natriuretic peptide receptor-A

Peptides 26 (2005) 985–1000

Internalization and trafficking of guanylyl cyclase/natriuretic peptide receptor-A Kailash N. Pandey ∗ Department of Physiology, Tulane University Health Sciences Center and School of Medicine, 1430 Tulane Avenue, New Orleans, LA 70112, USA Received 4 December 2004; accepted 20 December 2004 Available online 15 April 2005

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. © 2005 Elsevier Inc. All rights reserved. Keywords: Natriuretic peptides; Natriuretic peptide receptors; Guanylyl cyclase; Internalization; Trafficking; Down-regulation; Metabolic processing; Degradation

1. 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 ∗

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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].

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The general topological structure of NPRA is consistent with GC receptor family with at least four distinct regions comprising ligand-binding, transmembrane, protein kinaselike 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 ligandbound 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.

2. 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 domain of the receptor. The KHD of NPRA is more closely related to protein tyrosine kinases than protein serine/threonine kinases. In fact, it is largely similar to the protein kinase domain of the platelet-derived growth factor receptor with approximately 31% amino acid sequence identity between the comparable regions of the kinase domains [25,73,100]. It has been demonstrated that the KHD of NPRA serves as an important mediatory role in transducing the ligand-induced signals to activate the GC catalytic domain of the receptor [24,34,44]. It has been suggested that an intervening step involving the KHD is necessary to the cyclase catalytic activation process [48,59,60]. Several studies have suggested that ATP serves as an intracellular allosteric regulator of the KHD for the activation of NPRA [26,34,63,65,128]. ATP is thought to function by interacting with the KHD because this region contains a glycine-rich nucleotide-binding motif and has been postulated to provide ATP-regulatory module for ANP signaling [35,110]. It has been also suggested that binding of ANP to NPRA activates ATP binding to the KHD in the intracellular cytoplasmic space, which, in turn, activates the GC catalytic domain of the receptor [34,48,54]. Indeed, previous studies as well as recent data have indicated that the KHD seems to be important for ANP-dependent activation of NPRA [66,104]. However, the exact mechanisms of activation and relay of signals from the KHD to the GC catalytic active site of the receptor remain to be established. The GC catalytic domain of NPRA contains an approximately 250-amino acid region that constitutes the catalytic active site of the receptor [71,118,122]. The transmembrane GC receptors contain a single cyclase catalytic active site per polypeptide molecule, however, based on the structure modeling data [124] two polypeptide chains seem to be required to activate NPRA, and receptor functions as a homodimer [64,127,129]. Initially, the dimerization region of the receptor was believed to be located between the KHD and GC domain that has been suggested to form an amphipathic alpha helix structure [45]. It has been reported that the crystal packing of the extracellular ligand-binding domain of NPRA contains two possible dimer pairs, the head-to-head and tailto-tail dimer pairs associated through the membrane-distal and membrane-proximal subdomains, respectively [106]. The tail-to-tail dimer of NPRA has been proposed previously [124]. The crystal structure of NPRC has also suggested that NPRC is dimerized in head-to-head configuration bound with ligand CNP [51]. It has been previously proposed that head-to-head dimer may represent the latent inactive state and the tail-to-tail dimer could represent the hormone-activated state [123]. It has also been indicated that ligand-dependent activation of NPRA stabilizes a membrane-distal dimer interface of this receptor protein suggesting that ANP

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binding stabilizes the NPRA dimer with more stringent spacing at the dimer interface [31]. However, more recently, the results of site-directed mutagenesis and chemical modification studies suggested that head-to-head dimmer structure reflects the physiological dimmer structure of NPRA [106].

3. 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 complexes were not processed intracellularly [58]. These authors suggested that a rapid dissociation of receptor-ligand complexes takes place upon ANP binding to NPRA at 37 ◦ C on the plasma membrane, and intact ligand is released into culture medium. However, it is difficult to contemplate the findings of those previous studies since the dissociation of ligand was carried out in a medium containing very high concentrations of unlabeled ANP (1 ␮M) to preclude the rebinding of dissociated ligand to receptors, which might have produced artifactual results. Additionally, the cell lines utilized in those previous studies expressed more than one ANP receptor subtype including both NPRA and NPRC. At the same time another study indicated that in neuroblastoma cells, bound-ligand to NPRA was degraded and released by a neutral metalloendopeptidase [32]; however, further studies have not been carried out to confirm those results. Nevertheless, our subsequent studies utilizing MA-10 cell line containing exclusively high density of endogenous

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NPRA, as well as COS-7 and human embryonic kidney-293 (HEK-293) cells expressing recombinant NPRA, firmly established that ANP/NPRA complexes are rapidly internalized and processed intracellularly in intact cells [89,93,96]. The kinetics of internalization and metabolic processing of ANP through NPRC, which does not contain GC catalytic activity, has been extensively studied [1,18,27,52,82,83,85,88,90,98]. Early studies of the post-binding events of NPRC were greatly facilitated due to its predominant presence in vascular smooth muscle cells, which provided a suitable model to study the metabolic fate of NPRC. Studies utilizing both COS-7 and HEK-293 cells expressing recombinant NPRA and MA-10 cells containing endogenous NPRA determined the kinetics of ligandbinding, internalization, and intracellular sequestration of NPRA to distinguish between cell surface-associated, internalized, and degraded ligand-receptor complexes in the intact cells [89,96,97,99]. Acid treatments at 4 ◦ C removed greater than 95% of cell surface-associated 125 I-ANP radioactivity, whereas at 37 ◦ C less than 50% radioactivity was removed [89,96,97]. Those previous observations suggested that after binding of 125 I-ANP to NPRA, the ligand-receptor complexes were internalized at physiological temperatures and both the degraded and intact ligands were released into culture medium. The distribution of 125 I-ANP radioactivity on the cell surface, in the intracellular compartments, and into culture medium provided a dynamic equilibrium between the rates of 125 I-ANP uptake, its degradation, and extrusion. It was observed that a major portion of the internalized 125 IANP was released into culture medium, which consisted approximately 70–75% degraded products and about 25–30% intact ligand (Fig. 1). During the initial incubation period, the release of both the degraded and intact ligands was blocked to a greater extent by lysosomotropic agents chloroquine, ammonium chloride, and monensin; however, after longer incubation period, the effect of chloroquine was only par-

Fig. 1. Effect of unlabeled ligand on the release of internalized 125 I-ANP in 293 cells. Confluent cells were preincubated in the absence or presence of 200 ␮M chloroquine and allowed to bind 125 I-ANP at 4 ◦ C for 1 h. One group of the chloroquine-treated cells was exposed to unlabeled extracellular ANP (10 nM). Both the treated and control cells were warmed to 37 ◦ C, and the released 125 I-ANP radioactivity was determined at the indicated times. Panels A and B represent the degraded and intact 125 I-ANP released into the culture medium, respectively, as a function of incubation time. (䊉) Control; (
) Chloroquine; and () Chloroquine with ANP (adapted from Pandey et al. [97] with permission).

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tially effective in blocking the release of both the degraded and intact ligands [89,94,96,97]. Overall, the available evidence supports the notion that a majority of internalized 125 I-ANP is processed through the degradative compartments. Nevertheless, an alternate mechanism also seems to exist for the release of intact ligand. Dual pathways for the intracellular processing of ligand-receptor complexes of insulin have been previously suggested [76,120]. Our previous studies have provided definitive evidence for the recycling of internalized NPRA from cell interior to the plasma membrane [89,96,97]. Those previous studies have also shown that a majority of the internalized ligand (>70%) is degraded in the lysosomes and released into culture medium. Nevertheless, 25–30% of ligand-receptor complexes escape the lysosomal pathway and are extruded intact into cell exterior. It is expected that the intact ligand can rebind the recycled receptors on the cell surface and re-enter the cell via repeated retroendocytosis mechanisms. It is predicted that the homeostatic regulation of NPRA and its cellular sensitivity to ANP would be dependent on dynamic equilibrium of endocytosis mechanisms and intracellular processing of ANP/NPRA complexes in a cell-specific manner. The rates of both the internalization and breakdown of 125 I-ANP in MA-10 cells containing endogenous NPRA as well as in COS-7 and HEK-293 cells containing recombinant receptor were markedly inhibited in the presence of metabolic inhibitors such as chloroquine and dinitrophenol [89,96].

Chloroquine is known to inhibit the lysosomal degradation and dinitrophenol disrupts the energy-dependent intracellular trafficking of various ligand-receptor complexes [19,78,89].

4. 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 HEK293 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 approximately 55–60% of the original levels (Fig. 2). The pretreatment of cells with cycloheximide also exhibited a return in the cell surface 125 IANP binding, however, 20–25% lower binding efficiency than the control cells without cycloheximide [97]. A parallel set of dishes exposed to unlabeled ANP was also incubated at 16 ◦ C, and under these conditions, the 125 I-ANP binding was not discernible, indicating that there was no recycling of NPRA at lower temperature. If cells were subsequently warmed to 37 ◦ C, there was a rapid return of 125 I-ANP binding, suggesting that internalized NPRA recycled to the plasma membrane (Fig. 2). In another experiment, HEK-293

Fig. 2. Recycling of internalized NPRA after treatment of 293 cells with unlabeled ANP. Confluent 293 cells stably expressing NPRA were incubated with cycloheximide (20 ␮g/ml) at 37 ◦ C for 1 h and then exposed to 100 nM unlabeled ANP. Cells were washed free of ANP with acid buffer (pH 3.5), reincubated with and without cycloheximide and 125 I-ANP binding was determined. One group of cells was exposed in the presence of ANP throughout (dotted line) as a control. A second group of cells was washed with acidic buffer (pH 3.5), incubated at 16 ◦ C for 45 min, and then temperature was shifted to 37 ◦ C (shown by the arrow). The solid bar represents the binding of 125 I-ANP in control cells (C), which were never exposed to ANP (adapted from Pandey et al. [97] with permission).

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Fig. 3. Trypsin-dependent loss of 125 I-ANP binding and recycling of internalized NPRA in 293 cells. The confluent 293 cells were incubated at 4 ◦ C in the absence or presence of 0.025% trypsin for 10 min. After trypsin treatment, cells were washed with medium containing 10% serum to stop the trypsin reaction. The cells were then further incubated at 37 ◦ C in fresh medium containing 10% serum, and 125 I-ANP was determined in the absence or presence of cycloheximide (20 ␮g/ml). The solid bar represents the binding of 125 I-ANP in control cells, which were never exposed to trypsin. (䊉) without cycloheximide; (
) with cycloheximide (adapted from Pandey et al. [97] with permission).

cells were treated with 0.025% trypsin at 4 ◦ C for 10 min, which abolished cell surface 125 I-ANP binding capacity of NPRA. The cells were washed free of trypsin and reincubated in fresh medium in the absence or presence of cycloheximide at 37 ◦ C. In cycloheximide-treated cells, binding of 125 I-ANP was 30–40% lower than in control cells without cycloheximide treatments (Fig. 3). The results from these experiments further strengthened the view that a return in 125 I-ANP binding in HEK-293 cells was due to the recycling of NPRA [97]. Because a complete return of 125 I-ANP binding did not occur and it remained lower in cycloheximide-treated cells than non-treated control cells, it was predicted that de novo protein synthesis might also be involved. To determine that NPRA was reinserted into the plasma membrane from pre-existing intracellular pool, the cell surface and the total receptors were measured in intact and solubilized extract of HEK-293 cells. To assess the proportion of receptor population localized in cell interior, 125 I-ANP binding was performed in solubilized extracts of both trypsin-treated and untreated cells. Receptor binding in trypsin-treated solubilized cell preparations indicated that most of the receptors were present on the plasma membrane and only 18–20% were assigned to pre-existing intracellular pool [97]. Receptors can be inactivated by trypsin on the cell surface; therefore, it can be assumed that some receptors might be present on the cytoplasmic side of the plasma membrane, and this could account for a significant proportion of the measured intracellular receptor pool. However, this possibility seems to be less likely because only 20% receptors of the total NPRA

population constituted intracellular pool, which cannot account for the replacement of receptors lost during ANP treatment. Photoaffinity labeling studies in the intact cells also demonstrated that internalized NPRA recycles back to the plasma membrane [89,92]. The recycling of endocytosed NPRA was analyzed by incubating MA-10 cells with photoaffinity ligand azidobenzoyl-125 I-ANP (AZB-125 I-ANP) at 37 ◦ C followed by tryptic proteolysis to digest the photoaffinity-labeled intact 135-kDa receptor on the cell surface. The labeled 135-kDa NPRA was visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) and autoradiography. The internalized NPRA became trypsin-resistant, and those, which returned to the cell surface, became susceptible to trypsin and produced the 68-kDa tryptic fragment. The control cells, which were not exposed to trypsin, showed exclusively the 135-kDa intact receptor band of NPRA. After trypsin treatment of the photoaffinitylabeled cells, the amount of tryptic fragment increased in a time-dependent manner [89]. Densitometric analysis of the radioactive-labeled bands indicated that approximately 60% of the intact receptor pool became trypsin-sensitive and produced 68-kDa tryptic fragments after recycling of NPRA. The concomitant increase in the 68-kDa tryptic fragments of the receptor suggested that the internalized NPRA recycles from intracellular compartments to the plasma membrane. The density of the intact 135-kDa NPRA band decreased in the cell interior and the 68-kDa tryptic fragment increased, which suggested that internalized receptor molecules be-

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came trypsin-sensitive after returning to the plasma membrane [89]. A decrease in the receptor population from the intracellular pool was impaired by chloroquine and dinitrophenol, which further supported the notion that a decrease in the intracellular pool of NPRA occurs due to recycling of receptor to the plasma membrane and not by trypsin entering inside the cell. The reinsertion of photoaffinity-labeled receptors in the plasma membrane was very rapid at physiological temperature of 37 ◦ C and reached completion in 10 min (t1/2 = 5 min). After initiation of the internalization of ligand-receptor complexes, approximately 50–60% labeled receptors recycled from the cell interior to the plasma membrane in 10 min. The recycling of NPRA was slower at low temperatures (22 ◦ C) as compared to 37 ◦ C and essentially negligible at 10 ◦ C [89]. It should be noted that the incubation of adrenal membranes at pH 3.5–5.6, resulted in the degradation of the 135-kDa intact NPRA and the generation of the 68to 70-kDa receptor fragment [81]. These authors suggested that the 70-kDa receptor fragments is generated because of an endogenous proteolytic degradation of the 135-kDa receptor protein. Both chloroquine and dinitrophenol are known to impair the recycling of various ligand-receptor complexes [43,120,125]. The results of our previous studies as well as recent experimental data are consistent with the notion that if recycling is inhibited, the internalization of the receptor continues, suggesting that the receptor will not be reinserted into the plasma membrane and a rapid loss in the number of cell surface receptor will occur [89,96,97]. The ANP binding studies clearly demonstrated that in the presence of chloroquine the amount of bound 125 I-ANP to cell surface receptors was significantly decreased. Similarly in the chloroquine- and dinitrophenol-treated cells, the return of internalized photoaffinity-labeled ligand-receptor complexes to the plasma membranes was severely impaired, and the production of 68-kDa tryptic receptor fragment was drastically inhibited [89,96]. Those previous studies suggested that both lysosomotropic agent chloroquine, and the metabolic inhibitor dinitrophenol which depletes cellular ATP, disrupted the internalization and recycling process of NPRA [89,96,97]. However, it has been reported that ATP is not required for internalization of the insulin receptor [5,112]; nevertheless, it seems to be essential for internalization of epidermal growth factor (EGF) receptor [19]. The internalized hormone-receptor complexes of the low-density lipoprotein (LDL) enter the acidic vesicular compartments where the ligand dissociates from the receptor. The dissociated LDL receptors recycle back to the plasma membrane and the ligand is degraded in the lysosomal compartments [15]. The studies utilizing COS-7 and HEK-293 cells expressing recombinant NPRA, and MA-10 cells containing endogenous NPRA, suggested that the dissociation of bound ANP from the receptor is not a prerequisite for the recycling of NPRA [89,93]. It is indicative that the recycling of NPRA probably also occurs constitutively in addition to the ligand-dependent regulatory mechanisms.

It has been postulated that after endocytosis of NPRA, some of the internalized pool of receptors could be diverted to degradation while the remainder could be subject to regulated recycling [89,96]. Clearly, further studies would be required to clarify the mechanisms that control the endocytosis and recycling of NPRA. Consideration should be given to the fact that internalized NPRA may provide signals that contribute to the regulation of receptor gene expression. The ligand-evoked internalization and partial degradation of GABA receptors are known to both enhance as well as repress the receptor gene-expression [8,39,56,80]. Intriguing was the finding that agonist-dependent endocytosis of ␤2adrenergic receptors is a necessary step in the activation of MAP kinases and mitogenic signals [68]. Those previous findings support the view that certain classes of receptors may regulate their own biosynthesis through putative intracellular signals by ligand-dependent endocytosis. Indeed, at present, this is a speculative notion; however, it may provide new direction for future investigations on the trafficking and functional regulation of NPRA as well as other GC family of receptors.

5. 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 ligand and receptor molecules in the lysosomes. In the course of time, the increased rate of degradation can exceed the rate at which the receptors are replaced by de novo synthesis and the total number of receptor population is correspondingly reduced [121]. This in turn renders the cell refractory to the hormone, and results in a state of hormone resistance whereby the prevailing ligand concentrations is not sufficient to effectively cause the biological responsiveness. After binding of ANP to NPRA, ligand-receptor complexes are internalized and sequestered into the intracellular compartments and degraded products are released into culture medium. It has been shown that the pretreatment of HEK-293 cells with unlabeled ANP caused a substantial decrease in the 125 I-ANP binding capacity of NPRA in both a time- and dose-dependent manner [97]. The results of those previous studies showed that the treatment of cells with 10 nM ANP markedly reduced the cell surface 125 I-ANP binding by 55–65% in 60 min, and the micromolar concentrations of ANP produced almost a complete loss of cell surface ligand-binding capacity of NPRA. The maximum effect of ANP on down-regulation of NPRA occurred within 60 min after treatment of cells with hormone. Internalization of ANP/NPRA complexes seems to

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play an important role in receptor down-regulation [96,97]. The ligand-dependent down-regulation by receptor degradation is usually homologous in nature since it only involves the receptor of one particular ligand and even though other receptors may be present on the cell surface. The rate of internalization of other receptors is unaffected. This mechanism differs from that of sequestration since it involves the total loss of cellular receptors rather than receptor redistribution into a compartment, which may be inaccessible to extracellular ligands. In large part, the mechanisms that lead to homologous down-regulation of receptors involve the complete removal of ligand-bound receptors from cell surface by an endocytic process. There could be several variations in this theme and that can result in receptor internalization, ligand degradation, and then recycling of the receptor to the cell surface, or alternatively both the receptor and the ligand can be degraded in the lysosomal compartments. If receptor recycles to the cell surface, to some extent the internalization can be compensated by the reappearance of the receptors in the plasma membrane and thus down-regulation can be delayed, until receptors begin to be degraded. Essentially, down-regulation may result in a loss of cellular NPRA by means of an accelerated rate constant for receptor internalization and presumably inactivation. Desensitization of a receptor can be defined as a process by which the function of receptor is lost, whether it is ANP binding affinity, autoregulatory efficacy of the KHD, and/or GC catalytic activity. In our metabolic processing studies of NPRA, ANP binding has been utilized as the index of NPRA activity. Degradation, on the other hand, is considered as the actual loss of the receptor protein from the cell by proteolysis into amino acids. Temporally, inactivation may precede degradation or both events may occur simultaneously. An invaluable experimental tool in the elucidation of NPRA inactivation would be to use ANPinduced receptor down-regulation. A change in the receptor phosphorylation state has been implicated in the process of desensitization. Interestingly, the desensitization does not require the large-scale removal of receptors from the cell surface and is probably achieved by a combination of both receptor phosphorylation and degradation. Desensitization and/or inactivation of NPRA have been suggested by ANPdependent dephosphorylation of receptor protein [102,103]. However, the exact mechanism of the dephosphorylationdependent inactivation of NPRA is not well understood. Previous studies as well as recent data have indicated that the NPRA phosphorylation seems to occur in a number of cell types [35,36,66,87,105,111]. It has also been suggested that the phosphorylation of kinase homology domain of NPRA is essential for its activation process [104]. We anticipate that the inactivation of a receptor occurs intracellularly, and inhibition of internalization should prevent this process. However, the biochemical nature of the inactivation process and subcellular location of the event have yet to be determined.

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6. 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 NH4 Cl2 inhibited the degradation of ANP, providing direct evidence that ANP is metabolized in lysosomes (Fig. 2). ANP-specific endopeptidase is also known to degrade ANP; however, its location in the endosomal compartments has not been established. The endosomal system requires ATP for the maintenance of pH gradient across the endosomal membrane. Since acidification is believed to augment the release of ligand from the receptors within the endosomes, the question can be raised as if the ANP-endopeptidase is active on ANP, bound to the receptor or ANP acting, as a substrate for endopeptidase, has to be free in the lumen of the endosome. The localization of ANP-endopeptidase-like activity into cytosol, plasma membrane, and endosomes would be of a great significance. If the physiologically relevant location of the endopeptidase is endosomal compartment and if the enzymes in all three compartments are identical, the question can be raised as by what mechanism the cytosolic enzyme is inserted into the endosome. Furthermore, whether this insertion process is a locus for cellular regulation of the degradation of ANP remains to be established.

7. 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 ligand to the cell surface receptor inserted into the plasma membrane. The receptors, through some intrinsic affinity or aggregation induced by protein binding must cluster into pit on the plasma membrane. The proposed itinerary would be consistent with the notion that ANP/NPRA complexes should be delivered to the lysosomal compartments. Acidification of lysosomes induces the dissociation of ligand from the receptor, and a population of receptor molecules may recycle back to the plasma membrane and the following sequence of events may take place: (a) a lysosomotropic agent such as chloroquine is unable to completely block the degradation of ANP/NPRA in lysosomes, (b) the release of intact ANP seems to occur through a lysosome-independent pathway,

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Fig. 4. Schematic representation of internalization, recycling, and intracellular degradation of 125 I-ANP bound to NPRA in 293 cells. The schematic diagram shown postulates the stoichiometric kinetics of internalization, subcellular sequestration, recycling, and ultimately metabolic turnover of ligand-receptor complexes from cell surface to cell interior and back to the plasma membrane. The scheme depicts that after synthesis: (i) the receptor is inserted in the plasma membrane; (ii) the ligand-receptor complex enters the cell via coated pits; and (iii) the complex is processed intracellularly through endosome-, lysosome-, and/or chloroquine-insensitive pathways. Sorting of bound ANP-NPRA complexes into the intracellular compartments may occur by (i) lysosomal degradative metabolic pathway; (ii) endosomal dissociation metabolic pathway, and/or (iii) release through the chloroquine-insensitive pathway (adapted from Pandey et al. [97] with permission).

and (c) recycling of endocytosed NPRA back to the plasma membrane occurs simultaneously with the process leading to the degradation of the majority of ligand-receptor complexes into the lysosomal compartments. It has been suggested that internalized ANP bifurcates into two major pathways: a degradative pathway, through which the 70–80% of the internalized ANP of all incoming ligand is processed in lysosomes, and a retroendocytotic pathway that accelerates the release of intact ligand [89,97]. This approach should provide a direct assessment of ligand-bound receptor trafficking. It is conceivable that some remarkable differences do exist with regard to the internalization, processing, and metabolic turnover among various types of membrane receptors. The phenomenon of ANP-NPRA degradation is similar to that reported for low-density lipoprotein receptors in human fibroblasts [16,22], insulin receptors in adipocytes [10,46,76] and transfected Chinese hamster ovary cells [86], and thyrotropin hormone receptors in GH3 cells [3]. However, the degradation of asialoglycoprotein and its receptor complexes is not observed until about 30 min after endocytosis in hepatoma cells [50]. Similarly, the degradation of EGF is not detectable for at least 20 min in hepatocytes [37]. Although there is no apparent mechanistic explanation to ac-

count for such differences, several possibilities can be considered. Because the internalization of receptor-bound ligand should not be the limiting factor, there may be multiple pathways leading to the eventual metabolic turnover of ligandreceptor complexes, perhaps utilizing different intermediate vesicles for the transfer of ligand to the site of degradation. If this were the case, the intracellular sequestration route could be determined by either intrinsic properties of ligand-receptor complexes or the way various cells process the incoming ligands. It is also possible that there may be a single metabolic pathway composed of several distinct processing steps, which should be unique for a specific ligand-receptor complex. Dual pathways for the intracellular processing of ligand-receptor complexes have been proposed previously for insulin and epidermal growth factor (EGF) receptors [77,115,116,120,126]. The previous findings demonstrating that chloroquine effectively interrupts the degradative processing of NPRA without exerting a deleterious effect on the retroendocytotic pathway is novel and intriguing [97]. Similar to NPRA, several other types of ligand-receptor complexes recycle through the chloroquine-insensitive pathway, including EGF, insulin, and asialoglycoprotein receptors [17,21,76,113,126]. This establishes the notion that after internalization, many

K.N. Pandey / Peptides 26 (2005) 985–1000

types of ligand-receptor complexes can recycle through the chloroquine-insensitive pathway and finally be degraded via chloroquine-sensitive lysosomal compartments.

8. 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 170-amino acid region at the carboxyl-terminal end of NPRA (
937-NPRA,
916-NPRA,
887-NPRA) diminished the internalization of ligand-receptor complexes by 60% [96]. Cells expressing wild-type NPRA released 40–45% 125 I-ANP radioactivity into culture medium, but only 10–15% 125 I-radioactivity was released from the cells that expressed mutant NPRA (
635-NPRA). Approximately 35–40% of the 125 I-ANP radioactivity was detected into the intracellular compartments of cells that expressed the wild-type NPRA and only less than 10% of the 125 I-ANP radioactivity was detected in cells expressing the
635-NPRA. These findings suggested that specific regions within the intracellular domains of NPRA determine the extent of ligand-binding efficiency, endocytosis, and intracellular sequestration of ligand-receptor complexes in transfected COS-7 cells expressing NPRA [96]. The role of NPRA cytoplasmic tail is comparable to the thyrotropin-stimulating hormone receptor [84], insofar as its disruption blocks its internalization. Interestingly, most of the internalization signals have been reported to be present in the cytoplasmic domains of membrane receptors [49,53,101,114,115]. Evidence suggests that a majority of the receptors that undergo endocytosis contain internalization signals such as NPxY sequence motif in the cytoplasmic portion, near the transmembrane domain of the receptor. However, NPRA including other members of GC-coupled receptor family do not seem to contain an intracellular NPxY sequence motif near the transmembrane domain. Therefore, it indicates that the internalization of ligand-receptor complexes may not be solely dependent on NPxY motif located near the transmembrane domain of the receptor proteins. Although the KHD of NPRA has been suggested to play an important role in the functional activity of the receptor, the exact mechanisms by which it controls GC catalytic activity and other function of NPRA are not well understood. The experiments were designed to determine the structural elements in murine cDNA sequences that are essential for ligand-dependent internalization of NPRA. The recent findings have provided new insights into the structure and

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function of NPRA by demonstration that the GDAY (Gly920 Asp921 -Ala922 -Tyr923 ) motif is important for the internalization of ligand-receptor complexes into the cell interior [94]. As described above, previously it has been shown that carboxyl-terminal region is required for ligand-dependent internalization of NPRA [96]; however, the involvement of a specific sequence motif in the internalization process was not determined. The studies have demonstrated that point mutations in GDAY motif within the carboxyl-terminal region of NPRA have a major effect on internalization of this receptor protein [94]. The mutation of GDAY to ADAA (residues 920–923) diminished the internalization of ligandbound NPRA by almost 50% (Fig. 5); however, at a rate significantly greater than that observed for receptor lacking the carboxyl-terminal domain, which are totally devoid of rapid endocytotic behavior of the receptor protein [96]. The quantitative determination of degraded and intact ligand released into the culture medium was made based on the amount of total initial bound 125 I-ANP on the cell surface. The treatment of HEK-293 cells expressing wild-type and GDAY/AAAA mutant receptors with lysosomotropic agents chloroquine, ammonium chloride, and monensin blocked significantly the degradation of internalized 125 I-ANP/NPRA complexes as compared with untreated control cells (Fig. 6A and B). It is noteworthy that after treatment with lysosomotropic agents, cells expressing wild-type NPRA (Fig. 6A) contained almost 50% more 125 I-ANP-bound receptor levels in the intracellular compartments as compared with HEK-293 cells

Fig. 5. Quantitative analysis of degraded and intact 125 I-ANP released into culture medium of HEK-293 cells expressing wild-type or GDAY/AAAA mutant receptors. HEK-293 cells expressing wild-type or GDAY/AAAA mutant receptors were allowed to bind 125 I-ANP at 4 ◦ C for 1 h. After which, cells were washed four times with ice-cold assay medium to remove unbound ligand and then reincubated in 2 ml fresh assay medium at 37 ◦ C for 30 min. At the end of incubation period, the culture medium was collected and the intact and degraded ligand products were quantified (adapted from Pandey et al. [94] with permission).

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Fig. 7. Quantitative analysis of trypsin resistant intracellular NPRA in HEK293 cells expressing wild-type or mutant receptors. Confluent HEK-293 cells expressing wild-type, G920/A , D921/A , Y923/A , or GDAY/AAAA mutant NPRA were exposed to 0.025% trypsin in DMEM assay medium for 10 min at 4 ◦ C. The trypsin reaction was stopped by adding the soybean trypsin inhibitor (200 ␮g/ml) and by washing the cells with medium containing 10% FBS. 125 I-ANP binding was determined in recombinant HEK-293 cells expressing either wild-type or different mutant receptors at indicated time periods. The solid bar represents the 125 I-ANP binding in control cells, which were never exposed to trypsin treatments (adapted from Pandey et al. [94] with permission).

Fig. 6. Quantitative analyses of cell surface-associated, internalized, and released 125 I-ANP radioactivity after treatments with lysosomotropic agents in HEK-293 cells expressing wild-type or GDAY/AAAA mutant NPRA. confluent HEK-293 cells expressing either (A) wild-type (B) or mutant receptors were preincubated with lysosomotropic agents; chloroquine (200 ␮M), ammonium chloride (10 mM), and monensin (50 ␮M)) at 37 ◦ C for 60 min. Cells were then incubated with 125 I-ANP in 2 ml of fresh assay medium for 60 min at 4 ◦ C. 125 I-ANP radioactivity in acid eluate, cell extract, and culture medium was counted to determine the cell surface-associated, internalized, and released 125 I-ANP radioactivity, respectively (adapted from Pandey et al. [94] with permission).

expressing GDAY/AAAA mutant receptor (Fig. 6B). Both wild-type and GDAY/ADAA mutant cDNA constructs were transfected into HEK-293 cells and stably selected recombinant clonal cell lines expressed almost comparable receptor

densities with a similar Kd values (Table 1). The results indicated that mutation in GDAY sequence did not alter the level of receptor expression or half maximal effective ligand concentrations as previously reported [97]. Furthermore, the GDAY motif plays a dual role in receptor internalization into the cell interior and in subsequent recycling of internalized receptor to the plasma membrane. The mutation of Gly920 and Tyr923 to alanines in NPRA cDNA markedly attenuated the internalization of mutant receptors by almost 50% (Fig. 7). The mutation of Asp921 to alanine showed only a minimal effect, however, Ala922 failed to attenuate internalization of NPRA. These findings demonstrated that receptor internalization relies largely on residues Gly920 and Tyr923 in the carboxyl terminal region of NPRA, whereas mutation of Asp921 to alanine inhibited the recycling of internalized receptor to the cell surface [94]. To examine the possibility that NPRA was reinserted into the plasma mem-

Table 1 Competition 125 I-ANP binding and comparative receptor parameters in HEK-293 cells expressing wild-type or GDAY/AAAA mutant NPRA Transfection

Exon sequence

Dissociation constant (Kd)

Receptor density (Bmax )

Wild-type NPRA GDAY/AAAA NPRA

VYKVETIGDAYMVSG VYKVETIAAAAMVSG

2.4 × 10−10 M 2.4 × 10−10 M

1.86 × 106 1.89 × 106

Kd, dissociation constant; Bmax , receptor sites/cell. Confluent HEK-293 cells expressing either wild-type or GDAY/AAAA mutant receptors were exposed to 125 I-ANP at 4 ◦ C for 1 h in the presence or absence of unlabeled ANP. The non-specific binding was determined by using 100 × excess molar concentrations of unlabeled ANP. Half maximal effective concentrations (EC50 ) or Kd values and receptor density (Bmax ) were determined from the binding competition and Scatchard analysis of the 125 I-ANP binding data. The amino acid sequence encoded by the exon regions for the wild-type and mutant GDAY/AAAA NPRA cDNAs are indicated with single letter amino acid residues (adapted from Pandey et al. [94] with permission).

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Table 2 Quantitation of the total and intracellular pool of wild-type and GDAY/AAAA mutant NPRA in intact and solubilized recombinant HEK-293 cells treated with and without trypsin Treatment

Wild-type NPRA % Specific

Untreated controls cells Trypsin-treated cells Triton X-100 solubilized cells Trypsin-treated Triton X-100 solubilized cells

20.64 1.48 25.73 6.28

± ± ± ±

125 I-ANP

GDAY/AAAA NPRA binding

2.92 0.16 2.81 0.30

% Relative binding

% Specific 125 I-ANP binding

100 7.17 100 24.40

21.03 1.76 23.92 4.46

± ± ± ±

3.01 0.14 2.54 0.28

% Relative binding 100 5.99 100 18.68

HEK-293 cells, expressing wild-type or GDAY/AAAA mutant receptors were allowed to bind 125 I-ANP in control and trypsin-treated (0.025%) groups. In parallel experiments, the control and trypsin-treated cells were solubilized in buffer containing 1% Triton X-100, 15% glycerol, and protease inhibitors. The percent relative binding was determined from the specific 125 I-ANP binding parameters (adapted from Pandey et al. [94] with permission).

brane from pre-existing intracellular pool, the cell surface and total receptors were measured in intact and solubilized preparations of HEK-293 cells expressing either wild-type or GDAY/AAAA mutant NPRA (Table 2). To assess the proportion of both wild-type and GDAY/AAAA mutant receptors in the cell interior, 125 I-ANP binding was measured in solubilized extracts of both trypsin-treated and untreated recombinant HEK-293 cells. 125 I-ANP binding in trypsin-treated and Triton X-100-solubilized HEK-293 cells expressing the wildtype and GDAY/AAAA mutant receptors indicated that most of the receptors were present on the plasma membrane and only a small fraction of receptor population (15–20%) could be assigned to the pre-existing intracellular pool (Table 2). Several sequence-specific motifs have been demonstrated to play critical role in the internalization and trafficking of a number of hormone receptor proteins. It has been shown that the consensus internalization sequence Asn-Pro-x-Tyr (NPxY) in the intracellular domain of low-density lipoprotein (LDL) receptor is necessary for internalization [22]. The NPxY is also present in the cytoplasmic domains of other receptors [4,12,119]. Specifically, tyrosine residue in the NPxY signal has been implicated to be important for internalization of a number of hormone receptors [55,72,79]. The internalization of platelet-activating factor is also regulated by a putative motif DPxxY [23] and that of vasopressin type-2 receptor by NPxxY sequence [13]. Similarly, Gly-Pro-Leu-Tyr (GPLY) signal has been implicated to be important for internalization of insulin receptor [107]. In addition, Tyr-x-Arg-Phe (YxRF) sequence has been identified in the tranferrin receptor in which Tyr is at the first position in this signal sequence [29]. Similarly, YxxL motif has also been suggested to play a role in the endocytosis of LDL receptor-related protein [70]. A common feature of all these internalization motifs, including the currently reported GDAY for NPRA internalization [94], to contain a tyrosine residue either in the beginning or at the end of tetrapeptide sequence. Tyrosine residue has also been suggested to play role in the endocytosis of mannose-6-phosphate receptor [72] and influenza virus hemagglutinin [23,67,72]. Therefore, if a universal internalization signal exists, it may not be based on a universal amino acid sequence. It has been suggested that the critical characteristics of all these sequences might be their specification of a particular conformation, a tight

beta-turn in protein structure [29]. The role of NPEY and GPLY sequence motifs in receptor internalization has been well recognized, however, GPLY has been suggested to play a wider role in rapid endocytosis of insulin receptor [11]. The internalization and sequestration of NPRA can be determined by quantitating the amount of intracellular-labeled ligand-receptor complexes. However, these measurements are made at early time periods in the presence of 125 I-ANP at 37 ◦ C. We have previously demonstrated that after 5 min incubation of 125 I-ANP-labeled cells at 37 ◦ C, the internalization of ligand-receptor complexes of NPRA reaches maximum levels, after which a large proportion of internalized ligand begins to be degraded and a population of internalized receptor recycles back to the plasma membrane [96,97]. Thus, measuring the amount of intracellular radioactivity at times beyond 5 min cannot be considered an accurate reflection of the amount of ANP that had initially been endocytosed into the cell interior. On the other hand, some useful information can be gained from determining intracellular levels of ANP beyond the 5 min internalization period, by a combination of the internalization rate, the degradation rate, and the recycling or retroendocytotic rate of the NPRA. Evidently, after 5 min, the intracellular amount of radiolabeled 125 I-ANP reaches the highest levels in cell lines expressing wild-type or GDAY/ADAA mutant NPRA. However, the amount of intracellular mutant receptor was significantly reduced, by almost 50%, as compared with wild-type NPRA [94]. Similarly, the amount of degraded ligand released into the culture medium was also significantly reduced in cells expressing mutant NPRA. This would imply that the disruption of internalization sequence did not alter the subsequent intracellular routing of the receptor. In retrospect, the initial internalization pathway that is taken may also determine the subsequent fate of the receptor and ligand after endocytosis into the intracellular compartments. In general, relatively little is known about the signals for intracellular routing of receptors after internalization, as in case of the polymeric immunoglobulin receptor, in which serine phosphorylation seems to play a role in targeting the receptor for transendocytosis [20]. The mutagenesis studies clearly showed that the presence of GDAY sequence is necessary for internalization of NPRA, but not absolutely sufficient since the mutation of GDAY to AAAA

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K.N. Pandey / Peptides 26 (2005) 985–1000

did not completely block the internalization of the receptor [94]. The most important elements of the GDAY internalization signal are Gly920 and Tyr923 residues, whereas, Asp921 and Ala922 having lesser roles. Similarly, Gly950 and Tyr953 have been shown to be critical residues in Gly950 -Pro951 -Leu952 Tyr953 motif, which has been implicated to play role in the internalization of insulin receptor [107]. It was suggested that a common internalization motif consists a sequence of four amino acids with an aromatic residue, specially a tyrosine in the fourth position of the consensus motif. Previously, it has been indicated that the similarity in three-dimensional placement of the critical amino acids in the 4- and 6-residue signals would allow both types of signals to interact with similar recognition structure in coated pits [28]. It has also been suggested that the tyrosine recognition signals may form a small surface loop structure [62], but this structure differs from that proposed by others [28] in terms of the positioning of tyrosine in the loop. Additional studies provided direct evidence that NPxY sequence in LDL receptor forms a beta-turn structure and showed that peptides containing the NPxY motif assume a reverse-turn structure conformation with the Tyr in the 4th position of the turn [6]. The substitution of Tyr with the residues known to be inactive in endocytosis resulted in a disruption of beta-turn conformation. It has been suggested that the PPGY sequence of the acid phosphatase cytoplasmic tail forms a type 1 beta-turn with the Tyr in position 4 of the turn [38]. Those previous studies indicated that Tyr in the fourth position of internalization signals is critical for receptor endocytosis. Seven transmembrane G-protein-linked receptors have a homologous motif NPxxY, and mutation of this sequence to NPxxA resulted in a complete loss of agonist-induced receptor sequestration [7,47]. The conserved Tyr residue has also been shown to be required for internalization of vasopressin receptor and NPxxY sequence served as sequestration motif for seven transmembrane G-proteinlinked receptors [13]. Nevertheless, there are some exceptions to this motif, as in case of mannose-6-phosphate and insulin-like growth factor receptors: the critical Tyr residue is located at the amino-terminal 1st position of the YRHV consensus internalization signal [62]. The single mutation Asp921 to Ala in GDAY motif did not exhibit a major effect on receptor internalization; however, it significantly attenuated the recycling of internalized receptor to the plasma membrane. On the other hand, mutation of Gly920 and Tyr923 to alanines, inhibited the internalization of NPRA, but these residues did not have any discernible effect on the recycling of this receptor protein [94]. The tyrosine-based motif of GDAY sequence seems to modulate early internalization process of NPRA, whereas Asp921 residue in GDAY mediates recycling or later sorting events of GC-coupled receptors (Fig. 7). Thus, two overlapping motifs within GDAY in carboxyl-terminal domain of NPRA exert dual effects; endocytosis and subsequent trafficking of NPRA. Previous studies have demonstrated that acidic motifs such as DSLL in G-protein-coupled receptors, act as

recycling signals [42,57,115]. Indeed, more studies should be carried out to dissect out the dual role of GDAY motif in the events involved in internalization, recycling, and sequestration of NPRA. Overall, it is speculated that after internalization and sequestration from the plasma membrane, ligand-bound NPRA enters the milieu of cell interior. A variety of structures purported to be involved in the uptake of extracellular substances are collectively termed as the endosomal apparatus. It is proposed that endosomal apparatus may contain three separate compartments; (i) the early stage can be in close association to plasma membrane, (ii) the larger intracellular structures may contain endocytosed ligand and/or receptor, which should be vesicular in nature, and (iii) a structural compartment should contain acid phosphatase activity, similar to lysosomes [9]. Presumably, it is this third compartment that should accumulate the weak base chloroquine and maintains low pH; it is considered that the discharge of ANP most likely should occur in this compartment. At present, no linker or adaptor proteins are known to be identified which could provide associations of NPRA with coated pit proteins essential for endocytosis.

9. 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, ligandbinding, and movement of the receptor protein through the multiple subcellular compartments in the cell. The assessment of the stoichiometric distribution of 125 I-ANP bound to NPRA from plasma membrane to the intracellular compartments and into culture medium has provided the definitive means to directly determine the dynamics of ANP-mediated translocation and redistribution of biologically active NPRA. In this process, ligand-bound NPRA is rapidly internalized, delivered to the endosomes, and a majority of ligand-receptor complexes are degraded in the lysosomal compartments. However, a small population of receptor is dissociated from the ligand in the intracellular compartments, recycles back to the plasma membrane, and intact ligand is released into culture medium. Recent findings have established that the specific intracellular cytoplasmic domains as well as specific sequence motifs promote internalization and trafficking of NPRA. The experimental data support the notion that several components may influence the internalization and intracellular trafficking of NPRA, which may include: (a) Interaction of ligand-receptor complexes of NPRA, which governs the rate at which receptor traverses through the intracellular compartments. (b) The receptor protein itself, which should be adequately processed for appropriate routing in various subcellular compartments. (c) The specific signals, which inherently control the routing and trafficking processes of the ligand-receptor complexes. The specific routing path

K.N. Pandey / Peptides 26 (2005) 985–1000

followed by the NPRA may be cell-specific in nature. These possibilities have ensured that cellular regulation and expression of NPRA activity involves the cellular trafficking and movement of activated receptor into the subcellular compartments. Nevertheless, the cellular pathway and routing of the receptor trafficking, as well as the rate at which it traverses into the cell interior, which influences the sensitivity of the cells to NPs, is not well understood. Similarly, our understanding of the receptor biosynthesis and subcellular assembly responsible for receptor function remains to be determined. More importantly, studies of the biosynthetic assemblies of GC-coupled NP receptors and the regulatory roles of these assemblies in the receptor endocytosis, trafficking, desensitization/inactivation, down-regulation, and metabolic degradation of GC family of receptors need to be accomplished. The molecular basis of these processes, as well as the relationship with the role of NPRA phosphorylation, dephosphorylation, and/or glycosylation requires additional experimentation.

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 [1] Anand-Srivastava MB. Down-regulation of atrial natriuretic peptide ANP-C receptor is associated with alteration in G-protein expression in A10 smooth muscle cells. Biochemistry 2000;39:6503–13. [2] Anand-Srivastava MB, Trachte GJ. Atrial natriuretic factor receptor and signal transduction mechanisms. Pharmacol Rev 1993;45:455–97. [3] Ashworth R, Yu R, Nelson EJ, Dermer S, Gershengorn MC. Visualization of the thyrotropin-releasing hormone receptor and its ligand during endocytosis and recycling. Proc Natl Acad Sci USA 1995;92:512–6. [4] Backer JM, Kahn CR, Cahill DA, Ullrich A, White MF. Receptormediated internalization of insulin requires a 12-amino acid sequence in the juxtamembrane region of the insulin receptor ␤ subunit. J Biol Chem 1990;265:16450–4. [5] Backer JM, Kahn CR, White MF. Tyrosine phosphorylation of the insulin receptor is not required for receptor internalization: studies in 2,4-dinitrophenol-treated cells. Pro Natl Acad Sci USA 1989;86:3201–13. [6] Bansal A, Gierasch LM. The NPXY internalization signal of the LDL receptor adopts a reverse-turn conformation. Cell 1991;67:1195–201. [7] Barak LS, Tiberi M, Freeman NJ, Kwatra MM, Lefkowitz RJ, Caron MG. A highly conserved tyrosine residue in G protein-coupled receptors is required for agonist-mediated beta 2adrenergic receptor sequestration. J Biol Chem 1994;269:2790–5. [8] Barnes EM. Intracellular trafficking of GABAA receptors. Int Rev Neurobiol 1996;39:53–76. [9] Bergeron JJM, Cruz J, Khan MN, Posher BI. Uptake of insulin and other ligands in receptor-rich endocytic components of target cells: the endosomal apparatus. Ann Rev Physiol 1985;47:383–403.

997

[10] Berhanu P. Internalized insulin-receptor complexes are unidirectionally translocated to chloroquine-senstitive degradative sites. J Biol Chem 1988;263:5961–9. [11] Berhanu P, Anderson C, Paynter DR, Wood WM. The amino acid sequence GPLY is not necessary for normal endocytosis of the human insulin receptor B isoform. Biochem Biophys Res Commun 1995;209:730–8. [12] Berhanu P, Iabrahim-Schneck H, Anderson C, Wood WM. The NPEY sequence is not necessary for endocytosis and processing of insulin-receptor complexes. Mol Endocrinol 1991;5:1827–35. [13] Bouley R, Sun TX, Chenard M, McLaughlin M, McKee M, Lin HY, et al. Functional role of the NPxxY motif in internalization of the type 2 vasopressin receptor in LLC-PK1 cells. Am J Physiol 2003;285:C750–62. [14] Brenner BM, Ballerman BJ, Gunning ME, Zeidel ML. Diverse biological actions of atrial natriuretic peptide. Physiol Rev 1990;70:665–99. [15] Brown MS, Anderson RGW, Goldstein JL. Recycling receptors: the round-trip itinerary of migrant membrane proteins. Cell 1983;32:663–7. [16] Brown MS, Goldstein JL. Receptor-mediated endocytosis: insights from the lipoprotein receptor system. Pro Natl Acad Sci USA 1979;76:3330–7. [17] Burwen SJ, Barker ME, Goldman IS, Hradek GT, Raper SE, Jones AL. Transport of epidermal growth factor by rat liver: evidence for a nonlysosomal pathway. J Cell Biol 1984;99:1259–65. [18] Cahill PA, Redmond EM, Keenan AK. Vascular atrial natriuretic factor receptor subtypes are not independently regulated by atrial peptides. J Biol Chem 1990;265:21896–906. [19] Carpenter G, Cohen S. 125 I-labeled human epidermal growth factor: binding, internalization, and degradation in human fibroblasts. J Cell Biol 1976;71:159–71. [20] Casanova JE, Breitfeld PP, Ross SA, Mostov KE. Phosphorylation of the polymeric immunoglobulin receptor required for its efficient transcytosis. Science 1990;248:742–5. [21] Chang T-M, Kullberg DW. Diacytosis of 125 I-asialoorosomucoid by rat hepatocytes: a non-lysosomal pathway insensitive to inhbibition by inhibitors of ligand degradation. Biochim Biophys Acta 1984;805:268–76. [22] Chen WJ, Goldstein JL, Brown MS. NPXY, a sequence often found in cytoplasmic tails, is required for coated pit-mediated internalization of the low density lipoprotein. J Biol Chem 1990;265:3116–23. [23] Chen Z, Dupre DJ, LeGouill C, Rola-Pleszczynski M, Stankova J. 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;277:7356–62. [24] Chinkers M, Garbers DL. The protein kinase of the ANP receptor is required for signaling. Science 1989;245:1392–4. [25] Chinkers M, Garbers DL, Chang MS, Lowe DG, Chin H, Goeddel DV, et al. A membrane form of guanylate cyclase is an atrial natriuretic peptide receptor. Nature 1989;338:78–83. [26] Chinkers M, Singh S, Garbers DL. Adenine nucleotides are required for activation of rat atrial natriuretic peptide receptor/guanylyl cyclase expressed in a baculovirus system. J Biol Chem 1991;266:4088–93. [27] Cohen D, Koh GY, Nikonova LN, Porter JG, Maack T. Molecular determinants of the clearance function of type-C receptor of natriuretic peptides. J Biol Chem 1996;271:9863–9. [28] Collawn JF, Kuhn LA, Liu L-FS, Tainer JA, Trowbridge IS. Transplanted LDL and mannose-6-phosphate receptor internalization signals promote high-efficiency endocytosis of the transferrin receptor. EMBO J 1991;10:3247–53. [29] Collawn JF, Stangel M, Kuhn LA, Esekogwu V, Jing S, Trowbridge IS, et al. Transferrin receptor internalization sequence YXRF implicates a tight turn as the structural recognition motif for endocytosis. Cell 1990;63:1061–72.

998

K.N. Pandey / Peptides 26 (2005) 985–1000

[30] de Bold AJ. Atrial natriuretic factor a hormone produced by the heart. Science 1985;230:767–70. [31] DeLean A, McNicoll N, Labrecque J. Natriuretic peptide receptorA activation stabilizes a membrane-distal dimer interface. J Biol Chem 2003;278:11159–66. [32] Delporte C, Poloczek P, Tastenoy M, Winard J, Christopher J. 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;227:247–56. [33] Drewett JG, Garbers DL. The family of guanylyl cyclase receptors and their ligands. Endocr Rev 1994;15:135–62. [34] Duda T, Goraczniak RM, Sharma RK. Core sequence of ATP regulatory module in receptor guanylate cyclases. FEBS Lett 1993;315:143–8. [35] Duda T, Goraczniak RM, Sharma RK. The glycine residue of ATP regulatory module in receptor guanylate cyclases that is essential in natriuretic factor signaling. FEBS Lett 1993;335:309–14. [36] Duda T, Yadav P, Jankowska A, Venkataraman V, Sharma RK. Three dimensional atomic model and experimental validation for the ATP-regulated module (ARM) of the atrial natriuretic factor receptor guanylate cyclase. Mol Cell Biochem 2001;217:165– 72. [37] Dunn WA, Hubbard AL. Receptor-mediated endocytosis of epidermal growth factor by hepatocytes in the perfused rat liver: ligand and receptor dynamics. J Biol Chem 1984;249:5153–62. [38] Eberle W, Sander C, Klaus W, Schmidt B, van Figura K, Peters C. The essential tyrosine of the internalization signal in lysosomal acid phosphatase is part of a beta-turn. Cell 1991;67:1203–9. [39] Elster L, Hansen GH, Belhage BO, Fritschy JM, Mohler H, Schousboe A. 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;13:417–28. [40] Foster DC, Garbers DL. Dual role for adenine nucleotides in the regulation of the atrial natriuretic peptide receptor guanylyl cyclaseA. J Biol Chem 1998;273:16311–8. [41] Fuller F, Porter JG, Arfsten AE, Miller J, Schilling JW, Scarborough RM, et al. Atrial natriuretic peptide clearance receptor. Complete sequence and functional expression of cDNA clones. J Biol Chem 1988;263:9395–401. [42] Gage RM, Kim KA, Cao TT, von Zastrow M. A transplantable sorting signal that is sufficient to mediate rapid recycling of Gprotein-coupled receptors. J Biol Chem 2001;276:44712–20. [43] Ganzalez-Noriega A, Grubb A, Talkad JT, Sly WS. Chloroquine inhibits lysosomal enzyme pinocytosis and enhances lysosomal enzyme secretion by impairing receptor recycling. J Cell Biol 1980;85:839–52. [44] Garbers DL. Guanylyl cyclase receptors and their endocrine, paracrine and autocrine ligands. Cell 1992;71:1–4. [45] Garbers DL, Lowe DG. Guanylyl cyclase receptors. J Biol Chem 1994;269:30714–44. [46] Garza LA, Birnbaum MJ. Insulin-responsive aminopeptidease trafficking in 3T3-L1 adipocytes. J Biol Chem 2000;275:2560–7. [47] Gobilondo AM, Krasel C, Lohse MJ. Mutations of Tyr326 in the ␤2-adrenoreceptor disrupt multiple receptor functions. Eur J Pharmacol 1996;307:243–50. [48] Goraczniak RM, Duda T, Sharma RK. A structural motif that defines the ATP-regulatory module of guanylate cyclase in atrial natriuretic factor signaling. Bichem J 1992;282:533–7. [49] Haft SK, Quinn AM, Taylor SI. Involvement of dileucine motif in the internalization and degradation of the insulin receptor. J Biol Chem 1994;269:26286–94. [50] Hartford J, Bridges K, Ashwell G, Klausner RD. Intracellular dissociation of receptor-bound asialoglycoproteins in cultured hepatocytes. J Biol Chem 1983;258:3191–7. [51] He X, Chow D, Martick MM, Garcia KC. Allosteric activation of a spring-loaded natriuretic peptide receptor dimer by hormone. Science 2001;293:1657–62.

[52] Hirata Y, Takata S, Tomita M, Takaichi S. Binding, internalization, and degradation of atrial natriuretic peptide in cultured vascular smooth muscle cells of rat. Biochem Biophys Res Commun 1985;132:976–84. [53] Huang Z, Chen Y, Nissenson RA. The cytoplasmic tail of the Gprotein-coupled receptor for parathyroid hormone and parathyroid hormone-related protein contains positive and negative signals for endocytosis. J Biol Chem 1995;270:151–6. [54] Jewett JR, Koller KJ, Goeddel DV, Lowe DG. Hormonal induction of low affinity receptor guanylyl cyclase. Embo J 1993;12:769– 77. [55] Jing S, Spencer T, Miller K, Hopkins C, Trowbridge IS. Role of the human transferrin receptor cytoplasmic domain in endocytosis: localization of a specific signal sequence for internalization. J Cell Biol 1990;110:283–94. [56] Kim HY, Sapp DE, Olsen RW, Tobin AJ. GABA alters GABAA receptor mRNAs and increases ligand-binding. J Neurochem 1993;61:2334–7. [57] Kishi M, Liu X, Hirakawa T, Reczek D, Bretscher A, Ascoli M. Identification of two distinct structural motifs that when added to the C-terminal tail of the rat LH receptor, redirect the internalized hormone-receptor complex from a degradation to a recycling pathway. Mol Endocrin 2001;15:1624–35. [58] Koh GY, Nussenzweig DR, Okolicany J, Price DA, Maack T. Dynamics of atrial natriuretic factor-guanylate cyclase recetpors and receptor-ligand complexes in cultured glomerular mesangial and renomedullary interstitial cells. J Biol Chem 1992;267:11987– 94. [59] Koller KJ, deSauvage FJ, Lowe DG, Goeddel DV. Conservation of the kinase-like regulatory domain is essential for activation of the natriuretic peptide receptor guanylyl cyclase. Mol Cell Biol 1992;12:2581–90. [60] Koller KJ, Lipari MT, Goeddel DV. Proper glycosylation and phosphorylation of the type A natriuretic peptide receptor are required for hormone-stimulated guanylyl cyclase activity. J Biol Chem 1993;268:5997–6003. [61] Koller KJ, Lowe DG, Bennett GL, Minamino N, Kangawa K, Matsuo H, et al. Selective activation of the B natriuretic peptide receptor by C-type natriuretic peptide (CNP). Science 1991;252:120–3. [62] Ktistakis NT, Thomas D, Roth MG. Characteristics of the tyrosine recognition signal for internalization of transmembrane surface glycoproteins. J Cell Biol 1990;111:1393–407. [63] Kurose H, Inagami T, Ui M. Participation of adenosine 5 triphosphate in the activation of membrane-bound guanylate cyclase by the atrial natriuretic factor. FEBS Lett 1987;219:375–9. [64] Labrecque J, McNicoll N, Marquis M, De Lean A. A disulfidebridged mutant of natriuretic peptide receptor-A displays constitutive activity. Role of receptor dimerization in signal transduction. J Biol Chem 1999;274:9752–9. [65] Larose L, McNicoll N, Ong H, De Lean A. Allosteric modulation by ATP of the bovine adrenal natriuretic factor R1 receptor functions. Biochemistry 1991;30:8990–5. [66] Larose L, Rondeau JJ, Ong H, De Lean A. Phosphorylation of atrial natriuretic factor R1 receptor by serine/threonine protein kinases. Evidence for receptor regulation. Mol Cell Biochem 1992;115:203–11. [67] Lazarovits J, Roth M. A single amino acid change in the cytoplasmic domain allows the influenza virus hemagglutinin to be endocytosed through coated pits. Cell 1988;53:743–52. [68] Lefkowitz RJ. G protein-coupled receptors. J Biol Chem 1998;273:18677–80. [69] Levin ER, Gardner DG, Samson WK. Natriuretic peptides. N Engl, J Med 1998;339:321–8. [70] Li Y, Marzolo MP, van Kerkhof P, Strous GJ, Bu G. 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;275:17187–94.

K.N. Pandey / Peptides 26 (2005) 985–1000 [71] Liu Y, Ruoho ER, Rao VD, Hurley JH. Catalytic mechanism of the adenylyl cyclase Modeling and mutational analysis. Proc Natl Acad Sci USA 1997;94:13414–9. [72] Lobel P, Fujimoto K, Ye RD, Griffiths G, Kornfeld S. Mutations in the cytoplasmic domain of the 275 kd mannose 6-phosphate receptor differentially alter lysosomal enzyme sorting and endocytosis. Cell 1989;57:787–96. [73] Lowe DG, Chang M-S, Hellmis R, Chen E, Singh S, Garbers DL, et al. Human atrial natriuretic peptide receptor defines a new paradigm for second messenger signal transduction. EMBO J 1989;8:1377–84. [74] Lucas KA, Pitari GM, Kazerousnian S, Ruiz-Stewart I, Park J, Schulz S, et al. Guanylyl cyclases and signaling by cGMP. Pharmacol Rev 2000;52:375–413. [75] Maack T. The role of atrial natriuretic factor in volume control. Kidney Int 1996;49:1732–7. [76] Marshall S. Dual pathways for the intracellular processing of insulin: relationship between retroendocytosis of intact hormone and the recycling of insulin receptors. J Biol Chem 1985;260:13524–31. [77] Marshall S, Green A, Olefsky JM. Evidence for recycling of insulin receptors in isolated rat adipocytes. J Biol Chem 1981;256:11464–70. [78] Maxifeld FR, Willingram MC, Davis PJ, Pastan I. Amines inhibit the clustering of alpha 2-macroglobulin and EGF on the fibroblast cell surface. Nature 1979;227:661–3. [79] Miettinen HM, Rose JK, Mellman I. Fc receptor isoforms exhibit distinct abilities for coated pit localization as a result of cytoplasmic domain heterogeneity. Cell 1989;58:317–27. [80] Miranda JD, Barnes EM. Repression of ␥-aminobutyric acid type A receptor a 1 polypeptide biosynthesis requires chronic agonist exposure. J Biol Chem 1997;272:16288–94. [81] Misono KS. 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;152:658–67. [82] Murthy KK, Thibault G, Cantin M. Binding and intracellular degradation of atrial natriuretic factor by cultured vascular smooth muscle cells. Mol Cell Endocrinol 1989;67:195–206. [83] Napier M, Arcuri K, Vandlen R. Binding and internalization of atrial natriuretic factor by high-affinity receptors in A10 smooth muscle cells. Arch Biochem Biophys 1986;248:516–22. [84] Nussenzveig DR, Heinflink M, Gershengorn MC. Agoniststimulated internalization of the thyrotropin-releasing hormone receptor is dependent on two domains in the receptor carboxyl terminus. J Bio Chem 1993;268:2389–92. [85] Nussenzveig DR, Lewicki JA, Maack T. Cellular mechanisms of the clearance function of type-C receptors of atrial natriuretic factor. J Biol Chem 1990;265:20952–8. [86] Paccuad JP, Siddle K, Carpentier JL. Internalization of the human insulin receptor: the insulin-independent pathway. J Biol Chem 1992;267:13101–6. [87] Pandey KN. Stimulation of protein phosphorylation by atrial natriuretic factor in plasma membranes of bovine adrenal cortical cells. Biochem Biophys Res Commun 1989;163:988–94. [88] Pandey KN. Kinetic analysis of internalization, recycling and redistribution of atrial natriuretic factor-receptor complex in cultured vascular smooth-muscle cells. Ligand-dependent receptor downregulation. Biochem J 1992;288:55–61. [89] Pandey KN. 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;268:4382–90. [90] Pandey KN. Vascular action natriuretic peptide receptor. In: Sowers JR, editor. Contemporary endocrinology: endocrinology of the vasculature. Totawa, NJ: Humana Press Inc.; 1996. p. 255– 67.

999

[91] Pandey KN. Physiology of the natriuretic peptides gonadal function. In: Samson WK, Levin ER, editors. Contemporary endocrinology: natriuretic peptides in health and disease. Totawa NJ: Humana Press Inc.; 1997. p. 171–91. [92] Pandey KN. Dynamics of internalization and sequestration of guanylyl cyclase/atrial natriuretic peptide receptor-A. Can J Physiol Pharmacol 2001;79:631–9. [93] Pandey KN. Intracellular trafficking and metabolic turnover of ligand-bound guanylyl cyclase/atrial natriuretic peptide receptorA into subcellular compartments. Mole Cell Biochem 2002;230: 61–72. [94] Pandey KN, Arise KK, Renu G, Nguyen HT. Mutations in the cytoplasmic domain of natriuretic peptide receptor-A impair receptor endocytosis and trafficking. FASEB J 2004;18:A1028. [95] Pandey KN, Inagami T, Misono KS. Atrial natriuretic factor receptor on cultured Leydig tumor cells: ligand-binding and photoaffinity labeling. Biochemistry 1986;25:8467–72. [96] Pandey KN, Nguyen HT, Li M, Boyle JW. 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;271:374–9. [97] Pandey KN, Nguyen HT, Sharma GD, Shi SJ, Kriegel AM. Ligandregulated internalization, trafficking, and down-regulation of guanylyl cyclase/atrial natriuretic peptide receptor-A in human embryonic kidney 293 cells. J Biol Chem 2002;277:4618–27. [98] Pandey KN, Pavlou SN, Inagami T. 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;263:13406–13. [99] Pandey KN, Pavlou SN, Kovacs WJ, Inagami T. Atrial natriuretic factor regulates steroidogenic responsiveness and cyclic nucleotide levels in mouse Leydig cells in vitro. Biochem Biophys Res Commun 1986;138:399–404. [100] Pandey KN, Singh S. Molecular cloning and expression of murine guanylate cyclase/atrial natriuretic factor receptor cDNA. J Biol Chem 1990;265:12342–8. [101] Parez HD, Holmes R, Bilander LR, Adams RR, Manzan W, Jolley D, et al. Formyl peptide receptor chimeras define domains involved in ligand-binding. J BiolChem 1993;268:2292–5. [102] Potter LR, Garbers DL. Dephosphorylation of the guanylyl cyclaseA receptor causes desensitization. J Biol Chem 1992;267:14531–4. [103] Potter LR, Garbers DL. Protein kinase C-dependent desensitization of the atrial natriuretic peptide receptor is mediated by dephosphorylation. J Biol Chem 1994;269:14636–42. [104] Potter LR, Hunter T. Identification and characterization of the major phosphorylation sites of the B-type natriuretic peptide receptor. J Biol Chem 1998;273:15533–9. [105] Potter LR, Hunter T. Phosphorylation of the kinase homology domain is essential for activation of the A-type natriuretic peptide receptor. Mol Cell Biol 1998;18:2164–72. [106] Qiu Y, Ogawa H, Miyagi M, Misono KS. Constitutive activation and uncoupling of the atrial natriuretic peptide receptor by mutations at the dimer interface: role of the dimer structure in signaling. J Biol Chem 2004;279:6115–23. [107] Rajagopalan M, Neidigh JL, McClain DA. Amino acid sequence Gly-Pro-Leu-Tyr and Asn-Pro-Glu-Tyr in the submembranous domain of the insulin receptor are required for normal endocytosis. J Biol Chem 1991;266:23068–73. [108] Rathinavelu A, Isom GE. Differential internalization and precessing of atrial natriuretic factor B and C receptors in PC-12 cells. Biochem J 1991;276:493–7. [109] Schulz S, Singh S, Bellet RA, Singh G, Tubb DJ, Chin H, et al. The primary structure of a plasma membrane guanylate cyclase demonstrates diversity within this new receptor family. Cell 1989;58:1155–62.

1000

K.N. Pandey / Peptides 26 (2005) 985–1000

[110] Sharma RK. Evolution of the membrane guanylate cyclase transduction system. Mol Cell Biochem 2002;230:3–30. [111] Sharma RK, Duda T. Plasma membrane guanylate cyclase: a multimodule transduction system. Adv Exp Med Biol 1997;407:271–9. [112] Smith RM, Jarett L. Differences in adenosine triphosphate dependency of receptor-mediated endocytosis of ␣2 macroglobulin and insulin correlate with separate routes of ligand-receptor complex internalization. Endocrinology 1990;126:1551–60. [113] Sorkin A, Krolenkom S, Kudrjavtceva N, Lazebnik J, Teslenko L, Soderquist AM, et al. Recycling of epidermal grwoth factorreceptor complexes in A431cells: identification of dual pathways. J Cell Biol 1991;112:55–63. [114] Sorkin A, Mohammadi M, Huang J, Slessinger J. Internalization of fibroblast growth factor receptor is inhibited by a point mutation at tyrosine 766. J Biol Chem 1994;269:17056–61. [115] Sorkin A, von Zastrow M. Signal transduction and endocytosis close encounters of many kinds. Nature Rev Mol Cell Biol 2002;3:600–14. [116] Sorkin A, Westermark B, Heldin CH, Claesson-Welsh L. Effect of receptor kinase inactivation on the rate of internalization and degradation of PDGF and the PDGF beta-receptor. J Cell Biol 1991;112:469–78. [117] Suga S, Nakao K, Itoh H, Komatsu Y, Ogawa Y, Hama N, et al. Endothelial production of C-type natriuretic peptide and its marked augmentation by transforming growth factor-beta possible existence of vascular natriuretic peptide system. J Clin Invest 1992;90:1145–9. [118] Sunahara RK, Beuve A, Tesmer JJG, Sprang SR, Garbers DL, Gilman AG. Exchange of substrate and inhibitor specificities between adenylyl and guanylyl cyclase. J Biol Chem 1998;273: 16332–8. [119] Thies RS, Webster NJ, McClain DA. A domain of the insulin receptor required for endocytosis in the rat fibroblasts. J Biol Chem 1990;265:10132–7.

[120] Tietze C, Schlesinger P, Stahl P. Mannose-specific endocytosis receptor of alveolar macrophages: demonstration of two functionally distinct intracellular pools of receptor and their roles in receptor recycling. J Cell Biol 1982;92:417–24. [121] Tsao P, Cao T, Von Zastrow M. Role of endocytosis in mediating down-regulation of G-protein-coupled receptor. Trends Pharmacol Sci 2001;22:91–6. [122] Tucker CL, Hurley JH, Miller TR, Hurley JB. Two amino acid substitutions convert a guanylyl cyclase, Ret GC-1 into an adenylyl cyclase. Proc Natl Acad Sci USA 1998;95:5993–7. [123] van den Akker F. Structural insights into the ligand-binding domains of membrane bound guanylyl cyclases and natriuretic peptide receptors. J Mol Biol 2001;311:923–37. [124] van den Akker F, Zang X, Miyagi H, Huo X, Misono KS, Yee VC. Structure of the dimerized hormone-binding domain of a guanylyl cyclase-coupled receptor. Nature 2000;406:101–4. [125] Van Leuvan F, Cassiman JJ, Van Den Berghe H. Primary amines inhibit recycling of ␣2 M receptors in fibroblasts. Cell 1980;20:37–43. [126] Well A, Wellsh JB, Lazer CS, Wiley HS, Rosenfeld MG. Ligandinduced transformation by a noninternalizing epidermal growth factor receptor. Science 1990;247:962–4. [127] Wilson EM, Chinkers M. Identification of sequences mediating guanylyl cyclase dimerization. Biochemistry 1995;34:4696– 701. [128] Wong SK, Ma CP, Foster DC, Chen AY, Garbers DL. The guanylyl cyclase-A receptor transduces an atrial natriuretic peptide/ATP activation signal in the absence of other proteins. J Biol Chem 1995;270:30818–22. [129] Yang RB, Garbers DL. Two eye guanylyl cyclase are expressed in the same photoreceptor cells and form homomers in preference to heteromers. J Biol Chem 1997;272:13738–42.