br Role of dynamin in NPRA endocytosis In recent
Role of dynamin in NPRA endocytosis In recent studies using 125I-ANP binding assay and confocal microscopy, we have examined the role of dynamin in the internalization and trafficking of NPRA in a stably transfected HEK-293 Etoposide . Our findings indicated that ANP treatment of high-density NPRA expressing recombinant HEK-293 cells time-dependently accelerates internalization of receptor from the plasma membrane to the cell interior. However, the internalization of ligand-receptor complexes of NPRA was significantly decreased by specific inhibitors of clathrin- and dynamin-dependent receptor internalization. These decreases were approximately 80%-90% using monodansylcadaverine (MDC), 75%-80% using chlorpromazine (CPZ), and 85%-90% using mutant dynamin, which are specific blockers of endocytic vesicle formation and internalization pathways (Fig. 4). Moreover, IF visualization of the internalization of NPRA and enhanced GFP-tagged NPRA in HEK-293 cells by confocal microscopy have demonstrated the formation of endocytic vesicles after 5 min of ANP treatment; this effect was blocked by clathrin inhibitors (MDC and CPZ) and mutant dynamin construct . These studies showed that NPRA undergoes internalization via clathrin-mediated endocytosis as part of its normal cellular itinerary, including receptor trafficking, signaling, and metabolic degradation. Our earlier findings suggested that ANP-mediated internalization of NPRA in the recombinant HEK-293 cells occurred via the clathrin-dependent pathway and involved initial clustering of receptor-ligand cargo in clathrin-coated pits, with plasma membrane invagination forming clathrin-coated vesicles. Blockade of ligand-induced endocytosis of the receptor by MDC and CPZ is the most effective means of the mechanistic action that inhibits the receptor internalization. Confocal microscopy clearly showed the formation of endocytic vesicles, which is a characteristic feature of internalized receptor within the cytoplasm. Several previous studies have shown that both CPZ and MDC, as well as mutant dynamin, have effectively blocked endocytosis of the membrane receptors and affected both the assembly of clathrin components and the formation of clathrin-coated pits in cells of various types [, , , , ]. The classical clathrin-mediated endocytic pathway clearly seems to be a major route for the endocytosis of NPRA. Demonstration of this provides a significant advance in our understanding of the role of this receptor in regulating hypertension and cardiovascular homeostasis. In recent studies, we have found that MDC and CPZ effectively blocked the internalization of ANP-NPRA complexes in recombinant HEK-293 cells by approximately 80%-90% . MDC acts as a competitive blocker of transglutaminase by creating an isopeptide bond between the glutamine and lysine amino-acid residues of two proteins, thus inhibiting the clathrin-coated vesicle-dependent endocytic process [96,99,100]. Earlier findings suggested that MDC blocks the internalization and trafficking of various types of ligand-receptor complexes [, , ]. Similarly, CPZ has been shown to block the assembly of clathrin-coated pits and to inhibit coated-vesicle-mediated internalization of membrane proteins [, , ]. We demonstrated that CPZ inhibits endocytosis of ANP-NPRA complexes by 80%-85%, which is consistent with the previous finding that CPZ inhibits the endocytosis of several different types of hormone receptors [, , , ]. CPZ, MDC, and dynamin-mutant have also been shown to play inhibitory roles for endocytosis in Wnt signaling . The expression of a dominant-negative mutant of dynamin (K44A/dynamin) has been shown to block the formation of coated vesicles during the internalization of various ligand-receptor complexes [67,, , ]. Previous studies have also indicated that dynamin, through its interaction with AP2, has a critical role in the localization of clathrin-coated pits and clathrin-mediated endocytosis [114,115]. After recruitment to coated pits, dynamin forms a ring structure, and then pinches off coated vesicles into the cytoplasm. This process, known as pinchase, through its intrinsic GTPase activity, generates clathrin-coated vesicles in the clathrin-mediated endocytosis [105,, , ]. It is thought that the GTPase-defective dominant mutant interferes with wild-type dynamin and blocks the formation of endocytic coated-vesicles and internalization of membrane receptors [, , , , ].