Internalization and trafficking of guanylyl cyclase/natriuretic peptide receptor-A
Introduction
The biological actions of natriuretic peptide (NP) hormones are triggered by the interaction with highly selective and specific NP receptors (NPRs). Atrial natriuretic peptide (ANP) and two complementary related peptides named brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) exert natriuretic, diuretic, antimitogenic, and vasorelaxant activities. Three subtypes of NPRs have been cloned and characterized, namely natriuretic peptide receptor-A, -B, and -C, (designated as NPRA, NPRB, and NPRC). Both NPRA and NPRB contain guanylyl cyclase (GC) catalytic domain and are also referred to as GC-A and GC-B, respectively [44], [59]. ANP and BNP selectively stimulate NPRA, whereas CNP activates primarily NPRB and all three NPs bind to NPRC [61], [117]. Under normal hemodynamic conditions, ANP is predominantly synthesized, stored, and secreted in a regulated fashion by atrial myocytes [14], [30], [69]. However, in response to hemodynamic overload such as congestive heart failure, the ventricular ANP and BNP contents are greatly increased, contributing significantly to the circulating pool of these peptides. Both ANP and BNP exert their biological effects by interacting with NPRA and lead to the synthesis and accumulation of intracellular second messenger cGMP [33], [74], [90].
The general topological structure of NPRA is consistent with GC receptor family with at least four distinct regions comprising ligand-binding, transmembrane, protein kinase-like homology and GC catalytic domains [100]. The NPRB has the overall domain structure similar to that of NPRA with the binding selectivity to CNP [109]. NPRB is localized mainly in the brain and endothelial cells and is thought to mediate the actions of CNP in the central nervous systems and in vasculature. Comparison of the amino acid sequence indicated a 62% identity among NPRA and NPRB and the intracellular regions appear to be more highly conserved than the extracellular domains of these two receptors (78% versus 43%). The extracellular domain of NPRA is homologous to the NPRC, which contains a short (35-residues) cytoplasmic tail [41], apparently not coupled to the GC activation. NPRA is the dominant form of the NP receptors found in pheripheral organs and mediates most of the known actions of ANP and BNP. NPRA is considered a primary ANP-signaling molecule because major cellular and physiological responsiveness of hormone is mimicked by cGMP and its cell permeable analogs [2], [40], [91]. Based on the experimental evidence, it appears that NPRA is not just a cellular static protein; rather it is a dynamic cellular macromolecule that traverses through different compartments of the cell throughout its lifetime [89], [96]. By utilizing the pharmacologic and physiological perturbants and genetic tools, the biological actions of ANP can be modulated by the functional integrity of receptor protein suggesting that the regulation of NPRA activity is of biological importance. This review addresses the receptor-mediated internalization and cellular distribution of ANP/NPRA complexes from cell surface to cell interior. It is implicated that after internalization, the ANP/NPRA complexes dissociate into the subcellular compartments and a population of receptor recycles back to the plasma membrane. This is an interesting area of research in the natriuretic peptide receptor field because there is currently debate over whether ANP/NPRA complexes internalize at all or whether cells utilize some other mechanisms to release ANP from the NPRA. Indeed, controversy exists since it has been reported by default that among the three natriuretic peptide receptors only NPRC internalizes with bound ligand [75]. Hence, from a thematic standpoint it is evident that there is a current need to review this subject and provide a consensus forum that establishes the cellular trafficking and processing of ligand-bound GC-coupled NP receptors with an example of NPRA in intact cells. Towards this aim, the cellular life cycle of NPRA will be described in the context of ANP binding, internalization, recycling, down-regulation, and metabolic processing and degradation of NPRA in model cell systems.
Section snippets
Topology and structural domains of NPRA
The general topological structure of the GC receptor family is consistent with at least four distinct regions, which include an extracellular ligand-binding domain, a single transmembrane spanning region, and the intracellular protein kinase-like homology domain (KHD) and GC catalytic domain. The integrity of these regions of NPRA is conserved among human, mouse and rat [25], [73], [100]. The KHD contains an approximately 280-amino acid region that immediately follows the transmembrane spanning
Equilibrium binding and internalization of NPRA
Initial studies on the post-binding events of NPRA were hampered due to the lack of suitable primary cells containing predominantly this receptor protein. Nevertheless, our initial studies in Leydig tumor (MA-10) cell line [95] as well as report by others in PC-12 cells [108] indicated that ANP/NPRA complexes were internalized and sequestered inside the cells. On the other hand, studies by Maack and coworkers suggested that in renomedullary interstitial as well as in mesangial cells, ANP/NPRA
Endocytosis, sequestration, and recycling of NPRA
Ligand-binding studies in the intact cells demonstrated that internalized NPRA recycles back to the plasma membrane [89], [96], [97]. To examine if the internalized NPRA is recycled back to the plasma membrane, recombinant HEK-293 cells were incubated with 100 nM ANP at 37 °C for 2 h to deplete the cell surface receptors. After pretreatment with unlabeled ANP, cells were washed with acid buffer (pH 3.5) to remove bound ligand. After warming the cells at 37 °C, cell surface binding returned to
Down-regulation of NPRA
Receptors that are degraded following internalization can have important physiological and pathophysiological implications. If the circulating hormone levels are maintained above normal, the cells of peripheral tissues are exposed to unusually high levels of the hormone, and thus the proportion of cell surface receptors, which contain bound ligand, will be increased. As a result, this would have an effect on promoting ligand-receptor internalization that would lead to the degradation of both
Degradation of ligand-receptor complex of NPRA
The studies on stoichiometric analyses and metabolic processing of ANP/NPRA complexes in MA-10 cell line and recombinant COS-7 and HEK-293 cells, provided the evidence that a large population of bound ANP/NPRA complexes entered into lysosomes and the degraded products released into culture medium [89], [96], [97]. Lysosomotropic agent chloroquine and NH4Cl2 inhibited the degradation of ANP, providing direct evidence that ANP is metabolized in lysosomes (Fig. 2). ANP-specific endopeptidase is
Sequestration pathways of ligand-receptor complexes of NPRA
In metabolic processing studies of NPRA, ANP binding has been used as an index of NPRA activity. It is envisioned that the receptor-mediated endocytosis of ANPNPRA complexes may involve a number of sequential sorting steps through which ligand-receptor complexes could be eventually degraded, recycled back to the cell surface, or released into the cell exterior [96], [97]. As shown in Fig. 4, a number of these events may take place sequentially. The first step would be the noncovalent binding of
Molecular signals and internalization and trafficking of NPRA
The transfection studies have relied on the loss of function of deletion mutations to identify the regions within the KHD and GC catalytic domain of NPRA [94], [96]. The findings of those previous studies have suggested that the truncation of NPRA at the carboxyl-terminus end significantly reduced the hydrolysis of ligand-receptor complexes compared with wild-type receptor. The complete deletion of both the KHD and GC catalytic domains abolished the internalization of NPRA. The deletion of a
Perspectives and conclusions
The substantial evidence support the premise that expression and cellular regulation of NPRA activity is accomplished by the insertion of receptor on the plasma membrane, ligand-binding, and movement of the receptor protein through the multiple subcellular compartments in the cell. The assessment of the stoichiometric distribution of 125I-ANP bound to NPRA from plasma membrane to the intracellular compartments and into culture medium has provided the definitive means to directly determine the
Acknowledgements
I wish to thank my wife Kamala Pandey for her generous assistance in the preparation of this manuscript. The research work in the authors’ laboratory is supported by the National Institutes of Health grant (HL 57531).
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