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  • Moreover we have demonstrated that internalization and


    Moreover, we have demonstrated that internalization and subcellular trafficking of NPRA, using IF staining (IFS) and co-IP of plasma membrane, endosomal, lysosomal, and recycling endosome markers to follow intracellular trafficking and signaling by confocal IF microscopy (CIF) and immunoblotting (IB) analyses in recombinant HEK-293 cells [42]. These functional subcellular compartments provide the physical basis for the assembly and turnover of intracellular kinetics of ligand-receptor complexes, which in turn define the specialized endosomal–lysosomal signaling platforms for hormone-dependent physiological functions of membrane receptors and other membrane proteins.
    Role of short-signal motif in the internalization and trafficking of NPRA Co-immunoprecipitation assays confirmed that colocalization of the mutant receptor (FQQI/AAAA) was significantly decreased in subcellular compartments during endocytosis and trafficking processes [26]. These earlier studies showed that the FQQI signal sequence motif is critically essential for the internalization and subcellular trafficking of NPRA during the hormone signaling process in intact primary MMCs [26]. The confocal immunofluorescence studies also showed that, after ligand binding, WT receptor eGFP-NPRA is rapidly internalized and trafficked from the cell surface to subcellular compartments. However, the mutated motif FQQI/AAAA significantly inhibited internalization, subcellular trafficking, and signaling processes of NPRA receptor protein [26]. These studies also demonstrated that FQQI has a significant role in sustaining receptor signaling in intact MMCs and thus advanced our understanding of the molecular mechanisms of vesicular transport, subcellular trafficking, and concurrent NPRA signaling. Similarly, another mutation in the GDAY motif (GDAY/AAAA) in the NPRA carboxyl-terminal domain has been shown to reduce internalization of mutant receptors by almost 40%-45% as compared with the WT receptor [62]. Small signal-sequence motifs have been shown to play pivotal roles in endocytosis and intracellular trafficking of membrane receptor proteins to direct cargo into trafficking vesicles [126,127]. Short signal sequence motifs constitute the short sequence of Picrotoxin in a linear array that consists of two-to-six amino-acid residues. Nevertheless, only two or three residues have critical roles in receptor endocytosis and trafficking pathways [126,128,129]. The most common small-signal sequence motifs found in various membrane receptors are shown in Table 1. Tetrameric sequence Gly920-Asp921-Ala922-Tyr923 (GDAY) motif in the carboxyl terminal-region of NPRA serves as a signal for endocytosis [62]. The amino acid residues Gly920 and Tyr923 constitute the critical elements for internalization of NPRA in the GDAY signal motif, however, Asp921 exhibits an acidic environment for efficient signaling of GDAY during the subcellular trafficking process. The mutation of Gly920 and Tyr923 to Ala prevented the internalization of NPRA by almost 50%, but had no effect on the recycling process. Although, the mutation of Asp921 to Ala did not exhibit a major effect on receptor endocytosis, but critically attenuated the recycling of internalized receptors to the plasma membrane. The NPxY signal sequence is known to facilitate the internalization of low density lipoprotein (LDL) receptor [130]. Endocytosis of platelet-activating factor is governed by a putative DPxxY signal motif and type-2 vasopressin receptor by NPxxY motif [131,132]. Similarly, YxxL signal motif also functions in endocytosis of LDL receptor-related protein [133]. A common feature of these internalization signal motifs, including DPxxy, NPxY, and GDAY, is the presence of an amino acid tyrosine at the end of the tetrapeptide short sequence motif [62,131]. Tyrosine residues are also involved in endocytosis of mannose-6-phosphate receptor and in the influenza virus hemagglutinin, even though they are not present in the context of NPxY or YxRF consensus sequence suggesting that if a universal internalization signal exists, it may not contain a universal peptide sequence [131,134]. The critical characteristics of all these sequences might be their specification of a particular conformation, such as a tight beta-turn in protein structure [135]. Tyr recognition short sequence signal motifs present a small surface loop but they differ in primary structure in the context of the positioning of Tyr residue in the loop structure [128,136,137]. The substitution of Tyr with amino acid known to be inert in endocytosis resulted in disruption of the beta-turn structure. The tyrosine-based motifs such as NPxY and YxxL recruit clathrin and adaptor protein molecules and act as a ligand-receptor cargo recognition sequence for their delivery to endosomes and lysosomes [134,138]. These motifs initially recruit AP-2 at the plasma membrane and activate mu2 subunit that facilitates the binding of beta-2 subunit of AP-2 to clathrin at the cell surface, leading to clathrin-mediated internalization of receptor molecules [128,134,139,140].