Elsevier

Peptides

Volume 26, Issue 6, June 2005, Pages 985-1000
Peptides

Internalization and trafficking of guanylyl cyclase/natriuretic peptide receptor-A

https://doi.org/10.1016/j.peptides.2004.12.020Get rights and content

Abstract

One of the principal loci involved in the regulatory action of atrial and brain natriuretic peptides (ANP and BNP) is guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA), whose ligand-binding efficiency and GC catalytic activity vary remarkably in different target cells and tissues. In its mature form, NPRA resides in the plasma membrane and contains an extracellular ligand-binding domain, a single transmembrane region, and the intracellular protein kinase-like homology domain (KHD) and guanylyl cyclase (GC) catalytic domain. NPRA is a dynamic cellular macromolecule that traverses through different compartments of the cell through its lifetime. Binding of ligand to NPRA triggers a complex array of signal transduction events and accelerates the endocytosis. The endocytic transport is important in regulating signal transduction, formation of specialized signaling complexes, and modulation of specific components of internalization events. The present review describes the experiments which reveal the internalization of ligand-receptor complexes of NPRA, receptor trafficking and recycling, and delivery of both ligand-receptor molecules into subcellular compartments. The ligand-receptor complexes of NPRA are finally degraded within the lysosomes. The experimental evidence provides a consensus forum, which establishes the endocytosis, cellular trafficking, sequestration, and metabolic processing of ANP/NPRA complexes in the intact cells. The discussion is afforded to address the experimental insights into the mechanisms that cells utilize in modulating the delivery and metabolic processing of ligand-bound NPRA into the cell interior.

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).

References (129)

  • Z. Chen et al.

    Agonist-induced internalization of the platelet-activating factor receptor is dependent on arrestins but independent of G-protein activation. Role of the C-terminus and (D/N) PXXY motif

    J Biol Chem

    (2002)
  • M. Chinkers et al.

    Adenine nucleotides are required for activation of rat atrial natriuretic peptide receptor/guanylyl cyclase expressed in a baculovirus system

    J Biol Chem

    (1991)
  • D. Cohen et al.

    Molecular determinants of the clearance function of type-C receptor of natriuretic peptides

    J Biol Chem

    (1996)
  • J.F. Collawn et al.

    Transferrin receptor internalization sequence YXRF implicates a tight turn as the structural recognition motif for endocytosis

    Cell

    (1990)
  • A. DeLean et al.

    Natriuretic peptide receptor-A activation stabilizes a membrane-distal dimer interface

    J Biol Chem

    (2003)
  • C. Delporte et al.

    Atrial natriuretic peptide binds to ANP-R 1 receptors in neuroblastoma cells or is degraded extracellularly at the Ser-Phe bond

    Eur J Pharmacol

    (1992)
  • T. Duda et al.

    Core sequence of ATP regulatory module in receptor guanylate cyclases

    FEBS Lett

    (1993)
  • T. Duda et al.

    The glycine residue of ATP regulatory module in receptor guanylate cyclases that is essential in natriuretic factor signaling

    FEBS Lett

    (1993)
  • W. Eberle et al.

    The essential tyrosine of the internalization signal in lysosomal acid phosphatase is part of a beta-turn

    Cell

    (1991)
  • L. Elster et al.

    Differential distribution of GABA receptor subunits in soma and processes of cerebellular granule cells: effects of maturation and a GABA agonist

    J Dev Neurosci

    (1995)
  • D.C. Foster et al.

    Dual role for adenine nucleotides in the regulation of the atrial natriuretic peptide receptor guanylyl cyclase-A

    J Biol Chem

    (1998)
  • F. Fuller et al.

    Atrial natriuretic peptide clearance receptor. Complete sequence and functional expression of cDNA clones

    J Biol Chem

    (1988)
  • R.M. Gage et al.

    A transplantable sorting signal that is sufficient to mediate rapid recycling of G-protein-coupled receptors

    J Biol Chem

    (2001)
  • L.A. Garza et al.

    Insulin-responsive aminopeptidease trafficking in 3T3-L1 adipocytes

    J Biol Chem

    (2000)
  • Y. Hirata et al.

    Binding, internalization, and degradation of atrial natriuretic peptide in cultured vascular smooth muscle cells of rat

    Biochem Biophys Res Commun

    (1985)
  • Z. Huang et al.

    The cytoplasmic tail of the G-protein-coupled receptor for parathyroid hormone and parathyroid hormone-related protein contains positive and negative signals for endocytosis

    J Biol Chem

    (1995)
  • G.Y. Koh et al.

    Dynamics of atrial natriuretic factor-guanylate cyclase recetpors and receptor-ligand complexes in cultured glomerular mesangial and renomedullary interstitial cells

    J Biol Chem

    (1992)
  • K.J. Koller et al.

    Proper glycosylation and phosphorylation of the type A natriuretic peptide receptor are required for hormone-stimulated guanylyl cyclase activity

    J Biol Chem

    (1993)
  • H. Kurose et al.

    Participation of adenosine 5′ triphosphate in the activation of membrane-bound guanylate cyclase by the atrial natriuretic factor

    FEBS Lett

    (1987)
  • J. Labrecque et al.

    A disulfide-bridged mutant of natriuretic peptide receptor-A displays constitutive activity. Role of receptor dimerization in signal transduction

    J Biol Chem

    (1999)
  • J. Lazarovits et al.

    A single amino acid change in the cytoplasmic domain allows the influenza virus hemagglutinin to be endocytosed through coated pits

    Cell

    (1988)
  • R.J. Lefkowitz

    G protein-coupled receptors

    J Biol Chem

    (1998)
  • Y. Li et al.

    The YxxL motif, but not the two NPXY motifs, serve as the dominant endocytosis signal for low density lipoprotein receptor-related protein

    J Biol Chem

    (2000)
  • P. Lobel et al.

    Mutations in the cytoplasmic domain of the 275 kd mannose 6-phosphate receptor differentially alter lysosomal enzyme sorting and endocytosis

    Cell

    (1989)
  • T. Maack

    The role of atrial natriuretic factor in volume control

    Kidney Int

    (1996)
  • S. Marshall

    Dual pathways for the intracellular processing of insulin: relationship between retroendocytosis of intact hormone and the recycling of insulin receptors

    J Biol Chem

    (1985)
  • S. Marshall et al.

    Evidence for recycling of insulin receptors in isolated rat adipocytes

    J Biol Chem

    (1981)
  • H.M. Miettinen et al.

    Fc receptor isoforms exhibit distinct abilities for coated pit localization as a result of cytoplasmic domain heterogeneity

    Cell

    (1989)
  • J.D. Miranda et al.

    Repression of γ-aminobutyric acid type A receptor a 1 polypeptide biosynthesis requires chronic agonist exposure

    J Biol Chem

    (1997)
  • K.S. Misono

    Acidic pH- and metal ion (Zn2+ or Mn2+)-dependent proteolysis of 140 kDa atrial natriuretic factor receptor in bovine adrenal cortex plasma membranes: evidence for membrane-bound acidic metallo-endopeptidase

    Biochem Biophys Res Commun

    (1988)
  • K.K. Murthy et al.

    Binding and intracellular degradation of atrial natriuretic factor by cultured vascular smooth muscle cells

    Mol Cell Endocrinol

    (1989)
  • M. Napier et al.

    Binding and internalization of atrial natriuretic factor by high-affinity receptors in A10 smooth muscle cells

    Arch Biochem Biophys

    (1986)
  • D.R. Nussenzveig et al.

    Agonist-stimulated internalization of the thyrotropin-releasing hormone receptor is dependent on two domains in the receptor carboxyl terminus

    J Bio Chem

    (1993)
  • D.R. Nussenzveig et al.

    Cellular mechanisms of the clearance function of type-C receptors of atrial natriuretic factor

    J Biol Chem

    (1990)
  • K.N. Pandey

    Stimulation of protein phosphorylation by atrial natriuretic factor in plasma membranes of bovine adrenal cortical cells

    Biochem Biophys Res Commun

    (1989)
  • K.N. Pandey

    Stoichiometric analysis of internalization, recycling, and redistribution of photoaffinity-labeled guanylate cyclase/atrial natriuretic factor receptors in cultured murine Leydig tumor cells

    J Biol Chem

    (1993)
  • K.N. Pandey et al.

    Natriuretic peptide receptor-A negatively regulates mitogen-activated protein kinase and proliferation of mesangial cells: role of cGMP-dependent protein kinase

    Biochem Biophys Res Commun

    (2000)
  • K.N. Pandey et al.

    Ligand-regulated internalization, trafficking, and down-regulation of guanylyl cyclase/atrial natriuretic peptide receptor-A in human embryonic kidney 293 cells

    J Biol Chem

    (2002)
  • K.N. Pandey et al.

    Identification and characterization of three distinct atrial natriuretic factor receptors. Evidence for tissue-specific heterogeneity of receptor subtypes in vascular smooth muscle, kidney tubular epithelium, and Leydig tumor cells by ligand-binding, photoaffinity labeling, and tryptic proteolysis

    J Biol Chem

    (1988)
  • K.N. Pandey et al.

    Atrial natriuretic factor regulates steroidogenic responsiveness and cyclic nucleotide levels in mouse Leydig cells in vitro

    Biochem Biophys Res Commun

    (1986)
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