Clathrin-dependent internalization, signaling, and metabolic processing of guanylyl cyclase/natriuretic peptide receptor-A

Abstract

Cardiac hormones, atrial and brain natriuretic peptides (ANP and BNP), have pivotal roles in renal hemodynamics, neuroendocrine signaling, blood pressure regulation, and cardiovascular homeostasis. Binding of ANP and BNP to the guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) induces rapid internalization and trafficking of the receptor via endolysosomal compartments, with concurrent generation of cGMP. However, the mechanisms of the endocytotic processes of NPRA are not well understood. The present study, using I-ANP binding assay and confocal microscopy, examined the function of dynamin in the internalization of NPRA in stably transfected human embryonic kidney-293 (HEK-293) cells. Treatment of recombinant HEK-293 cells with ANP time-dependently accelerated the internalization of receptor from the cell surface to the cell interior. However, the internalization of ligand-receptor complexes of NPRA was drastically decreased by the specific inhibitors of clathrin- and dynamin-dependent receptor internalization, almost 85% by monodansylcadaverine, 80% by chlorpromazine, and 90% by mutant dynamin, which are specific blockers of endocytic vesicle formation. Visualizing the internalization of NPRA and enhanced GFP-tagged NPRA in HEK-293 cells by confocal microscopy demonstrated the formation of endocytic vesicles after 5 min of ANP treatment; this effect was blocked by the inhibitors of clathrin and by mutant dynamin construct. Our results suggest that NPRA undergoes internalization via clathrin-mediated endocytosis as part of its normal itinerary, including trafficking, signaling, and metabolic degradation.

Emerging concepts of receptor endocytosis and concurrent intracellular signaling: Mechanisms of guanylyl cyclase/natriuretic peptide receptor-A activation and trafficking

“…In recent studies using 125 I-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 cells [69]. 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.…”

“…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 [69]. 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.…”

“…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% [69]. 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].…”

“…Signaling from inside the cell is persistent and appears to trigger specific downstream effects. We have recently demonstrated that intracellular accumulation of cGMP significantly decreases after treatment with endocytic inhibitors MDC and CPZ and transfection with dynamin mutant GTPase blocker [69]. Diminution in the intracellular accumulation of cGMP indicates that these same endocytic inhibitors and dynamin GTPase blocker (dynamin mutant) have a concerted regulatory role in NPRA trafficking and signaling process.…”

“…Clathrin-mediated endocytosis is a well-established mechanism for numerous membrane-bound hormone receptors containing glucagon, insulin, platelet-derived growth factor (PDGF), and epidermal growth factor (EGF), which are also internalized by ligand-mediated endocytosis [35,67,68]. Our recent studies have demonstrated that NPRA is dynamin-dependently endocytosed in clathrin-coated vesicles [69]. Here, we provide a comprehensive summary of past and present findings on the internalization, intracellular trafficking, and concurrent signaling mechanisms of NPRA focused on cell-based studies as well as in intact live cells and animals in vivo to establish the actions of NPRA in the dynamic physiological context.…”