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  • Multiple studies from our laboratory and others

    2022-06-27

    Multiple studies from our laboratory and others, which have used stoichiometric kinetic analyses of ANP/NPRA, have provided strong evidence that bound ligand-receptor complexes are internalized and processed intracellularly, and that degraded products are released into the culture medium of various cell types harboring endogenous receptors [34,61,63,64,74], The same events have been demonstrated in transfected COS-7 and HEK-293 octanoic acid expressing recombinant NPRA [62,75,76]. Moreover, in response to CNP-binding, NPRB has been shown to be internalized and recycled back to the cell surface in cultured glioma cells and hippocampus neurons [77]. The prevailing consensus now is that, in response to ligand binding, both NPRA and NPRB are internalized into the cell interior and redistributed in subcellular compartments. To obtain unequivocal evidence, it became important to visualize cell trafficking and redistribution of ANP/NPRA complexes in intact cells. Recent findings from our laboratory have resolved this important issue, unequivocally demonstrating ligand-dependent endocytosis and intracellular trafficking of NPRA by confocal immunofluorescence (IF) and co-immunoprecipitation (Co-IP) analyses with organelle-specific marker proteins in eGFP/NPRA-transfected HEK-293 cells and primary MMCs [26,42]. Our findings have facilitated direct visualization of ligand-dependent endocytosis, trafficking, and redistribution of eGFP-NPRA into subcellular compartments with concurrent production of intracellular second-messenger cGMP. Earlier we have also demonstrated the kinetics of 125I-ANP-NPRA internalization in recombinant Cos-7 and HEK-293 cells [34,75]. However, in recent studies, we examined immunofluorescence-based NPRA internalization in MMCs, a cell system with physiological relevance [26]. We used eGFP-NPRA to capture immunofluorescence images generated by this receptor protein only during the processes of endocytosis and trafficking, without any interference from either NPRB or NPRC. Also, since ANP binds to both NPRA and NPRC, we have used c-ANF to block ANP binding to octanoic acid NPRC in the 125I-ANP binding assay. The peptide c-ANF binds only to NPRC, not to either NPRA nor NPRB. Thus, all the binding parameter for ligand-dependent internalization of ligand-receptor complexes were accounted due to NPRA as previously reported [78]. The mechanisms of NPRA signaling during the glycosylation of NPRA is not well understood. Previously, it was suggested that glycosylation sites in guanylyl cyclase-coupled receptors might be important for proper folding and stability of the receptor protein, as well as essential to facilitate ligand binding of NPRA and NPRB [18,79,80]. However, one study has suggested that glycosylation is not essential for ligand-binding of NPRA [81]. More experimentations are needed to confirm the functional role of glycosylation in the ligand binding and trafficking of both NPRA and NPRB GC-coupled receptors.
    Ligand-stimulated internalization of NPRA After ANP binding, NPRA dimerizes, while the GC catalytic domain of the receptor becomes activated [82]. Studies have shown that after ANP binding, ligand-receptor complexes are internalized, redistributed in the cell interior, and metabolized in lysosomes [40,61,63,64,74]. It has been suggested that internalized receptor traffic into intracellular compartments, as well as a population of receptor, escapes the lysosomal compartments and recycles back to plasma membranes [34,35,42,61,75,83]. Our classical studies using Leydig tumor (MA-10) cells containing a high density of endogenous NPRA and recombinant human embryonic kidney-293 (HEK-293), as well as COS-7 cells transfected with NPRA cDNA construct, firmly established that ligand-receptor complexes of ANP-NPRA are rapidly and ligand-dependently internalized and redistributed intracellularly [61,84,85]. Those findings demonstrated that after ligand-binding NPRA is internalized with bound hormone by an energy- and temperature-dependent mechanism, after which degraded ligand is released into the culture medium [34,42,61,62,86].