Role of FQQI motif in the internalization, trafficking, and signaling of guanylyl-cyclase/natriuretic peptide receptor-A in cultured murine mesangial cells

Abstract

Binding of the cardiac hormone atrial natriuretic peptide (ANP) to transmembrane guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA), produces the intracellular second messenger cGMP in target cells. To delineate the critical role of an endocytic signal in intracellular sorting of the receptor, we have identified a FQQI (Phe(790), Gln(791), Gln(792), and Ile(793)) motif in the carboxyl-terminal region of NPRA. Mouse mesangial cells (MMCs) were transiently transfected with the enhanced green fluorescence protein (eGFP)-tagged wild-type (WT) and mutant constructs of eGFP-NPRA. The mutation FQQI/AAAA, in the eGFP-NPRA cDNA sequence, markedly attenuated the internalization of mutant receptors by almost 49% compared with the WT receptor. Interestingly, we show that the μ1B subunit of adaptor protein-1 binds directly to a phenylalanine-based FQQI motif in the cytoplasmic tail of the receptor. However, subcellular trafficking indicated that immunofluorescence colocalization of the mutated receptor with early endosome antigen-1 (EEA-1), lysosome-associated membrane protein-1 (LAMP-1), and Rab 11 marker was decreased by 57% in early endosomes, 48% in lysosomes, and 42% in recycling endosomes, respectively, compared with the WT receptor in MMCs. The receptor containing the mutated motif (FQQI/AAAA) also produced a significantly decreased level of intracellular cGMP during subcellular trafficking than the WT receptor. The coimmunoprecipitation assay confirmed a decreased level of colocalization of the mutant receptor with subcellular compartments during endocytic processes. The results suggest that the FQQI motif is essential for the internalization and subcellular trafficking of NPRA during the hormone signaling process in intact MMCs.

Keywords: guanylyl cyclase/natriuretic peptide receptor-A; immunofluorescence; mouse mesangial cells; receptor internalization; trafficking.

Fig. 1.

Schematic representation of the intracellular trafficking motif, expression, and amino acid residue requirements (AAAA, AQQA, AQQI, and FQQA) for internalization of wild-type (WT) and mutant (natriuretic peptide receptor-A; NPRA) in intact mouse mesangial cells (MMCs). A: topology indicating the extracellular, transmembrane (TM), and cytoplasmic regions of NPRA. The internalization motif analyzed corresponds to amino acids 790–793. eGFP, enhanced green fluorescent protein. B: amino acid sequence of the WT internalization motif in the carboxyl-terminal domain of NPRA as indicated by the single letter code. Δ790–793 indicates the internal substitution of residues Phe⁷⁹⁰, Gln⁷⁹¹, Gln⁷⁹², and Ile⁷⁹³ with alanine. C: lanes a, b, c, d, and e represents the expressed WT and mutant receptor (AAAA, AQQA, AQQI, and FQQA) bands in MMCs, respectively. The arrow indicates the position of 162-kDa eGFP-NPRA fusion protein band. D: alanine substitutions at amino acid positions 788–795 and in different combinations in the FQQI motif. Confluent MMCs in 6-cm² dishes transiently transfected with WT and mutant receptors were pretreated with 100 nM C-atrial natriuretic factor receptor ligand (C-ANF) to block the NPRC, and then treated with ¹²⁵I-labeled atrial natriuretic peptide (ANP) at 4°C for 1 h in the absence or presence of unlabeled ANP. After 15 min of incubation, the internalization of ligand-receptor complexes was quantified as described in materials and methods. Values are means ± SE of 6 separate experiments in triplicate. **P < 0.01, ***P < 0.001 relative to WT receptor.

Fig. 2.

Kinetics of cell surface-associated, internalized, and released ¹²⁵I-ANP radioactivity in MMCs transiently expressing WT or FQQI/AAAA mutant receptors. Cells were transiently transfected with pcDNA-6.2-GW/GFP-microRNA (miR) expression vector containing Npr1-miRNA inserts pCMVNpr1miRNA-1595 and pCMV-Npr1miRNA-2931 to inhibit the endogenous expression of NPRA. After knockdown of the endogenous expression of NPRA, cells were transiently transfected with WT and mutated eGFP-NPRA. After transient transfection, confluent cells in 6-cm² dishes were pretreated with 100 nM C-ANF to block the NPRC, then labeled with ¹²⁵I-ANP at 4°C for 1 h in the absence or presence of unlabeled ANP. Cells were washed 4 times with assay medium and incubated in 2 ml of fresh medium at 37°C. At the indicated time points, the cell surface-associated (acid-sensitive) radioactivity was eluted with acetate buffer (pH 3.5), and cells were dissolved in 1 N NaOH to measure the internalized (acid-resistant) radioactivity. Cell surface-associated (A), internalized (B), and released (C) ¹²⁵I-ANP radioactivity was determined in the acid eluate, cell extract, and culture medium, respectively, as described in materials and methods. In the culture medium, the intact and degraded ligand (D) products were quantified as described in materials and methods. Values are means ± SE of 4 independent experiments in triplicate dishes. :***P < 0.001 relative to WT receptor.

Fig. 3.

Colocalization of internalized eGFP-NPRA-WT and mutated motif FQQI/AAAA with μ₁B subunit of adaptor protein-1 (AP-1) in MMCs. Colocalization experiments were performed 48 h after transfection of cells with eGFP-NPRA-WT and mutated motif FQQI/AAAA. Cells were treated with 100 nM ANP for different times. A: colocalization of eGFP-NPRA-WT with μ₁B of AP-1 was observed after 2.5- and 5-min treatment with ANP. B: colocalization of eGFP-NPRA mutated motif FQQI/AAAA with μ₁B of AP-1 decreased by 50% compared with WT receptor. C: quantification of the percent colocalization of eGFP-NPRA-WT and mutated motif FQQI/AAAA with μ₁B of AP-1. The images shown are typical of 5 independent experiments. Values are means ± SE.

Fig. 4.

Colocalization of internalized eGFP-NPRA mutated motif FQQI/AQQI or FQQI/AQQA with μ₁B of AP-1 in cultured MMCs. Colocalization experiments were performed 48 h after transfection of cells with eGFP-NPRA mutated motifs FQQI/AQQI and FQQI/AQQA. Cells were treated with 100 nM ANP for different times. The mutation FQQI/AQQA decreased the receptor interaction with the adapter protein. Colocalization of eGFP-NPRA mutated motif FQQI/AQQI with μ₁B of AP-1 was decreased by 23% (A) and FQQI/AQQA was decreased by 45% (B) compared with the WT receptor. C: quantification of the percent colocalization of eGFP-NPRA-WT, mutated motif FQQI/AQQI, and FQQI/AQQA with μ₁B of AP-1. The images shown are typical of 5 independent experiments. Values are means ± SE.

Fig. 5.

Colocalization of internalized eGFP-NPRA-WT and mutated motif FQQI/AAAA with early endosome antigen-1 (EEA-1) in MMCs. Colocalization experiments were performed 48 h after transfection of cells with eGFP-NPRA-WT and mutated motif FQQI/AAAA. Cells were treated with 100 nM ANP for different times. A: colocalization of eGFP-NPRA-WT and EEA-1 marker was observed after 5 and 10 min of treatment with ANP, after which the fluorescence intensity gradually decreased from 15 to 30 min. B: colocalization of mutant receptor FQQI/AAAA into endosomes. Colocalization of mutated eGFP-NPRA and EEA-1 marker was observed with a marked decrease by 57% compared with the WT receptor. C: quantification of the percent colocalization of eGFP-NPRA-WT and mutated motif FQQI/AAAA with EEA-1. The images shown are typical of the images obtained in 5 independent experiments. Values are means ± SE.

Fig. 6.

Colocalization of internalized eGFP-NPRA mutated motif FQQI/AQQI or FQQI/AQQA with EEA-1 in cultured MMCs. Colocalization experiments were performed 48 h after transfection of cells with eGFP-NPRA mutated motifs FQQI/AQQI and FQQI/AQQA. Cells were treated with 100 nM ANP for different times. The mutation of FQQI/AQQA significantly decreased the receptor colocalization into endosomes. Colocalization of eGFP-NPRA mutated motif FQQI/AQQI with EEA-1 was decreased by 31% (A) and FQQI/AQQA was decreased by 55% (B) compared with the WT receptor. C: quantification of percent colocalization of eGFP-NPRA-WT, mutated motif FQQI/AQQI, and FQQI/AQQA with EEA-1. The images shown are typical of 4–5 independent experiments. Values are means ± SE.

Fig. 7.

Colocalization of internalized eGFP-NPRA-WT and mutated motif FQQI/AAAA with Rab 11 in MMCs. Colocalization experiments were performed 48 h after transfection of cells with eGFP-NPRA-WT and mutated motif FQQI/AAAA. Cells were treated with 100 nM ANP for different times. A: colocalization of eGFP-NPRA-WT with Rab 11 was observed after 5- and 15-min treatment with ANP. B: colocalization of eGFP-NPRA mutated motif FQQI/AAAA with Rab 11 marker decreased by 40%. C: quantification of percent colocalization of eGFP-NPRA-WT and mutated motif FQQI/AAAA with Rab 11. The images shown are typical of 5 independent experiments. Values are means ± SE.

Fig. 8.

Colocalization of internalized eGFP-NPRA-WT and mutated motif FQQI/AAAA with lysosome-associated membrane protein-1 (LAMP-1) in MMCs. Cells were treated with 100 nM ANP for different times. A: colocalization of eGFP-NPRA-WT with LAMP-1 marker was gradually increased from 5 to 30 min after ANP treatment. B: colocalization of eGFP-NPRA mutated motif FQQI/AAAA with LAMP-1 marker was decreased by 31%. C: quantification of the percent colocalization of eGFP-NPRA-WT and mutated motif FQQI/AAAA with LAMP-1. The images shown are typical of the images obtained in 4 independent experiments. Values are means ± SE.

Fig. 9.

Coimmunoprecipitation of eGFP-NPRA-WT with pan-cadherin, μ₁B, EEA-1, Rab 11, and LAMP-1 in MMCs. To determine the association of NPRA with pan-cadherin, μ₁B, EEA-1, Rab 11, and LAMP-1, cells were stimulated with 100 nM ANP for different time points. Anti-eGFP antibody was utilized for immunoblotting (IB) of eGFP-NPRA fusion protein. A: immunoblot of NPRA after immunoprecipitation (IP) of pan-cadherin showed a decreased association with increasing time points. B: densitometric Western blot quantification of NPRA association with pan-cadherin relative to untreated cells. C: coimmunoprecipitation of NPRA with μ₁B showed maximum association at 2.5 min, and after that it gradually decreased. D: densitometric Western blot quantification of NPRA with μ₁B relative to untreated cells. E: coimmunoprecipitation of NPRA with EEA-1 showed maximum association at 5 min, and after that it gradually decreased. F: densitometric Western blot quantification of NPRA with EEA-1 relative to untreated cells. G: strong association of receptor and recycling endosomes was observed at 10 min, and after that it gradually decreased. H: densitometric Western blot quantification of NPRA with Rab 11 relative to untreated cells. I: association of NPRA with lysosomes gradually increased after 5 min, and it was maximum at 30 min. J: densitometric Western blot quantification of NPRA with LAMP-1 relative to untreated cells. Values are means ± SE of 4 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 relative to untreated cells.

Fig. 10.

Coimmunoprecipitation of mutant eGFP-NPRA with pan-cadherin, μ₁B, EEA-1, Rab 11 and LAMP-1 in MMCs. Anti-eGFP antibody was utilized for immunoblotting of eGFP-NPRA fusion protein. A: immunoblot of WT and mutant eGFP-NPRA after immunoprecipitation of pan-cadherin showed an increased association with mutated motifs FQQI/AAAA and FQQI/AQQA at 30 min and decreased association with other mutated residue of FQQI/AQQI and FQQI/FQQA. B: densitometric Western blot quantification of mutant eGFP-NPRA association with pan-cadherin relative to the WT receptor. C: coimmunoprecipitation of mutant eGFP-NPRA (mutated motifs FQQI/AAAA and FQQI/AQQA) with μ₁B showed decreased association at 2.5 min and increased association with mutated motifs FQQI/AQQI and FQQI/FQQA. D: densitometric Western blot quantification of mutant eGFP-NPRA with μ₁B relative to the WT receptor. E: coimmunoprecipitation of mutant eGFP-NPRA (mutated motifs FQQI/AAAA and FQQI/AQQA) with EEA-1 showed decreased association at 5 min and increased association with mutated motifs FQQI/AQQI and FQQI/FQQA. F: densitometric Western blot quantification of mutant eGFP-NPRA with EEA-1 relative to the WT receptor. G: association of the WT receptor and recycling endosomes was observed at 10 min. However, mutant receptors (mutated motifs FQQI/AAAA and FQQI/AQQA) showed a decreased association with mutated motifs FQQI/AQQI and FQQI/FQQA. H: densitometric Western blot quantification of mutant eGFP-NPRA with Rab 11 relative to the WT receptor. I: association of mutant eGFP-NPRA (mutated motifs FQQI/AAAA and FQQI/AQQA) with lysosomes showed decreased association at 30 min; however, it was increased with FQQI/AQQI and FQQI/FQQA motifs. J: densitometric Western blot quantification of mutant eGFP-NPRA with LAMP-1 relative to the WT receptor. Values are means ± SE of 5 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 relative to WT receptor.

Fig. 11.

Immunofluorescence localization of cGMP and schematic representation of internalization, intracellular trafficking, and signaling pathway of NPRA in MMCs. Immunofluorescence localization of cGMP was performed 48 h after transfection of cells with eGFP-NPRA-WT and mutated motif FQQI/AAAA. To inhibit the endogenous expression of NPRA, previously cells were transiently transfected with pcDNA-6.2-GW/GFP-miR expression vector containing Npr1-miRNA insert pCMVNpr1miRNA-1595 and pCMV-Npr1miRNA-2931. The cells were treated with 100 nM ANP for 10 min at 37°C in the presence of IBMX, as described in materials and methods. A: after treatment with ANP for 10 min, eGFP-NPRA-WT showed significantly more diffused fluorescence intensities in the cytoplasm than mutated motif FQQI/AAAA. B: to assay the stimulation of intracellular accumulation of cGMP, cells were transiently transfected with eGFP-NPRA-WT and mutated motif FQQI/AAAA. Intracellular accumulation of cGMP through eGFP-NPRA-WT and mutated motif FQQI/AAAA was quantitated by ELISA. The images shown are typical of 4 independent experiments. Values are means ± SE. C: scheme depicting the sequential events of internalization, trafficking, recycling, and degradation of ligand-receptor complexes in the intracellular compartments. After binding of ANP to NPRA, ligand-receptor complexes enter the cell via clathrin-coated pits, and the ligand-bound receptor complex traffics intracellularly through the endosomes, lysosomes, and a population of receptor recycles back to the plasma membrane through the recycling endosomes, with concurrent generation of intracellular cGMP. Sorting of the bound ANP-NPRA complex occurs by endosomal dissociation metabolic and lysosomal degradative pathways. On the other hand, the mutation of FQQI residues to AAAA (FQQI/AAAA) significantly inhibited the receptor internalization and intracellular trafficking. Note that the multivesicular body formation likely places the receptor in the lumen of the lysosomes. ECD, extracellular domain; KHD, kinase homology domain; GCD, guanylyl cyclase domain.