfected with FLAG-GC-A were labeled with anti-FLAG-M2 antibody and anti-mouse ¹²⁵I-IgG before incubation at 37°C in the absence or presence of 1 μM ANP for the periods of time shown. Samples were then acid-washed and counted. Values represent average ± S.E.M., where n = 6. C, ¹²⁵I-transferrin is rapidly internalized in tTA-HeLa cells. Aliquots of the tTA-HeLa cells transfected with FLAG-GC-A were labeled with ¹²⁵I-transferrin at 4°C. Internalized ¹²⁵I-transferrin was determined after the indicated periods of time at 37°C. Values represent the average ± S.E.M., where n = 8.

Fig. 5.

GC-A is rapidly internalized in 293 PMA cells. A, 293 cells stably expressing FLAG-GC-A were incubated at 4°C with either ¹²⁵I-ANP or anti-FLAG antibody followed by anti-mouse ¹²⁵I-IgG. Cells were incubated at 37°C for the indicated times before acid-washing and counting. Values represent average ± S.E.M., where n = 14. B, untransfected 293 cells or 293 cells stably expressing FLAG-GC-A were labeled at 4°C with ¹²⁵I-ANP. Internalized radioactivity as a function of time at 37°C is shown. Values represent average ± the range of two determinations. C, untransfected or 293 cells stably expressing FLAG-GC-A were labeled at 4°C with anti-FLAG antibody followed by ¹²⁵I-IgG secondary antibody. The cells were incubated at 37°C in the presence or absence of 1 μM ANP for the indicated periods of time. Values represent the average ± the range determinations, where n = 2.

Fig. 6.

NPR-C is rapidly and constitutively internalized in HeLa and 293 cells. A, tTA-HeLa cells were transiently transfected with FLAG-NPR-C. Cells were then labeled with anti-FLAG antibody followed by anti-mouse ¹²⁵I-IgG at 4°C. Cells were incubated at 37°C for the indicated times before acid washing. Values represent the average ± S.E.M., where n = 14. B, 293 cells were transfected with FLAG-NPR-C and labeled with either ¹²⁵I-ANP or ¹²⁵I-IgG at 4°C. Aliquots were incubated at 37°C for the times shown and before acid washing. Values represent the average ± S.E.M., where n = 6. C, 293 cells transiently transfected with FLAG-NPR-C were labeled with anti-FLAG antibody and ¹²⁵I-IgG secondary antibody at 4°C. Aliquots were incubated at 37°C in the presence or absence of 1 μM ANP, BNP, or CNP for the times indicated, where n = 4.

Fig. 7.

Concomitant down-regulation of extracellular and intracellular FLAG-GC-A in 293 cells. The 293 cells stably expressing FLAG-GC-A were incubated with 10 μg/ml cycloheximide in the absence or presence of 200 nM ANP for the period of times indicated. In one experiment, crude membranes were prepared and then assayed for guanylyl cyclase activity in the presence of 1% Triton X-100 and Mn²⁺-GTP. In a second experiment, cells were incubated with ANP as described above and then labeled with anti-FLAG antibody followed by ¹²⁵I-IgG at 4°C. Total ¹²⁵I-IgG radioactivity and guanylyl cyclase activities were normalized to activities obtained from cells not incubated with ANP (control) and plotted as a function of time of ANP exposure.