Subcellular trafficking of guanylyl cyclase/natriuretic peptide receptor-A with concurrent generation of intracellular cGMP

Atrial natriuretic peptide (ANP) activates guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA), which lowers blood pressure and blood volume. The objective of the present study was to visualize internalization and trafficking of enhanced GFP (eGFP)-tagged NPRA (eGFP–NPRA) in human embryonic kidney-293 (HEK-293) cells, using immunofluorescence (IF) and co-immunoprecipitation (co-IP) of eGFP–NPRA. Treatment of cells with ANP initiated rapid internalization and co-localization of the receptor with early endosome antigen-1 (EEA-1), which was highest at 5 min and gradually decreased within 30 min. Similarly, co-localization of the receptor was observed with lysosome-associated membrane protein-1 (LAMP-1); however, after treatment with lysosomotropic agents, intracellular accumulation of the receptor gradually increased within 30 min. Co-IP assays confirmed that the localization of internalized receptors occurred with subcellular organelles during the endocytosis of NPRA. Rab 11, which was used as a recycling endosome (Re) marker, indicated that ∼20% of receptors recycled back to the plasma membrane. ANP-treated cells showed a marked increase in the IF of cGMP, whereas receptor was still trafficking into the intracellular compartments. Thus, after ligand binding, NPRA is rapidly internalized and trafficked from the cell surface into endosomes, Res and lysosomes, with concurrent generation of intracellular cGMP.     

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

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.     

Intracellular trafficking and metabolic turnover ofligand-bound guanylyl cyclase/atrial natriuretic peptide receptor-A into subcellular compartments

Atrial natriuretic peptide (ANP) is the first described member of the natriuretic peptide hormone family. ANP elicits natriuretic, diuretic, vasorelaxant and antiproliferative effects, important factors in the control of blood pressure homeostasis. One of the principal loci involved in the regulatory action of ANP is the guanylyl cyclase-linked ANP-receptor which has been designated as NPRA, also referred to as GC-A, whose ANP-binding efficiency and guanylyl cyclase activity vary remarkably in different target tissues. However, the cellular and molecular basis of these activities and the functional expression and regulation of NPRA are not well understood. The mature form of receptor resides in the plasma membrane and consists of an extracellular ligand-binding domain, a single transmembrane-spanning region, and intracellular protein kinase-like homology and guanylyl cyclase catalytic domains. In this review, emphasis has been placed on the interaction of ANP with NPRA, the ligand-mediated endocytosis, trafficking, and subcellular distribution of ligand-receptor complexes from cell surface to the intracellular compartments. Furthermore, 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 cell utilizes some other mechanisms to release ANP from the bound receptor molecules. Indeed, controversy exist since it has been previously reported by default that among the three natriuretic peptide receptors only NPRC internalizes with bound ligand. Hence, from a thematic standpoint it is clearly evident that there is a current need to review this subject and provide a consensus forum that establishes the cellular trafficking, sequestration and processing of ANP/NPRA complexes in intact cells. Towards this aim the cellular life-cycle of NPRA will be described in the context of ANP-binding, internalization, metabolic processing, and/or inactivation, down-regulation, and degradation of ligand-receptor complexes in model cell systems.     

The functional genomics of guanylyl cyclase/natriuretic peptide receptor-A: perspectives and paradigms

The cardiac hormones atrial natriuretic peptide and B-type natriuretic peptide (brain natriuretic peptide) activate guanylyl cyclase (GC)-A/natriuretic peptide receptor-A (NPRA) and produce the second messenger cGMP. GC-A/NPRA is a member of the growing family of GC receptors. The recent biochemical, molecular and genomic studies on GC-A/NPRA have provided important insights into the regulation and functional activity of this receptor protein, with a particular emphasis on cardiac and renal protective roles in hypertension and cardiovascular disease states. The progress in this field of research has significantly strengthened and advanced our knowledge about the critical roles of Npr1 (coding for GC-A/NPRA) in the control of fluid volume, blood pressure, cardiac remodeling, and other physiological functions and pathological states. Overall, this review attempts to provide insights and to delineate the current concepts in the field of functional genomics and signaling of GC-A/NPRA in hypertension and cardiovascular disease states at the molecular level.     

Dynamics of internalization and sequestration of guanylyl cyclase/atrial natriuretic peptide receptor-A

The guanylyl cyclase/natriuretic peptide receptor-A (NPRA), also referred to as GC-A, is a single polypeptide molecule. In its mature form, NPRA resides in the plasma membrane and consists of an extracellular ligand-binding domain, a single transmembrane-spanning region, and intracellular cytoplasmic domain that contains a protein kinase-like homology domain (KHD) and a guanylyl cyclase (GC) catalytic active site. The binding of atrial natriuretic peptide (ANP) to NPRA occurs at the plasma membrane; the receptor is synthesized on the polyribosomes of the endoplasmic reticulum, and is presumably degraded within the lysosomes. It is apparent that NPRA is a dynamic cellular macromolecule that traverses through different compartments of the cell through its lifetime. This review describes the experiments addressing the interaction of ANP with the NPRA, the receptor-mediated internalization and stoichiometric distribution of ANP-NPRA complexes from cell surface to cell interior, and its release into culture media. It is hypothesized that after internalization, the ligand-receptor complexes dissociate inside the cell and a population of NPRA recycles back to plasma membrane. Subsequently, some of the dissociated ligand molecules escape the lysosomal degradative pathway and are released intact into culture media, which reenter the cell by retroendocytotic mechanisms. By utilizing the pharmacologic and physiologic perturbants, the emphasis has been placed on the cellular regulation and processing of ligand-receptor complexes in intact cells. I conclude the discussion by examining the data available on the utilization of deletion mutations of NPRA cDNA, which has afforded experimental insights into the mechanisms the cell utilizes in modulating the expression and functioning of NPRA.     

Ligand-mediated endocytosis and intracellular sequestration of guanylyl cyclase/natriuretic peptide receptors: role of GDAY motif

The guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA), also referred to as GC-A, is a single polypeptide molecule having a critical function in blood pressure regulation and cardiovascular homeostasis. GC-A/NPRA, which resides in the plasma membrane, consists of an extracellular ligand-binding domain, a single transmembrane domain, and an intracellular cytoplasmic region containing a protein kinase-like homology domain (KHD) and a guanylyl cyclase (GC) catalytic domain. After binding with atrial and brain natriuretic peptides (ANP and BNP), GC-A/NPRA is internalized and sequestered into intracellular compartments. Therefore, GC-A/NPRA is a dynamic cellular macromolecule that traverses different subcellular compartments through its lifetime. This review describes the roles of short-signal sequences in the internalization, trafficking, and intracellular redistribution of GC-A/NPRA from cell surface to cell interior. Evidence indicates that, after internalization, the ligand–receptor complexes dissociate inside the cell and a population of GC-A/NPRA recycles back to the plasma membrane. Subsequently, the disassociated ligands are degraded in the lysosomes. However, a small percentage of the ligand escapes the lysosomal degradative pathway, and is released intact into culture medium. Using pharmacologic and molecular perturbants, emphasis has been placed on the cellular regulation and processing of ligand-bound GC-A/NPRA in terms of receptor trafficking and down-regulation in intact cells. The discussion is concluded by examining the functions of short-signal sequence motifs in the cellular life-cycle of GC-A/NPRA, including endocytosis, trafficking, metabolic processing, inactivation, and/or down-regulation in model cell systems.     

Human natriuretic peptide receptor-A guanylyl cyclase. Hormone cross-linking and antibody reactivity distinguish receptor glycoforms.

Most of the physiological actions of atrial natriuretic peptide (ANP) may be attributed to activation of the natriuretic peptide receptor-A (NPR-A) guanylyl cyclase. We report here that truncation of the NPR-A cytoplasmic domain results in increased expression of cell surface ANP binding sites. The truncated receptor exhibited a hyperbolic time course for ANP binding and had a high affinity for [125I]hANP, Kd = 8 pM. Cells expressing truncated NPR-A were used as an immunogen to obtain monoclonal antibodies against the native conformation of the extracellular domain. These antibodies were used to select for high levels of stable NPR-A expression in 293 cells, by fluorescence-activated cell sorting. Disuccinimidyl suberate cross-linked [125I]ANP to 135-kDa NPR-A on intact cells. Monoclonal antibody immunoprecipitation of 35S-labeled proteins revealed NPR-A size heterogeneity, with 135- and 125-kDa species. A synthetic peptide antibody directed against the extracellular domain immunoprecipitated 125-kDa NPR-A, but recognized both sizes of receptor by Western blotting. The 125-kDa NPR-A did not bind to or cross-link ANP. NPR-A size variants were expressed on the cell surface, and heterogeneity was removed by deglycosylation with protein:N-glycosidase F. Our results suggest that the degree of N-linked glycosylation of the NPR-A extracellular domain influences the ability to bind ANP.     

C-type natriuretic peptide and guanylyl cyclase B receptor

Guanylyl cyclases (GC) are widely distributed enzymes that signal via the production of the second messenger cGMP. The particulate guanylyl cyclases share a similar topology: an extracellular ligand binding domain and intracellular regulatory kinase-homology and cyclase catalytic domains. The natriuretic peptide receptors GC-A and -B mediate the effects of a family of peptides, atrial, B- and C-type natriuretic peptide (ANP, BNP and CNP, respectively), with natriuretic, diuretic and vasorelaxant properties. ANP and BNP, through the activation of GC-A, act as endocrine hormones to regulate blood pressure and volume, and inhibit cardiac hypertrophy. CNP, on the other hand, acts in an autocrine/paracrine fashion to induce vasorelaxation and vascular remodeling, and to regulate bone growth through its cognate receptor GC-B. GC-B, like GC-A, is phosphorylated in the basal state, and undergoes both homologous and heterologous desensitization, reflected by dephosphorylation of specific sites in the kinase-homology domain. This review will examine the structure and function of GC-B, and summarize the physiological processes in which this receptor is thought to participate.     

Human natriuretic peptide receptor-A guanylyl cyclase is self-associated prior to hormone binding.

The human natriuretic peptide receptor-A (NPR-A) guanylyl cyclase is specifically activated to synthesize cGMP by binding of atrial natriuretic peptide (ANP) to the receptor's extracellular domain. In this report, NPR-A monoclonal and polyclonal antibodies were used to assess the aggregation status of wild-type NPR-A and a truncation mutant lacking most of the NPR-A cytoplasmic domain. On intact human embryonic kidney 293 cells, in the absence of ANP, recombinant human NPR-A is self-aggregated through disulfide bonds in an M(r) > 500,000, possibly tetrameric, complex. Under nonreducing conditions, truncated NPR-A was a monomer, indicating that the cytoplasmic domain is necessary for NPR-A self-association. In the presence of the homobifunctional cross-linker dithiobis(succinimidyl propionate), or disuccimidyl suberate, truncated NPR-A could be cross-linked as a dimer and trimer only in the presence of ANP. Wild-type NPR-A was cross-linked with disuccinimidyl suberate to an M(r) > 500,000 species in the absence of ANP, and with ANP, a smaller, M(r) approximately 400,000 receptor trimer cross-linking product was observed, together with the larger, possibly tetrameric complex. When whole cell stimulation of cGMP production by ANP was tested on the low level of endogenous 293 cell NPR-A, maximal stimulation was observed regardless of truncated NPR-A overexpression. The absence of a dominant negative effect by the truncated NPR-A, together with the cross-linking data, demonstrates that preassociated NPR-A is the functionally relevant form of this receptor.     

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

We examined the kinetics of internalization, trafficking, and down-regulation of recombinant guanylyl cyclase/natriuretic peptide receptor-A (NPRA) utilizing stably transfected 293 cells expressing a very high density of receptors. After atrial natriuretic peptide (ANP) binding to NPRA, ligand-receptor complexes are internalized, processed intracellularly, and sequestered into subcellular compartments, which provided an approach to examining directly the dynamics of metabolic turnover of NPRA in intact cells. The translocation of ligand-receptor complexes from cell surface to intracellular compartments seems to be linked to ANP-dependent down-regulation of NPRA. Using tryptic proteolysis of cell surface receptors, it was found that approximately 40-50% of internalized ligand-receptor complexes recycled back to the plasma membrane with an apparent t(12) = 8 min. The recycling of NPRA was blocked by the lysosomotropic agent chloroquine, the energy depleter dinitrophenol, and also by low temperature, suggesting that recycling of the receptor is an energy- and temperature-dependent process. Data suggest that approximately 70-80% of internalized (125)I-ANP is processed through a lysosomal degradative pathway; however, 20-25% of internalized ligand is released intact into the cell exterior through an alternative mechanism involving an chloroquine-insensitive pathway. It is implied that internalization and processing of bound ANP-NPRA complexes may play an important role in mediating the biological action of hormone and the receptor protein. In retrospect, this could occur at the level of receptor regulation or through the initiation of ANP mediated signals. It is envisioned that the endocytotic pathway of ligand-receptor complexes of ANP-NPRA would lead to termination and/or diminished responsiveness of ANP in target cells.     

Central role of guanylyl cyclase in natriuretic peptide signaling in hypertension and metabolic syndrome

Studied for nearly 30 years for its ability to control many parameters, such as vascular smooth muscle cell relaxation, heart fibrosis, and kidney function, the natriuretic peptide (NP) system is now considered to be a key element in several other major metabolic pathways. After stimulation by NPs, natriuretic peptide receptors (NPR) convert GTP to the second messenger cGMP. In addition to its vasodilatory effects and natriuretic and diuretic functions, cGMP has been positively associated with fat cell function, apoptosis, and NPR expression/activity modulation. The NP system is also closely linked to metabolic syndrome (MetS) progression and obesity control. A new era is now on its way targeting the NP system to not only treat high blood pressure, but to also assist in the fight against the obesity pandemic. Here, we summarize recent data on the role of NPs in hypertension and MetS.     

Guanylyl cyclase / atrial natriuretic peptide receptor-A: role in the pathophysiology of cardiovascular regulation

Atrial natriuretic factor (ANF), also known as atrial natriuretic peptide (ANP), is an endogenous and potent hypotensive hormone that elicits natriuretic, diuretic, vasorelaxant, and anti-proliferative effects, which are important in the control of blood pressure and cardiovascular events. One principal locus involved in the regulatory action of ANP and brain natriuretic peptide (BNP) is guanylyl cyclase / natriuretic peptide receptor-A (GC-A/NPRA). Studies on ANP, BNP, and their receptor, GC-A/NPRA, have greatly increased our knowledge of the control of hypertension and cardiovascular disorders. Cellular, biochemical, and molecular studies have helped to delineate the receptor function and signaling mechanisms of NPRA. Gene-targeted and transgenic mouse models have advanced our understanding of the importance of ANP, BNP, and GC-A/NPRA in disease states at the molecular level. Importantly, ANP and BNP are used as critical markers of cardiac events; however, their therapeutic potentials for the diagnosis and treatment of hypertension, heart failure, and stroke have just begun to be realized. We are now just at the initial stage of molecular therapeutics and pharmacogenomic advancement of the natriuretic peptides. More investigations should be undertaken and ongoing ones be extended in this important field.     

Overexpression of dimeric guanylyl cyclase cores of an atrial natriuretic peptide receptor.

Using a bacterial expression system, large amounts of the catalytic core of an atrial natriuretic peptide receptor guanylyl cyclase were produced and purified. After refolding the protein from a buffer containing urea, the enzyme had positively cooperative kinetics with a Hill coefficient, nH = 1.42 +/- 0.08. Size exclusion chromatography and denaturing polyacrylamide gel electrophoresis demonstrated that the enzyme is composed of homodimers with interacting catalytic sites.     

Chromosomal distribution of three members of the human natriuretic peptide receptor/guanylyl cyclase gene family

Chromosomal localization of the genes encoding three homologous human proteins, the ANPRA, ANPRB, and ANPRC cell surface receptors, was determined by polymerase chain reaction (PCR) analysis of genomic DNA from somatic cell hybrids. The ANPRA gene was assigned to 1q12----qter by intron-specific PCR. The ANPRB gene was assigned to 9p11----p22 using species-specific length variation in PCR fragments. The ANPRC gene was assigned to chromosome 5 using human-specific PCR primers identified by screening a human primer panel on parental DNA samples (shotgun primer screening). Chromosomal assignments based on PCR analysis were confirmed and the genes further sublocalized by in situ hybridization of cloned cDNA probes to human metaphase chromosomes. The ANPRA gene was sublocalized to 1q21----q22, the ANPRB gene to 9p12----p21, and the ANPRC gene to 5p13----p14.