Ligand-Mediated Endocytosis and Intracellular Sequestration of Guanylyl Cyclase/Natriuretic Peptide Receptors: Role of GDAY Motif

Functional domains of GC-A/NPRA

The primary structure of GC-A/NPRA is consistent with that of the GC receptor family, with at least four distinct regions comprising ligand-binding domain, transmembrane region, the protein kinase-like homology domain (protein KHD) and the GC catalytic domain. The integrity of these regions are conserved among humans, mice, and rats [34-36]. The GC-B/NPRB has an overall domain structure similar to that of GC-A/NPRA and is localized mainly in the brain and endothelial cells, which is thought to mediate the action of CNP in the central nervous system and vasculature [37, 38]. Comparison of the amino-acid sequence indicated a 62% identity between GC-A/NPRA and GC-B/NPRB, with the intracellular regions appearing to be more highly conserved than are the extracellular domains of these two receptors [36]. The extracellular domain of GC-A/NPRA is homologous to the NPRC, which contains a short (35-residue) cytoplasmic tail, that apparently is not coupled to GC activation [39]. GC-A/NPRA mediates most of the known actions of ANP and BNP because major cellular and physiological responsiveness is mimicked by cGMP and its cell permeable analogs [5, 6, 9, 14, 17, 40, 41]. The intracellular signaling mechanism of GC-A/NPRA elicits effects in a cGMP-dependent manner (Fig. 1). The protein KHD of GC-A/NPRA contains an approximately 280-amino acid region that immediately follows the transmembrane spanning domain of the receptor and is more closely related to protein tyrosine kinases than it is to protein serine/threonine kinases [36]. It has been demonstrated that the protein KHD has as an important mediatory role in transducing ligand-induced signals to activate the GC catalytic domain of the receptor [11, 12, 34, 42-45]. The GC catalytic domain of the receptor contains an approximately 250-amino acid region that constitutes the catalytic active site of the receptor [46-48]. The transmembrane GC receptors contain a single cyclase catalytic active site per polypeptide molecule, however, based on structure modeling data, two polypeptide chains seem to be required to activate GC-A/NPRA, and the receptor functions as a homodimer [49-52]. Initially, the dimerization region of the receptor was thought to be located between the protein KHD and GC domain, which has been suggested to form an amphipathic alpha helix structure [53]. It has been reported that the crystal packing of the extracellular ligand-binding of NPRA contains two possible dimer pairs, the head-to-head and tail-to-tail dimer pairs, respectively, associated through the membrane-distal and membrane-proximal subdomains [54, 55]. The tail-to-tail dimer of GC-A/NPRA has been proposed [52]. The crystal structure suggests that NPRC is dimerized in a head-to-head configuration bound with ligand [56, 57]. It has been proposed that a head-to-head dimer may represent the latent inactive state, while the tail-to-tail dimer could represent the hormone-activated state [58]. The ligand-dependent activation of NPRA stabilizes a membrane-distal dimer interface of this receptor protein, suggesting that ANP binding stabilizes the GC-A/NPRA dimer with more stringent spacing at the dimer interface [59]. Nevertheless, the results of site-directed mutagenesis and chemical modification studies suggest that the head-to-head dimmer structure reflects the physiological dimmer structure of GC-A/NPRA [54, 55].

The gene encoding GC-A/NPRA is designated as Npr1, which has been cloned and characterized in humans [35, 60, 61], mice [36, 62], rat [34, 63], bullfrogs [64], euryhaline eels [65], and medaka fish [66]. The primary structure of this gene is essentially similar in all these species, containing 22 exons interrupted by 21 introns [62]. The sequence analysis of the promoter region has identified putative cis-acting binding sites for the known transcription factors, but the functional significance of most of these sites remains to be elucidated. Although transcriptional regulation of the Npr1 gene is poorly understood, the activity and expression of NPRA is regulated by various factors, including natriuretic peptides [67-74], hormones such as endothelin [75], glucocorticoids [76-79], angiotensin II [80-85], growth factors [67, 86, 87], extracellular ion composition [88-93], pregnancy [94-98], physiological and pathophysiological conditions [99-108], and transcription factors [109-111]. A better understanding of the regulation of NPRA expression depends on more extensive functional characterization of its promoter region and elucidation of the functional significance of the potential cis-elements in this region, which are responsible for the binding of known transacting factors.

Receptor binding properties and ligand-mediated internalization of GC-A/NPRA

ANP binding to intact cells containing endogenous NPRA or to purified receptor preparations evaluated by scatchard plot analysis have revealed a single class of binding sites with a kD value of 1×10⁻¹⁰ −1×10⁻⁹M [28, 112-115]. Initial studies on the post-binding events of GC-A/NPRA were hampered by a lack of suitable primary cells predominantly containing this receptor protein. Nevertheless, our studies using a Leydig tumor (MA-10) cell line [28, 29], as well as the work of others with PC-12 cells [116] indicated that ANP/NPRA complexes are internalized and sequestered in cells. On the other hand, Koh et al., [117] indicated that ANP/NPRA complexes are not processed intracellularly in renomedullary interstitial cells. These authors suggested that rapid dissociation of receptor-ligand complexes takes place upon ANP binding to NPRA at 37°C on the plasma membrane. However, in those studies the dissociation of ligand was performed in cell culture medium containing very high concentrations of unlabeled ANP (1μM) to preclude the rebinding of dissociated ligand to receptors, which might have produced artifactual results. In addition, the cell lines used in those studies expressed more than one ANP receptor subtype, including both NPRA and NPRC. The kinetics of internalization and metabolic processing of ANP through NPRC, which does not contain GC catalytic activity, have been extensively studied [8, 113, 118-126]. Early studies of the post-binding events of NPRC were greatly facilitated due to its predominance in vascular smooth muscle cells, which provided a suitable model for study of the metabolic fate of NPRC. Studies using the MA-10 cell line containing exclusively high-density endogenous GC-A/NPRA, as well as COS-7 and human embryonic kidney-293 (HEK-293) cells expressing recombinant GC-A/NPRA, firmly established that ANP/NPRA complexes are rapidly internalized and intracellularly processed in intact cells [5, 6, 29, 67, 127]. Earlier, it has been indicated that in neuroblastoma cells bound-ligand to GC-A/NPRA was degraded and released by a neutral metalloendopeptidase [128], but further studies have not been carried out to confirm this finding.

The stoichiometric kinetic analyses of ligand-binding, internalization, and sequestration of GC-A/NPRA have been studied to distinguish between cell-surface associated, internalized, and degraded ligand-receptor complexes in MA-10 cells containing endogenous receptors and both COS-7 and HEK-293 cells expressing recombinant receptors [29, 31, 32, 115]. Early observations suggested that after binding of ¹²⁵I-ANP to GC-A/NPRA, ligand-receptor complexes were internalized at physiological temperatures and both the degraded and intact ligands were released into culture medium [29, 31]. The distribution of ¹²⁵I-ANP radioactivity on the cell surface, in intracellular compartments, and in culture medium provided a dynamic equilibrium among the rates of ¹²⁵I-ANP uptake, its degradation, and its extrusion in intact cells (Fig. 2). It was observed that a major portion of the internalized ¹²⁵I-ANP was released into culture medium, which consisted of approximately 70%-75% degraded products and about 25%-30% intact ligand [29, 30, 115, 127]. During the initial incubation period, the release of both degraded and intact ligands was blocked by the lysosomotropic agents chloroquine, ammonium chloride, and monensin (Table 1). However, after longer incubation, chloroquine was only partially effective in blocking the release of both degraded and intact ligands [29, 31, 32, 115]. Previous studies have provided the evidence supporting the notion that most of the internalized ¹²⁵I-ANP is processed through the degradative compartments in intact cells [30-32, 115]. Nevertheless, an alternate mechanism also seems to exist for the release of intact ligand to cell exterior. Dual pathways for the intracellular processing of ligand-receptor complexes of insulin have been suggested [129, 130]. Our previous studies have provided definitive evidence for the recycling of internalized GC-ANPRA to the plasma membrane [29-32]. Those studies also showed that majority of the internalized ligand (>70%) is degraded in lysosomes and released into culture medium. Nevertheless, 25%-30% of ligand-receptor complexes escape the lysosomal pathway and are extruded intact to the cell exterior. It is expected that the intact ligand can rebind recycled receptors on the cell surface and reenter the cell via repeated retroendocytosis.

It is envisioned that the homeostatic regulation of GC-A/NPRA and its cellular sensitivity to ANP are dependent on the dynamic equilibrium of endocytosis mechanisms and intracellular processing in a cell-specific manner. The rates of both the internalization and breakdown of ¹²⁵I-ANP in MA-10 cells containing endogenous GC-A/NPRA, as well as in COS-7 and HEK-293 cells containing recombinant receptor, were markedly inhibited in the presence of lysosomtropic inhibitors [29, 31]. Chloroquine and monensin inhibit lysosomal degradation, while dinitrophenol disrupts the energy-dependent intracellular trafficking of various ligand-receptor complexes [29, 31, 32, 130-135]. Ligand-binding studies in intact cells demonstrated that internalized GC-A/NPRA recycles back to the plasma membrane [29, 31, 32]. Receptor binding in trypsin-treated solubilized cell preparations have indicated that most of the receptors were present on the plasma membrane; only 18%–20% were assigned to the preexisting intracellular pool [32, 115]. Since receptors can be inactivated by trypsin on the cell surface, it can be assumed that some receptors might be present on the cytoplasmic side of the plasma membrane, accounting for a significant proportion of the measured intracellular receptor pool. However, this possibility seems to be unlikely because only 20% of the total GC-A/NPRA population of receptors constituted the intracellular pool, which cannot account for the replacement of receptors lost during ANP treatment [32]. Previous studies using photoaffinity labeling of intact cells have also demonstrated that internalized GC-A/NPRA recycles back to the plasma membrane [29, 118]. Trypsin treatment of the photoaffinity-labeled cells produced the 68 kDa tryptic fragment, which increased in a time-dependent manner [17, 29]. The concomitant increase in the 68-kDa tryptic fragments of the receptor suggested that the internalized GC-A/NPRA recycles from intracellular compartments to the plasma membrane. The density of the intact 135-kDa receptor band decreased in the cell interior and the 68-kDa tryptic fragment increased, suggesting that internalized receptor molecules became trypsin-sensitive after returning to the plasma membrane [29]. 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 GC-A/NPRA occurs due to recycling of receptor to the plasma membrane rather than as a result of trypsin entering the cell interior. The results of our earlier studies, as well as recent experimental findings, are consistent with the notion that if recycling is inhibited, internalization of the receptor continues, suggesting that the receptor will not be reinserted into the plasma membrane and that a rapid loss in the number of cell surface receptors will occur [29, 31, 32, 115].

ANP binding experiments have clearly demonstrated that in the presence of chloroquine the amount of ¹²⁵I-ANP bound to GC-A/NPRA was significantly decreased [29, 31]. Those previous studies suggested that both the lysosomotropic agent chloroquine and the metabolic inhibitor dinitrophenol, which depletes cellular ATP, disrupted the internalization and recycling of GC-A/NPRA [29, 31, 32]. Although it has been reported that ATP is not required for the internalization of insulin receptor [136, 137], it is essential for the internalization of epidermal growth factor (EGF) receptor [131]. Studies using COS-7 and HEK-293 cells expressing recombinant GC-A/NPRA and MA-10 cells containing endogenous receptors suggested that the dissociation of bound ANP from the receptor is not a prerequisite for the recycling of GC-A/NPRA [29, 127]. The internalized hormone-receptor complexes of the low-density lipoprotein (LDL) enter the acidic vesicular compartments where the ligand dissociates from the receptor. Subsequently the dissociated LDL receptors recycle back to the plasma membrane and the ligand is degraded in the lysosomal compartments [138]. This indicates that the recycling of GC-A/NPRA probably occurs constitutively as well as via the ligand-dependent regulatory mechanisms. It has been postulated that after endocytosis of GC-A/NPRA, some of the internalized pool of receptors could be diverted to degradation, while the remainder are subject to regulated recycling [29, 31, 133]. Clearly, further studies are needed to clarify the mechanisms that control the endocytosis and recycling of GC-A/NPRA. The possibility that internalized GC-A/NPRA provides signals that contribute to the regulation of receptor gene expression should be considered. The ligand-evoked internalization and partial degradation of GABA receptors both enhance and repress receptor gene expression [139-142]. One Intriguing finding was that agonist-dependent endocytosis of ß2-adrenergic receptors is a necessary step in the activation of mitogenic signals [143]. The early studies support the view that a certain class of receptors may regulate their own biosynthesis through intracellular signals involving ligand-dependent endocytosis. At present, this notion remains speculative; however, it may provide a new direction for investigations of the trafficking and functional regulation of GC-A/NPRA, as well as other members of the GC family of receptors.

Down-regulation, desensitization, and metabolic processing of GC-A/NPRA

Receptors that are metabolically processed and degraded after internalization can have important physiological and pathophysiological implications, including an effect on promoting ligand-receptor internalization that would lead to the degradation of both ligand and receptor molecules in 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, so that the total number of receptors is correspondingly reduced [32, 144]. This in turn, renders the cell refractory to the hormone. In this state of hormone resistance the prevailing ligand concentrations is not sufficient to cause biological responsiveness. After binding of ANP to GC-A/NPRA, ligand-receptor complexes are internalized and sequestered into intracellular compartments, while degraded products are released into culture medium [127, 133]. The pretreatment of HEK-293 cells with unlabeled ANP caused a substantial decrease in the ¹²⁵I-ANP binding capacity of GC-A/NPRA in both time- and dose-dependent manner [32, 133]. Internalization of ANP/NPRA complexes seems to be important in receptor down-regulation [31, 32, 133]. The ligand-dependent down-regulation by receptor degradation is usually homologous, since it involves only the receptor of one particular ligand, even though other receptors may be present on the cell surface. This mechanism differs from that of sequestration since it involves the total loss of cellular receptors rather than receptor redistribution into a subcellular compartment, which may be inaccessible to extracellular ligands. Usually the mechanisms leading to the homologous down-regulation of receptors involve complete removal of ligand-bound receptors from the cell surface by an endocytic processes. There could be several variations on this theme, resulting in receptor internalization, ligand degradation, and recycling of the receptor to the cell surface; alternatively, both the receptor and the ligand can be degraded in lysosomal compartments. If a receptor recycles to the cell surface, the internalization can be compensated for to some extent by reappearance of the receptors in the plasma membrane. Thus, down-regulation can be delayed until receptors begin to be degraded. Essentially, down-regulation may result in a loss of cellular GC-A/NPRA by means of an accelerated rate constant for receptor internalization and, presumably, inactivation.

Desensitization of receptor can be defined as a process whereby the function of a receptor is lost, whether that function is ANP-binding affinity, autoregulatory efficacy of the protein KHD, and/or GC catalytic activity and intracellular accumulation of cGMP. In our metabolic processing studies of GC-A/NPRA, we have used ANP binding as the index of receptor activity [32, 115]. Degradation, on the other hand, has been considered to be the actual loss of the receptor protein from the cell surface by proteolysis into amino acids. Inactivation can precede degradation or both events can occur simultaneously. ANP-induced receptor down-regulation would be an invaluable experimental tool for elucidation of GC-A/NPRA inactivation or desensitization. A change in the receptor phosphorylation state has been implicated in the process of desensitization [16]. Interestingly, desensitization does not require 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 GC-A/NPRA has been suggested by ANP-dependent dephosphorylation of receptor protein [145]. However, the exact mechanism of the dephosphorylation-dependent inactivation of GC-A/NPRA is not well understood. Both early and recent results have indicated that GC-A/NPRA phosphorylation seems to occur in various cell types [42, 146-150].

It is anticipated that the desensitization or inactivation of receptor occurs intracellularly and that inhibition of internalization should prevent this process. The stoichiometric analyses and metabolic processing of ANP/NPRA complexes in the MA-10 cell line and recombinant COS-7 and HEK-293 cells provided evidence that a large population of bound ANP/NPRA complexes enters into lysosomes and the degraded products are released into the culture medium [29, 31, 32, 133]. The lysosomotropic agent chloroquine and NH₄Cl₂ inhibited the degradation of ANP, providing direct evidence that ligand is metabolized in lysosomes. Specific endopeptidases are also known to degrade ANP, but their location in the endosomal compartments has not been established. The endosomal system requires ATP for the maintenance of a pH gradient across the endosomal membrane. Since acidification is believed to augment the release of ligand from receptors within the endosomes, it must be asked whether ANP-endopeptidase is active on ANP; bound to the receptor or ANP, acting as a substrate for endopeptidase; or has to be free in the lumen of the endosome. The localization of ANP-endopeptidase-like activity in the cytosol, plasma membrane, and endosomes would be of great significance. If the physiologically relevant location of the endopeptidase is the endosomal compartment and the enzymes in all three compartments are identical, a question can be raised about the mechanism whereby the cytosolic enzyme is inserted into the endosome. Furthermore, if this insertion process is a locus for cellular regulation of the degradation of ANP, that remains to be established. Inactivation of GC-A/NPRA might occur intracellularly, but inhibition of internalization should prevent this process. However, the molecular and biochemical aspects of the process of inactivation of GC-A/NPRA, as well as the subcellular localization of the events, have yet to be determined.

We envision that receptor-mediated endocytosis of ANP/NPRA 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 [5, 30-32]. Some 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 coated pits on the plasma membrane. This proposed itinerary would be consistent with the notion that ANP/NPRA complexes are be delivered to lysosomal compartments. Acidification of lysosomes induces the dissociation of ligand from the receptor, after which a population of receptor molecules may recycle back to the plasma membrane, initiating the following events: i) as lysosomotropic agent such as chloroquine is unable completely to block the degradation of ANP/NPRA in lysosomes; ii) the release of intact ANP occurs through a lysosome-independent pathway; and iii) recycling of endocytosed GC-A/NPRA back to the plasma membrane occurs simultaneously with this process, leading to degradation of the majority of ligand-receptor complexes into the lysosomal compartments. Some findings suggest that after the internalization of ANP-receptor complexes, ligand bifurcates into a degradative pathway, through which the 70%-80% of internalized ANP of all incoming ligand is processed in lysosomes, and a retroendocytotic pathway that accelerates the release of intact ligand [29, 32]. An investigative approach based on these hypotheses should provide a direct assessment of ligand-bound receptor trafficking and sequestration into intracellular compartments.

It is conceivable that some remarkable differences do exist with regard to 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 [151, 152], insulin receptors in adipocytes [129, 153, 154], transfected Chinese hamster ovary cells [155], and thyrotropin hormone receptors in GH3 cells [156]. However, the degradation of asialoglycoprotein and its receptor complexes is not observed until about 30 min after endocytosis in hepatoma cells [157]. Similarly, the degradation of EGF-receptor complexes is not detectable for at least 20 min in hepatocytes [158]. Although there is no apparent mechanistic evidence to account 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 ligand-receptor complexes, perhaps through the use of different intermediate vesicles for the transfer of ligand to the site of degradation. If this is the case, then it should be possible to determine the intracellular sequestration route by either intrinsic properties of ligand-receptor complexes or the pathways by which various cells process the incoming ligands. It is also possible that there is 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 for insulin and epidermal growth factor (EGF) receptors [130, 159-162]. Similarly, the finding that chloroquine effectively interrupts the degradative processing of GC-A/NPRA without exerting a deleterious effect on the retroendocytotic pathway is novel and intriguing [32, 133], giving rise to the notion that after internalization, many types of ligand-receptor complexes can recycle through the chloroquine-insensitive pathway and finally be degraded via chloroquine-sensitive lysosomal compartments. Several types of ligand-receptor complexes, including EGF, insulin, and asialoglycoprotein receptors, recycle through the chloroquine-insensitive pathway, [129, 160, 163-165].

Short sequence motifs and internalization of GC-A/NPRA

Using deletion mutagenesis, the specific regions in the intracellular domain of GC-A/NPRA have been determined for internalization, sequestration, and recycling [31, 133]. The loss of function of deletion and/or site-directed mutations have been utilized to identify the regions within the protein KHD and GC catalytic domains of this receptor protein (Fig. 3). The results of immunoprecipitation studies indicated that wild-type and carboxyl-terminal-deleted receptors are expressed on the cell surface without a significant decrease in the protein contents of mutant receptors (Fig. 4). It has been demonstrated that the truncation of GC-A/NPRA at the carboxyl-terminal end significantly reduced the hydrolysis and metabolic degradation of ligand-receptor complexes as compared with the wild-type receptor [31]. The complete deletion of both the protein KHD and GC catalytic domains abolished the internalization of GC-A/NPRA. The deletion of 170 amino acids at the carboxyl-terminal end of the receptor ( 937-NPRA, 916-NPRA, 887-NPRA) reduced the internalization of ligand-receptor complexes by 60% [31]. Our findings suggested that specific regions within the intracellular domains of GC-A/NPRA determine the extent of ligand binding efficiency, endocytosis, and intracellular sequestration of ligand-receptor complexes in transfected COS-7 cells [31]. Those previous studies indicated that with increasing deletions of amino acid residues at the carboxyl-terminal end of the receptor, a large proportion of ligand-receptor complexes did not internalize and remained on the cell surface (Fig. 5). Analysis of the intact and degraded ligands demonstrated that the release of ¹²⁵I-ANP consisted of a higher amount of degraded products and a lesser amount of intact ligand in the culture medium of cells expressing either wild-type or carboxyl-terminal truncated mutant GC-A/NPRA [31]. Interestingly, most of the internalization signals have been reported to reside in the cytoplasmic domains of endocytosed receptors [30, 166-169]. Evidence suggests that a majority of the receptors that undergo endocytosis contain internalization signals such as Asn-Pro-x-Tyr (NPXY) sequence motif in the cytoplasmic portion, near the transmembrane domain of the receptor. However, NPRA including other members of the GC-A receptor family do not seem to contain an intracellular NPXY sequence motif near the transmembrane domain. Nevertheless, examples exist where this motif is not essential for endocytosis and processing of ligand-receptor complexes; insulin-receptor complexes in CHO cells are one such case [170]. Therefore, the internalization of ligand-receptor complexes may not be solely dependent on the NPXY motif located near the transmembrane domain of the receptor protein. Although it has been suggested that the protein KHD of GC-A/NPRA is critical to the functional ability of the receptor, the exact mechanisms whereby it controls GC catalytic activity and other functions of GC-A/NPRA are not well understood.

A variety of structures, known collectively as the endosomal apparatus, are considered to be involved in the uptake of extracellular substances. The endosomal apparatus is nonlysosomal and is characterized by subcellular fractionation, morphological markers, and enzyme activities. It has been suggested that the endosomal apparatus may contain three separate compartments: the early stage is close to plasma membrane; the larger intracellular structures contain endocytosed ligand and/or receptor, which is vesicular and tubular; and a structural compartment contains acid phosphatase activity, much as do the lysosomes [171]. Most likely, it is this third compartment that accumulates the weak base chloroquine, maintains low pH, and contains an ATP-dependent pump. Presumably, the discharge of ANP from the bound ligand-receptor complexes occurs in this compartment.

Role of GDXY motif in the internalization and trafficking of GC-A/NPRA

The sequence motif GDAY in the carboxyl terminal-domain of GC-A/NPRA serves as an internalization signal for endocytosis and trafficking of this receptor protein [115]. The residues Gly⁹²⁰ and Tyr⁹²³ constitute the important elements in the GDAY internalization signal sequence motif (Fig. 6). However, it is thought that the residue Asp⁹²¹ provides an acidic environment for efficient signaling of the GDAY motif during GC-A/NPRA internalization. The mutation of Asp⁹²¹ to alanine did not have a major effect on internalization, but significantly attenuated the recycling of internalized receptors back to the plasma membrane [30, 115]. On the other hand, mutation of Gly⁹²⁰ and Tyr⁹²³ residues to alanines inhibited the internalization of NPRA, although these residues had no discernible effect on the receptor recycling. These findings suggested that the tyrosine-based GDAY motif modulates the early internalization of GC-A/NPRA, whereas Asp residue in the GDAY sequence seems to mediate recycling or later sorting of the receptor. Two overlapping motifs within the GDAY sequence may exert different but specific effects on endocytosis and subsequent trafficking of GC-A/NPRA. More studies are needed to define the dual role of GDAY motif in receptor internalization and recycling [6, 115].

It is conceivable that the GDAY motif has a dual function in receptor internalization and in subsequent recycling of internalized receptor back to the plasma membrane. The mutations of Gly⁹²⁰ and Tyr⁹²³ to alanine in NPRA attenuated the internalization of mutant receptors by almost 50% as compared with wild-type receptor [115]. However, the mutation of Asp⁹²¹ to alanine had only a minimal effect. It is possible that the regulation of receptor internalization relies largely on residues Gly⁹²⁰ and Tyr⁹²³ in the carboxyl-terminal domain of the receptor protein. On the other hand, mutation of Asp⁹²¹ to alanine significantly attenuated the recycling of internalized receptor back to the plasma membrane. Thus, the functional role of the cytoplasmic tail of GC-A/NPRA is important, being comparable to the thyrotropin-stimulating hormone receptor in that its disruption attenuates receptor internalization [172]. The point and deletion mutations within the carboxyl-terminal region of GC-A/NPRA also seem to have a major effect on internalization [31]. Replacement of the selected Gly⁹²⁰, Asp⁹²¹, and Try⁹²³ residues with alanine in the GDAY showed that the internalization of mutant receptor was significantly reduced compared with wild-type receptor [115]. The GDAY/AAAA mutant receptor displayed a dose-response curve similar to that of wild-type receptor. The recombinant HEK-293 cells harboring both wild-type and GDAY/AAAA mutant receptors, respectively, expressed 1.87×10⁶ and1.89×10⁶ receptor sites/cell and the binding affinity was comparable in both cell lines, with Kd = 2.4 × 10⁻⁸ M [115].

The GC-A/NPRA is internalized into subcellular compartments in the ligand-dependent manner [28, 29, 116, 118, 127, 133]. The ligand-dependent endocytosis and sequestration of GC-A/NPRA involves a series of sequential sorting steps through which ligand-receptor complexes can eventually be degraded, receptor recycled back to the plasma membrane, and intact ligand released into the cell exterior [29, 31, 127, 133]. The recycling of endocytosed receptor to the plasma membrane and the release of intact ANP into the cell exterior occur simultaneously with processes leading to degradation of the majority of ligand-receptor complexes into lysosomes, which appears to be controlled by receptor sequences in the carboxyl-terminal domain of GC-A/NPRA [31, 115]. Furthermore, a return in ¹²⁵I-ANP binding in trypsin-treated cells indicated that ¹²⁵I-ANP binding in trypsinized cells was due to recycling of the receptor protein [115, 127]. As shown in Fig. 7, after trypsin treatment, the greater return of ¹²⁵I-ANP binding in HEK-293 cells expressing wild-type receptors was due to a large population of internalized receptors and subsequent recycling to the plasma membrane compared with cells expressing GDAY mutant receptors. These findings provided direct evidence that treatment of cells with unlabeled ANP accelerates the disappearance of surface receptors, indicating that ANP-dependent down-regulation of GC-A/NPRA involves the internalization of the receptor [115]. The internalization of platelet-activating factor is also regulated by DPXXY motif [173] and that of type-2 vasopressin receptor by the NPXXY sequence [174]. It has been suggested that, similarly, the YXXL motif functions in endocytosis of LDL receptor-related protein [175]. A common feature of these internalization signal motifs, including NPXY and GDAY, is the presence of a tyrosine residue at the end of the tetrapeptide sequence motif [115, 173]. Moreover, tyrosine residues in the mannose-6-phosphate receptor and the influenza virus hemagglutinin are also involved in endocytosis and trafficking, even though they are not present in the context of NPXY or YXRF consensus sequence motifs. Therefore, if a universal internalization signal exists, it may not be based on a universal amino acid sequence [173, 176-179].

The critical features of the internalization sequences might be their specification of a particular conformation, such as a tight beta-turn in protein structure [180]. A compilation of some of the membrane receptors with known signal sequence has been provided in Table 2. The Gly⁹²⁰ residue constitutes the important element in conjunction with the Tyr⁹²³ residue of the GDAY internalization signal motif [30, 115]. Similarly, the Gly⁹⁵⁰-Pro⁹⁵¹-Leu⁹⁵²-Tyr⁹⁵³ motif has been implicated in internalization of the insulin receptor, in which Gly⁹⁵⁰ and Tyr⁹⁵³ residues have been shown to be the critical components [181]. A common internalization motif consists of four amino acids with an aromatic residue, specifically a tyrosine in the fourth position. The tyrosine recognition signals form a small surface loop [182], but one differing in terms of the positioning of tyrosine in the loop structure [179, 183]. Earlier studies have provided direct evidence that the NPXY sequence in the LDL receptor forms a beta-turn structure, but that peptides containing the NPXY motif assume a reverse-turn conformation with Tyr in the fourth position of the turn [184]. Using residues known to be inactive in receptor endocytosis to substitute for Tyr resulted in disruption of the beta-turn conformation. A similar approach was used to obtain evidence that the PPGY sequence of the acid phosphatase in the cytoplasmic tail forms a type 1 beta turn with Tyr in the fourth position [185]. Thus, the presence of Tyr in the fourth position of internalization signals is critical for receptor endocytosis. Similarly, seven transmembrane G-protein-linked receptors have a homologous motif, NPXXY. Mutation of this sequence to NPXXA resulted in a complete loss of agonist-induced receptor sequestration [186, 187]. The conserved Tyr was required for internalization of vasopressin receptor [174]. However, there are exceptions to this rule. For example, in YXXF (Tyr-X-Arg-Phe) motif, the critical Tyr residue is included at the amino-terminal first position and provides general consensus of YXXphi motifs for internalization of insulin-like growth factor receptors, mannose-6-phosphate receptors, and transferrin receptors [179, 180, 182].

Internalization of GC-B/NPRB

Interestingly, Brackmann et al. 2005 [188] have shown that guanylyl cyclase-B/natriuretic peptide receptor-B (GC-B/NPRB) is internalized and recycled in hippocampus neurons and C6 glioma cells cultures.. These authors suggested that the trafficking of GC-B/NPRB occurs ligand-dependently in response to CNP binding and stimulation of the receptor protein. The internalization and trafficking of GC-B/NPRB has been suggested to involve a clathrin-dependent mechanism. Our recent work indicates that the internalization of GC-A/NPRA also involves clathrin-dependent pathways [189]. Receptor internalization is severely diminished by inhibitors of clathrin proteins such as chlorpromazine and monodensyl cadaverine. However, interaction of the GDAY motif in GC-A/NPRA and GC-B/NPRB with clathrin adaptor proteins remains to be established.