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    • Distribution of Iodine labeled ANP I ANP radioactivity on th

    2022-05-27

    Distribution of 125Iodine-labeled-ANP (125I-ANP) radioactivity on the cell surface, in the intracellular compartments, and in culture medium established a dynamic equilibrium of receptor-mediated 125I-ANP uptake, degradation, and release outside of BV6 (Fig. 2). A major proportion of internalized 125I-ANP was released into the culture medium, which consisted of approximately 75%-80% degraded products and about 20%-30% intact ligand [26,34,42,61,62]. The release of both degraded and intact ligands was prevented by introducing the lysosomotropic agents such as ammonium chloride (NH4Cl2), chloroquine, or nigericin in the culture media [40,42,61]. Early work suggested that most of the internalized 125I-ANP was metabolized through the pathways of lysosomal degradative compartments in intact cells; however, a population of internalized ligand-receptor complexes recycled back to the plasma membrane and ligand was extruded outside the cell (Fig. 3). It is believed that the released intact ligand could rebind to the receptor and re-enter the cell by the pathway of the retro-endocytosis mechanism [35,61,84]. Although, after internalization, most of the endocytosed ligand was degraded in the lysosomes and then released into the culture medium, a population of ligand-receptor complexes escaped the lysosomal degradative pathway and extruded intact outside of cells [34,62,87]. By using an antibody-tracking method, one study indicated that, in a cell-specific manner, both NPRA and BV6 NPRB are ligand-independently internalized [70]. However, antibody-tracking could determine the internalization kinetics of ligand-receptor complexes only qualitatively. Our recent studies using confocal immunofluorescence microscopy clearly visualized and unequivocally established that ligand-dependent internalization and subcellular trafficking of eGFP-tagged-NPRA (eGFP-NPRA) occurs in stably transfected recombinant HEK-293 cells and transiently NPRA-transfected primary murine mesangial cells (MMCs) [26,42]. Furthermore, NPRB has been shown, in response to CNP binding, to be internalized and recycled back to the cell surface in cultured hippocampal neurons and C6 glioma cells [77].
    Role of clathrin adaptor proteins in the endocytosis of NPRA It is thought that the tyrosine-based Yxxphi sorting signals located in the carboxyl-terminus domain direct the internalization and trafficking of various membrane receptors by interacting with mu 1, mu 2, mu 3, and mu 4 subunits of adaptor proteins (APs), including, respectively, AP-1, AP-2, AP-3, and AP-4 [26,88,89]. Recent studies have demonstrated the role of adaptor protein 2 in endocytosis of insulin-like growth factor-I receptor (IGF-IR), which interacts with insulin-receptor substrate (IRS)-1 with the clathrin adaptor complex AP2 [90]. However, IRS-1 inhibits recruitment of IGF-IR into clathrin-coated structures; for this reason, IGF-IR avoids rapid endocytosis and prolongs its activity on the cell surface. Beta-arrestins are known to act as endocytic adaptors by recruiting the AP-2 complex to G-protein-coupled receptors (GPCRs), linking them to clathrin-coated pits (CCPs) for internalization [91]. Upon activation by Wnt, the Frizzled receptor is internalized in a process that requires the recruitment of Dishevelled, a family of proteins involved in the Wnt signaling pathways [92]. Interaction between Dishevelled2 (Dvl2) and μ2-adaptin, a subunit of the clathrin adaptor AP-2, is required to engage activated Frizzled4 with the endocytic machinery for its internalization. Our recent studies have shown that the μ1B subunit of AP-1 directly binds to a phenylalanine-based FQQI motif in the cytoplasmic tail of NPRA receptor protein [26]. However, more studies are needed to establish the exact role of adapter proteins in the endocytotic pathways of NPRA. To delineate the critical role of endocytic signals in the intracellular sorting and signaling of NPRA, immunofluorescence staining and Co-IP of eGFP-NPRA with the AP-1 marker have been used to follow the intracellular trafficking and concurrent signaling of mutated and WT receptors by confocal immunofluorescence microscopy and immunoblotting in MMCs (Fig. 3). Subcellular trafficking of receptor has shown that the immunofluorescence colocalization of mutated receptors with μ1B markers was decreased by 50% for the subunit of AP-1 as compared with the WT receptor in intact MMCs [26]. It has been shown that the autosomal recessive hypercholesterolemia protein (ARH) is important in clathrin-mediated endocytosis of low-density lipoprotein receptors (LDLRs) [93]. In addition to its role in endocytosis, ARH unites with AP-1B in basolateral exocytosis of LDLR from recycling endosomes (REs) to plasma membrane.