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  • br Intracellular trafficking with concurrent signaling of NP

    2022-07-30


    Intracellular trafficking with concurrent signaling of NPRA Our recent studies have shown internalization and concurrent signaling of NPRA in subcellular compartments; this had not been previously demonstrated [26,42]. Preparation of the enhanced GFP (eGFP)-tagged NPRA (eGFP-NPRA) construct has greatly helped to visualize the internalization, intracellular trafficking, and subsequent signaling of receptor in the subcellular compartments. These studies delineated the molecular mechanisms of NPRA trafficking with ANP/NPRA/cGMP signaling in recombinant HEK-293 JNJ 5207852 dihydrochloride receptor and primary MMCs in the physiological context [26,42]. Earlier studies showed that cGMP production is not exclusively activated at the cell surface, but also occurs after receptors have been internalized and continued trafficking in subcellular locations [26,42]. However, endocytic inhibitors MDC and CPZ and dynamin GTPase blocker (dynamin mutant) significantly decrease the intracellular accumulation of cGMP [69]. Moreover, the receptor containing the mutated short signal motif (FQQI/AAAA) also produced a significantly lower level of intracellular cGMP during subcellular trafficking than do the WT receptor without this mutation [26]. Interestingly, early studies reported that G-protein-coupled receptor (GPCR) continued, using their bound ligands, to produce signals by generating intracellular second-messenger cAMP throughout the internalization and trafficking itinerary [124]. Moreover, cGMP regulates various processes, including hypertension, cardiovascular homoeostasis, antimitogenic effects vascular reactivity, sensory transduction, anti-inflammation responses, neuronal plasticity, and learning [124,168]. Generally, cGMP interacts with three distinct kinds of intracellular effector protein molecules, including cGMP-dependent protein kinases (PKG), cGMP-activated phosphodiesterases (PDEs), and cGMP-regulated ion channels (CNGs) [169]. It is possible that cGMP-binding proteins transduce the cGMP signal to alter cell function through different mechanisms, comprising an inhibitory effect and/or by stimulating protein phosphorylation [170,171]. Using immunofluorescence microscopy of cGMP, it was possible to clearly visualize that ligand-receptor complexes of ANP/NPRA continuously produce intracellular cGMP at various time points after treatment with ANP during the internalization and trafficking of this receptor protein [26,42,69]. Based on our previous studies, it became evident that signaling from inside the cell into subcellular compartments is persistent. This signaling appears to trigger specific downstream effects of cGMP [26,42]. Attenuation of the intracellular accumulation of cGMP suggested that endocytic JNJ 5207852 dihydrochloride receptor inhibitors and dynamin GTPase blocker may have a concerted regulatory role in NPRA trafficking and signaling in intracellular compartments [69].
    Internalization and subcellular trafficking are critical for most membrane receptors with approaches in different subcellular compartments and some receptors that are recycled back to the plasma membrane during the signaling process. Live-cell imaging eliminates the possible effects of fixation on cell volume, as well as the loss reduction, or redistribution of GFP-chimeric receptors in physiological and pathophysiological contexts. Live-cell imaging permits investigation of receptor trafficking while avoiding confounding variables associated with fixation, particularly the loss or artifactual movement of eGFP-tagged membrane receptors. As individual cells are tracked in real time, the subcellular localization of receptors can be imaged and measured. Live-cell imaging allows the study of protein trafficking, migration, proliferation, apoptosis, cell-cycle pathway, autophagy, and protein-protein interactions and dynamics [172]. To understand the mechanistic pathway of receptor endocytosis, trafficking, and signaling of live cells and animal in vivo have been used to determine the roles of various receptors in their physiological milieu.