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

Abstract

SynopsisAtrial 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 .

Clathrin-dependent internalization, signaling, and metabolic processing of guanylyl cyclase/natriuretic peptide receptor-A

“…The fusion of the C-terminus of murine NPRA cDNA [10] to the N-terminus of enhanced green fluorescent protein (eGFP) was done by subcloning it into p cGFP-NT vector (Clontech, San Diego, CA) for its expression as an eGFP-NPRA fusion protein, as previously reported [13]. HEK-293 cells were grown in 6-well culture plates containing Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS).…”

“…Initially, subconfluent cells were transfected with eGFP-NPRA cDNAs using Lipofectamine™ reagents. To establish the stably expressing receptor cell lines, 400 μg/ml of geneticin was added to the culture medium after transfection as previously described [13, 21]. Antibiotic-resistant clones were isolated and established for receptor expression, using ANP as a ligand [21].…”

“…Unbound 125 I-ANP was completely removed by washing the cells 4x (2ml each week) with ice-cold DMEM assay medium as described above. The total cell-associated 125 I-ANP radioactivity was quantitated by dissolving cells in 0.5 N NaOH and counting the radioactivity in the cell lysate as previously reported [13, 21]. The initial zero-time control value of 125 I-ANP binding was represented by 100%.…”

“…NPRA and NPRB show similar homology and both also contain an extracellular ligand-binding domain, a single transmembrane region, an intracellular protein kinase-like homology domain (protein KHD), and a guanylyl cyclase (GC) catalytic domain [8–11]. ANP and BNP activate NPRA, which produces second-messenger cGMP in response to hormone binding [12, 13]. CNP activates NPRB, which also produces cGMP.…”

“…This controversy arose in response to reports that of the three natriuretic peptide receptors only NPRC is internalized with bound ligand [43–45]. In our recent study using confocal immunofluorescence microscopy, we clearly visualized the internalization and subcellular trafficking of eGFP-tagged NPRA (eGFP-NPRA) in HEK-293 cells and murine mesangial cells [12, 13, 46]. NPRB, in response to CNP binding, has also been shown to be internalized and recycled back to the cell surface in cultured hippocampus neurons and C6 glioma cells [47].…”