Báo cáo khoa học: Cellular refractoriness to the heat-stable enterotoxin peptide is associated with alterations in levels of the differentially glycosylated forms of guanylyl cyclase C pdf

Cellular refractoriness to the heat-stable enterotoxin peptide is associated with alterations in levels of the differentially glycosylated forms of guanylyl cyclase C Yashoda Ghanekar, Akhila Chandrashaker and Sandhya S. Visweswariah Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore, India The heat-stable enterotoxin peptides (ST) produced by enterotoxigenic Escherichia coli are one of the major causes of transitory diarrhea in the developing world. Toxin bind- ing to its receptor, guanylyl cyclase C (GC-C), results in receptor activation and the production of high intracellular levels of cGMP. GC-C is expressed in two differentially glycosylated forms in intestinal epithelial cells. Prolonged exposure of human colonic cell lines to ST peptides induces cellular refractoriness to the ST peptide, in terms of intra- cellular cGMP accumulation. We have investigated the mechanism of cellular desensitization in human colonic Caco2 cells, and observe that exposure of cells to ST leads to a time and dose-dependent inability of cells to respond to the peptide in terms of GC-C stimulation, both in whole cells and membranes prepared from desensitized cells. This is concomitant with a 50% reduction in ST-binding activity in desensitized cells. Desensitization was correlated with a loss of the plasma membrane-associated, hyperglycosylated 145 kDa form of GC-C, while the predominant 130 kDa form, localized both on the plasma membrane and the endoplasmic reticulum, continued to be present in ST-trea- ted cells. Desensitized cells recovered ST-responsiveness on removal of the ST peptide, which was correlated with a reappearance of the 145 kDa form on the cell surface, fol- lowing processing of the endoplasmic reticulum-associated pool of the 130 kDa form. Selective internalization of the 145 kDa form of the receptor was required for cellular desensitization, as ST-treatment of cells at 4 °C did not lead to refractoriness. We therefore show a novel means of regulation of cellular responsiveness to the ST peptide, whereby altering cellular levels of the differentially glycos- ylated forms of GC-C can lead to differential ligand-medi- ated activation of the receptor. Keywords: guanylyl cyclase C; desensitization; Caco2 cells; heat-stable enterotoxin; glycosylation. The regulation of any receptor-mediated signaling pathway is integral for maintaining normal homeostasis of a cell. Initial activation of a receptor by its ligand results in the triggering of a cellular response, which is then usually attenuated by various cellular mechanisms. These can involve receptor internalization and/or modification, ligand degradation, or the activation of cellular pathways that counteract the initial response, and can lead to cellular refractoriness. Membrane-associated forms of guanylyl cyclase serve as receptors for a variety of peptide ligands that mediate their response by increases in intracellular cGMP levels [1]. Guanylyl cyclase A (GC-A), the receptor for atrial natriuretic peptides (ANP), is basally phos- phorylated in cells and a rapid dephosphorylation of the receptor is correlated with a desensitization of the receptor to its ligand, and contributes to the cellular refractoriness that is observed in cells that are exposed to ANP [2]. In addition, ligand-mediated internalization of GC-A has been described, but only a fraction of the ligand- bound receptor is directed for degradation, with a signifi- cant amount is recycled to the plasma membrane surface [3]. We have been studying guanylyl cyclase C (GC-C), the receptor for the guanylin/uroguanylin family of peptides. GC-C is predominantly expressed in intestinal cells, where it was initially described as being the mediator of the action of the bacterial heat-stable enterotoxin peptides (ST) [4–6]. In addition, robust GC-C expression is also observed in the regenerating rat liver [7] and in extraintestinal tissues [8]. Ligand binding to GC-C leads to accumulation of intracel- lular cGMP, followed by the activation of cyclic nucleotide- dependent protein kinases resulting in the phosphorylation of the cystic fibrosis transmembrane conductance regulator [9,10]. Cystic fibrosis transmembrane conductance regulator is a chloride channel and phosphorylation increases chloride ion efflux resulting in loss of fluid from the cell and characteristic watery diarrhea that is associated with the ST peptides. Recently, GC-C has also been shown to be involved in regulation of colonic cell proliferation [11] and apoptosis [12], and modulation of a cGMP-gated ion channel that then regulates DNA synthesis [13]. Correspondence to S. S. Visweswariah, Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore 560012, India. Fax: + 91 80 3600999, Tel.: + 91 80 3942542, E-mail: sandhya@mrdg.iisc.ernet.in Abbreviations: ANP, atrial natriuretic peptide; GC-A, guanylyl cyclase A; GC-C, guanylyl cyclase C; IBMX, isobutylmethyl xanthine; PDE5, cGMP-binding, cGMP-specific phosphodiesterase; PDZ, PSD-95, Disc-large, ZO-1; ST, stable toxin; STh, stable toxin of the human variety; ST Y72F , STh with tyrosine-19 replaced by phenylalanine. Enzyme: guanylyl cyclase (4.6.1.290) (Received 8 May 2003, revised 17 July 2003, accepted 4 August 2003) Eur. J. Biochem. 270, 3848–3857 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03779.x Several cell lines that express GC-C are available which can be potentially used as a model to study GC-C signaling [14]. These cell lines include T84, Caco2, HT29, NCI H508 and SW 116, all derived from different types of colonic carcinomas. T84 cells are derived from lung metastasis of a patient with colonic carcinoma and exhibit many charac- teristics of polarized epithelial cells [15]. Caco2 cells, on the other hand exhibit an enterocyte-like morphology, although they were derived from a colonic adenocarcinoma [16]. Upon reaching confluency, Caco2 cells spontaneously differentiate in culture and resemble villus cells and thus provide a good in vitro system to study regulation of cellular pathways in differentiating enterocytes [17]. GC-C has been cloned [18] and characterized from Caco2 cells [19], and Caco2 cells express lower levels of GC-C in comparison to T84 cells [14]. GC-C expression levels increase after the cells differentiate in culture [19], and unlike T84 cells, differentiated Caco2 cells also express guanylin [20,21]. Recent studies indicate that Caco2 cells express soluble guanylyl cyclase as well as protein kinase G and activation of the soluble guanylyl cyclase leads to inhibition of Na + /H + exchanger NHE3 [22] and apical Cl – /OH – exchange activity by activation of protein kinase G [23]. The transient nature of ST-induced diarrhea suggests that the GC-C signaling pathway is modulated in vivo in response to ligand. In T84 cells, 18 h ST treatment led to cellular refractoriness to further ST stimulation and this refractoriness was contributed by both down-regulation of GC-C leading to reduced cGMP synthesis, as well as activation of the type 5 phosphodiesterase (PDE5A) and increased cGMP degradation [24]. On desensitization, there was a decrease in the V max of the guanylyl cyclase catalytic activity of GC-C with no change in the S 0.5 of the enzyme for its substrate, MgGTP [25]. There did not appear to be an appreciable change in the total receptor content in desen- sitized cells, as measured by Scatchard analysis, which could account for the reduction in catalytic activity. In the current study, we have explored the phenomenon of the induction of cellular refractoriness to the ST peptide in Caco2 cells postdifferentiation, when GC-C levels are relatively high and expressed at a uniform level over the time period of the experiments as conducted here. As shown below, alterations in the levels of the differentially glycosylated forms of GC-C regulate the cellular response to the ST-peptide, providing a novel means of inducing desensitization, as well as suggesting that the glycosylation state of GC-C determines its ability to be activated by its ligands. Materials and methods Tissue culture media and all fine chemicals were from Sigma-Aldrich, USA. Protein A agarose and ECL Plus TM Western blotting detection reagent were obtained from Amersham Biosciences, UK. 125 Iodine and Western blot chemiluminescence reagent were from NEN Life Science Products, USA. Stable toxin of the human variety (STh) and a mutant form of the STh peptide, ST Y72F ,were purified as described earlier [26]. Caco2 cells were obtained from M. C. Rao, Department of Physiology and Biophys- ics, University of Illinois at Chicago. Culture and maintenance of cells Caco2 cells were cultured in Dulbeccos’s modified Eagle’s medium (DMEM) and Ham’s nutrient mixture F12 in the ratio 1 : 1 (DMEM/F12) containing 10% fetal bovine serum, nonessential amino acids, 120 mgÆL )1 penicillin and 270 mgÆL )1 streptomycin. To allow differentiation of Caco2 cells into intestinal villus cells, cells were kept in culture for 7–10 days after they were confluent [19]. To confirm differentiation, sucrase isomaltase gene expression was monitored by reverse transcriptase and poymerase chain reaction, using RNA prepared from 15-day-old Caco2 cells [27] (data not shown). Desensitization of Caco2 cells In standard desensitization experiments, 14 to 20-day-old Caco2 cells were washed with serum-free DMEM/F12 and incubated with or without 10 )7 M SThinDMEM/F12for 9 h. Monolayers were then washed and re-stimulated with fresh STh (10 )7 M ) in the presence or absence of 500 l M isobutylmethyl xanthine (IBMX) for 30 min in serum-free medium for 15 min. Cell monolayers were washed, and cells lysedin0.1 M citric acid or 0.1 M HCl. Cyclic GMP in the lysates was measured by radioimmunoassay as described previously [28]. To monitor the recovery of cellular responsiveness following desensitization induced by ST, desensitized cells were washed with serum-free DMEM/F12 and then incu- bated with DMEM/F12, 10% fetal bovine serum and nonessential amino acids for 12 h in the absence of ST peptide, and in the presence of cycloheximide (100 lgÆmL )1 ) or swainsonine (10 lgÆmL )1 ). Cells were either re-stimulated with ST peptide, or membranes prepared for in vitro guanylyl cyclase assays as described below. In some cases, membranes were prepared from cells and taken for immuno- precipitation and Western blot analysis as described above. Preparation of cell membranes Membranes were prepared essentially as described previ- ously [25]. Briefly, Caco2 cells were washed with chilled phosphate buffered saline (NaCl/P i ,10m M sodium phos- phate buffer, pH 7.2, 0.9% sodium chloride) and scraped into homogenization buffer (50 m M Hepes, pH 7.5, 100 m M NaCl, 5 m M EDTA, 1 m M dithiothreitol, 5 lgÆmL )1 soybean trypsin inhibitor, 5 lgÆmL )1 leupeptin, and 5 lgÆmL )1 aprotinin). The cell lysate was sonicated and centrifuged at 10 000 g for 1 h at 4 °C. The pellet obtained was resuspended in buffer containing 50 m M Hepes, pH 7.5, 5 lgÆmL )1 soybean trypsin inhibitor, 5 lgÆmL )1 leupeptin, 5 lgÆmL )1 aprotinin and 100 l M sodium ortho- vanadate. The protein was estimated by using a modifica- tion of the Bradford protein assay [29]. In vitro guanylyl cyclase assays Assays were carried out as described earlier [25]. Membrane protein (20 lg) was incubated in presence or absence of 10 )7 M ST in assay buffer consisting of 60 m M Tris-Cl, pH 7.6, 500 l M IBMX, and a GTP regeneration system Ó FEBS 2003 Glycosylation of GC-C and desensitization (Eur. J. Biochem. 270) 3849 consisting of 10 lg creatine kinase and 7.5 m M creatine phosphate. The assay was initiated by adding 4 m M MgCl 2 and 1 m M GTP as substrate and incubated at 37 °C for 5–10 min. The reaction was terminated by addition of 400 lLof 50 m M sodium acetate buffer, pH 4.6. The samples were boiled in a water bath, centrifuged at 10 000 g for 5 min and cGMP in the supernatant was assayed by radioimmuno- assay, as described earlier [28]. To measure the manganese- mediated activation of GC-C, 4 m M manganese and 1 m M GTP was used as substrate. In experiments performed to monitor Lubrol-PX mediated activation, membranes were incubated in 0.3% Lubrol-PX for 10 min at 37 °Cwith 4m M MgCl 2 and 1 m M GTP as substrate. Receptor binding analysis ST Y72F was iodinated using Na 125 I as described earlier [30] and was available in the laboratory. Membrane protein (100–200 lg) was incubated with increasing concentrations of 125 I-labeled ST Y72F for 1 h at 37 °C in binding buffer (50 m M Hepes, pH 7.5, 4 m M MgCl 2 , 0.1% bovine serum albumin, 10 lgÆmL )1 leupeptin, 10 lgÆmL )1 aprotinin). Following incubation, the reactions were filtered through GF-C filters, filters dried and associated radioactivity monitored in an LKB gamma counter. The data was analyzed using GRAPHPAD PRISM (San Diego, CA, USA). Immunodetection of GC-C Western blot analysis was performed with 50 lgofmem- brane protein and monoclonal antibody GCC:C8 (500 ngÆmL )1 ) raised to the protein kinase like domain of GC-C, as described earlier [25]. Bound antibody was detected by enhanced chemiluminescence according to the manufacturer’s instructions. Immunofluorescence of Caco2 cells Immunocytochemistry was carried out as described earlier [31]. Cells were plated on coverslips, washed with NaCl/P i and fixed in NaCl/P i containing 4% paraformaldehyde for 20–30 min. Cells were washed and incubated with 2% bovine serum albumin and 0.1% Triton X-100 in NaCl/P i for 1 h at room temperature to block nonspecific sites and permeabilize cells. Cells were then incubated overnight with 5 lgÆmL )1 GC-C:4D7, an antibody raised to the protein kinase-like domain of GC-C [32] or with GC-C:4D7 antibody preadsorbed with a fusion protein of the kinase- like domain of GC-C and glutathione S-transferase, in blocking buffer [8]. After washing, FITC-conjugated anti- mouse antibody (Life Technologies, USA) was added for 1 h at room temperature. Cells were washed and mounted in Vectashield mounting medium (Vector Laboratories, USA). Cells were visualized under a Zeiss fluorescence microscope using standard filters for FITC and DAPI at 63 · magnification. Immunoprecipitation of GC-C Membranes prepared from Caco2 cells were solubilized at a concentration of 1 mgÆmL )1 in immunoprecipitation buffer (20 m M Tris-Cl pH 7.5, 100 m M NaCl, 2 m M EDTA, 1% Triton X-100, 5 lgÆmL )1 soybean trypsin inhibitor, 5 lgÆmL )1 aprotinin, 5 lgÆmL )1 leupeptin, and 100 l M sodium orthovanadate) for 1 h at 4 °C. The soluble fraction was incubated overnight with a polyclonal antibody raised to the C-terminal domain of GC-C (CTD antibody) [32] at a concentration of 20 lgÆmL )1 . Ten microliters of protein A agarose was added to collect the immunocomplex. The immunoprecipitate was washed thrice with immunoprecip- itation buffer, boiled in sample buffer and subjected to polyacrylamide gel electrophoresis and Western blot ana- lysis, as described earlier. For complete deglycosylation of GC-C with PNGaseF, approximately 300 lg of membrane protein was solubilized and immunoprecipitated as described above. The immuno- precipitate was washed twice with 50 m M sodium phosphate buffer, pH 7.2 and then boiled in 50 m M sodium phosphate buffer with 0.1% SDS and 50 m M 2-mercaptoethanol for 5 min. The reaction was cooled to room temperature and NP-40 was added to a final concentration of 0.75%. N-Glycosidase F (200 mU; Roche, Germany) was added and the reaction incubated at 37 °C for 8 h. After incuba- tion, the reaction was stopped by addition of Laemmli buffer, boiled and subjected to SDS gel electrophoresis and Western blot analysis. For Endo H treatment of cells, GC-C was immunopre- cipitated as described above and the immunoprecipitate treated with Endo H (500 U; NEB, USA) as per the manufacturer’s instructions. Incubation at 37 °Cwasper- formed for 6 h and samples were then subjected to SDS gel electrophoresis and Western blot analysis asdescribedabove. Surface biotinylation of Caco2 cells Cells were washed with NaCl/P i (pH 8.0) containing 1 m M CaCl 2 and 0.5 m M MgCl 2 (NaCl/P i -CM), and then incu- bated in NaCl/P i -CM containing 500 lgÆmL )1 sulfo-NHS- biotin (Sigma) for 30 min at room temperature. Excess biotin was quenched by incubation with 50 m M Tris-Cl, pH 7.5, for 5 min. Cells were briefly washed with NaCl/P i and then scraped in homogenization buffer and membranes were prepared. Membrane protein was solubilized and then immunoprecipitated as described above. Beads were washed three times with immunoprecipitation buffer containing Triton X-100 and twice with immunoprecipitation buffer without Triton X-100 and proteins were resolved on 7.5% SDS, transferred onto poly(vinylidene difluoride) mem- brane and subjected to Western blot analysis with strept- avidin–peroxidase. Results Homologous desensitization of GC-C in Caco2 cells We studied this phenomenon of GC-C desensitization in Caco2 cells by treating cells with ST for 18 h, following which the cells were washed and restimulated with 10 )7 M ST. Significant cGMP production was elicited by ST application to control cells as a consequence of GC-C activation. In cells that were preincubated with ST for 18 h, only a slight increase in cGMP synthesis was observed after fresh ST stimulation (Fig. 1A), indicating that similar to T84 cells, Caco2 cells also showed cellular refractoriness to 3850 Y. Ghanekar et al. (Eur. J. Biochem. 270) Ó FEBS 2003 ST. Interestingly, even when we inhibited PDE activity in cells by the addition of a phosphodiesterase inhibitor to desensitized cells, increased cGMP accumulation was not observed, in contrast to our results with T84 cells (Fig. 1A). PDE5 is expressed in Caco2 cells, and as we have reported earlier in T84 cells [24], PDE5 activation was observed as a consequence of increased cGMP accumulation in Caco2 cells during the initial ST application (unpublished obser- vations). This suggested that the refractoriness to the ST peptide observed in Caco2 cells must be attributed to down- regulation of GC-C activity on ligand addition. ST was applied to cells for varying times and we measured the ability of these cells to respond to ST on fresh stimulation. As seen in Fig. 1B, desensitization was observed after 3 h ST treatment and at least 6 h ST treatment was required for maximum down-regulation of GC-C activity. This requirement for prolonged treatment of cells to ST to observe desensitization, is in contrast to the rapid inactivation that is seen for other members of the guanylyl cyclase receptors, such as GC-A and the sea urchin sperm receptor [2,33,34]. As shown in Fig. 1C, high concentrations of ST were required to induce desensitization, suggesting that the mechanism of desensitization appeared to be coupled to a high occupancy of the receptor by the ligand. Increases in intracellular cGMP alone could not trigger desensitization, as addition of 8-Br cGMP to Caco2 cells did not result in cellular refractoriness to ST (data not shown). In vitro guanylyl cyclase assay after ST-induced desensitization In vitro guanylyl cyclase assays were performed with membranes prepared from control and ST-treated Caco2 cells. Consistent with the results seen in whole cells, there was a 15-fold stimulation of guanylyl cyclase activity in membranes prepared from control cells on addition of ST (Fig. 2A). In contrast, there was a dramatic loss of ST-mediated activation of GC-C in the membranes pre- pared from cells that had been exposed to ST earlier. Interestingly, receptor-binding analysis performed with membranes prepared from control and ST-treated cells showed only a 50% loss in receptor content (Fig. 2B). These results indicate that although ST binding sites were present in the desensitized cells, ligand binding to these sites was not coupled to cGMP production, as significant loss of ST- mediated activation of GC-C was observed in desensitized cells. We monitored guanylyl cyclase activity in membranes prepared from control and ST-treated cells using MnGTP as a substrate, where guanylyl cyclase activity can be observed even in the absence of ligand. Again, in contrast to the significant loss of ST-mediated activation of guanylyl cyclase activity, 50% of the activity seen in control cells was still observed, representing the amount of GC-C detected by ST binding (Fig. 2C). Therefore, the fraction of GC-C present in the membranes of desensitized cells possessed guanylyl cyclase activity, but this form of the receptor could not respond to ligand stimulation. Expression of differentially glycosylated forms of GC-C in Caco2 cells Western blot analysis with a monoclonal antibody to GC-C using membranes prepared from control Caco2 cells revealed the presence of two immunoreactive bands of 145 and 130 kDa in size. Treatment of immunoprecipitated GC-C with protein N-glycosidase F resulted in the genera- tion of an immunoreactive band of M r 120 kDa, a size predicted from the cDNA sequence of GC-C without glycosylation, indicating that the two forms of GC-C represented alternately glycosylated forms of the receptor (Fig. 3A). EndoH treatment of the immunoprecipitate led to a reduction in size of the 130 kDa form and not the 145 kDa form, showing that the 130 kDa form represented Fig. 1. Prolonged ST treatment leads to cellular refractoriness to further ST-stimulation in Caco2 cells. (A) Caco2 monolayers were treated with 10 )7 M ST for 18 h, monolayers were washed and restimulated with 10 )7 M ST for 15 min in the presence or absence of 500 l M IBMX. Cells were lysed in 0.1 M citric acid and intracellular cGMP was measured by radioimmunoassay. (B) Cells were treated with 10 )7 M ST for the indicated times. At the end of incubation, cells were washed and restimulated with 10 )7 M ST in the presence of 500 l M IBMX for 15 min. (C) Caco2 cells were treated with varying concentrations of ST for 9 h. Following incubation, cells were washed and restimulated with 10 )7 M ST for 15 min in the presence of IBMX. Values represent mean ± SEM of duplicate determinations with each experiment performed at least twice. Ó FEBS 2003 Glycosylation of GC-C and desensitization (Eur. J. Biochem. 270) 3851 the high mannose containing form of GC-C. It was likely therefore that the predominant 130 kDa form was residing in the endoplasmic reticulum. Indeed, immunofluorescence with GC-C:D7 monoclonal antibody showed that a signi- ficant fraction of GC-C was present inside the cell, presumably in the endoplasmic reticulum (Fig. 3B). This large fraction of GC-C present intracellularly appears to be a property of all cells that express the receptor, as has been reported earlier [31,35,36]. No fluorescence was observed with cells incubated with GC-C:4D7 antibody preadsorbed with the fusion protein comprising the kinase-like domain of GC-C and glutathione S-transferase, as has been reported earlier (Fig. 3B). Western blot analysis was carried out using membrane protein prepared from control and desensitized cells. Most interestingly, while both the 130 and 145 kDa forms were detected in control cells, only the 130 kDa form of GC-C was detected in desensitized Caco2 cells (Fig. 4A). As shown earlier, membranes prepared from desensitized cells did not show ligand-stimulated activation, even thought they were able to bind the ST peptide (Fig. 2). Therefore, there appeared to be a correlation between the presence of the 145 kDa form of GC-C, which represents the mature glycosylated form of GC-C, and the ability of GC-C to be stimulated by ST. Cells regain their ability to be stimulated by ST following the reappearance of the 145 kDa form of GC-C. Cells were cultured for 18 h in the presence of ST, ST was then removed and cells fed with serum-containing medium without ST. At various times after renewal of the medium, cells were harvested and membranes subjected to Western blot analysis and stimulation with ST peptide. As shown in Fig. 4B, loss of ST-induced stimulation was correlated with the absence of the 145 kDa form. Following ST removal, Fig. 2. GC-C activity and content in desensitized cells. (A) Twenty micrograms of membrane protein prepared from control and ST-treated Caco2 cells were incubated with or without 10 )7 M ST in the presence of Mg-GTP (4 : 1 m M ) for 10 min and the cGMP synthesized was measured by radioimmunoassay. Values represent mean ± SEM of duplicate determinations with the experiment performed at least twice. (B) Receptor binding analysis of control and desensitized cells. Membrane protein prepared from control (right panel) and desensitized cells (left panel) were subjected to 125 I-labeled ST binding. Membrane protein was incubated with increasing amounts of 125 I-labeled ST Y72F at 37 °C for 1 h. At the end of incu- bation, the reaction was filtered through GF-C filters, filters were dried and radioactivity associated with the filter was measured. Data was analyzed using GRAPHPAD PRISM . The experiment was performed at least twice and the values shown represent data from a single experi- ment. (C) Five micrograms of membrane protein prepared from control and desensitized cells were incubated with MnGTP (4 m M MnCl 2 and 1 m M GTP) as a substrate. Reaction was carried out for 5 min at 37 °C,andcGMPsynthesizedwasmonitoredbyRIA.Values represent mean ± SEM of duplicate determinations with each experiment performed at least twice. Fig. 3. Expression of differentially glycosylated forms of GC-C in Caco2 cells. (A) GC-C was immunoprecipiated from Caco2 cells using the CTD antibody. The immunoprecipitate was incubated with or without PNGase F or Endo H, and separated by 6% SDS/PAGE. (B) Immunocytochemistry of Caco2 cells. Cells cultured on coverslips were blocked, permeabilized and incubated with 10 lgÆmL )1 GC-C:4D7 or with normal mouse IgG (data not shown) and then with FITC-tagged anti-(mouse IgG). The cells were mounted in Vectashield mounting medium and visualized using a standard filter for FITC at 63 · mag- nification. 3852 Y. Ghanekar et al. (Eur. J. Biochem. 270) Ó FEBS 2003 the 145 kDa form reappeared, and along with that, ST-induced stimulation was restored. These results there- fore show that the extent of glycosylation of GC-C determines its ability to be ligand-stimulated, and respon- siveness of cells to the ST peptide can be controlled by the presence or absence of differentially glycosylated forms of GC-C. It is possible that the reappearance of the 145 kDa form following removal of ST peptide was a consequence of further glycosylation of the ER-associated 130 kDa form, and subsequent transport to the plasma membrane. There- fore, cells were allowed to recover in the absence and presence of cycloheximide. As shown in Fig. 4C, the presence of cycloheximide did not hinder the reappearance of the 145 kDa form of GC-C, nor prevent restoration of ST-responsiveness in cells, indicating that de novo protein synthesis was not required, and a pool of ER-associated GC-C is available to replenish the ligand-activable form of the receptor, lost from the cell surface on ST addition. The levels of cGMP accumulation achieved during recovery in the presence of cycloheximide was slightly greater than in its absence across (P < 0.05), suggesting that the synthesis of a factor that could destabilize GC-C expression in cells was inhibited in cycloheximide-treated cells. To determine whether inhibition of glycosylation could prevent recovery, desensitized cells were treated with swainsonine, an inhibitor of a-mannosidase II, during the recovery process, and then stimulated with ST. As shown in Fig. 4D, swainsonine inhibited the reacquisition of respon- siveness by 50%. This shows that modification of the a-1,6 arm of the mannose residues was essential to allow formation of the 145 kDa form of GC-C and ligand responsiveness. Western blot analysis of cells cultured in the presence of swainsonine showed the presence of a band of 136 kDa, representing the partially glycosylated form of GC-C. Desensitization requires GC-C internalization and receptor activation Removal of the 145 kDa form from the surface of cells could be either through selective proteolysis or internaliza- tion and degradation. It is unlikely that selective proteolysis had occurred, as we do not detect any low molecular weight Fig. 4. Alterations of differentially glycosylated forms of GC-C in Caco2 cells. (A) One hundred micrograms of membrane protein from control and desensitized cells was subjected to Western blot analysis with GC-C:C8 antibody. (B) GC-C was immunoprecipitated using the CTD antibody and immunoprecipitates were subjected to Western blot analysis using GC-C:C8 antibody. Lane 1, control cells; lane 2, desensitized cells; lane 3, recovery. (C) Desensitized Caco2 cells were washed and incubated without ST for 12 h in culture medium in the absence or presence of cycloheximide. Membranes were prepared from these cells. Twenty micrograms of membrane protein was incubated with MgGTP (4 : 1 m M ) in the presence or absence of 100 n M ST for 10 min. Values represent mean ± SEM of duplicate determinations with each experiment performed at least twice. (D) Desensitized cells were incubated in medium containing 10% serum and swainsonine as indicated, for 12 h. Monolayers were then washed and restimulated with ST peptide for 15 min and cGMP produced monitored by radi- oimmunoassay. Values represent the mean ± SEM of duplicate determinations with the experiment performed twice. In addition, membrane protein prepared from control or swainsonine treated cells (200 lg) was solubilized and taken for immunoprecipitation and Western blot analysis. Lane 1, desensitized cell membrane; lane 2, membrane after recovery; lane 3, membrane after recovery in the presence of swainsonine. Ó FEBS 2003 Glycosylation of GC-C and desensitization (Eur. J. Biochem. 270) 3853 fragment of GC-C in desensitized cells, using multiple monoclonal and polyclonal antibodies (data not shown). If internalization and subsequent degradation of the 145 kDa form of GC-C was the cause for the desensitization, prolonged exposure of cells to ST may be required. We performed desensitization experiments at 4 °C, and as shown in Fig. 5A, no receptor desensitization was observed, and no loss of the 145 kDa fraction of GC-C was seen in cells treated at 4 °C with ST (Fig. 5B). These results therefore suggest that internalization of the 145 kDa form followed by degradation may account for the inability of cells to respond to the ST peptide, and this process is inhibited at 4 °C. We surface biotinylated control and desensitized cells, immunoprecipitated the receptor using a GC-C antibody and probed immunoprecipitates with streptavidin peroxi- dase. As shown in Fig. 5C, interestingly, a significant amount of 130 kDa protein was localized on the surface of the cells, along with the 145 kDa form of GC-C, perhaps representing a form of the receptor that had reached the surface in a Golgi-independent manner [37]. The presence of a biotinylated 130 kDa form of the receptor was not due to biotinylation of the intracellular 130 kDa protein as a consequence of leaky or damaged cells during the biotiny- lation reaction. This was judged by trypan blue exclusion, where more than 99% of the cells were viable following biotinylation (data not shown). Interestingly, on ST treat- ment of cells, the 130 kDa form was retained on the cell surface, as monitored by immunoprecipitation of GC-C using the CTD antibody followed by Western blot analysis with streptavidin-peroxidase (Fig. 5C). Therefore, there is a specific down-regulation of the 145 kDa form of GC-C on prolonged ligand treatment, indicating that only the ligand- responsive form of the receptor is perhaps routed to the lysosomal compartment for degradation. The continued presence on the surface of cells of the 130 kDa form, even on ST-treatment, indicates that this form is clearly ligand- unresponsive, and is either not internalized, or recycled efficiently to the surface. Discussion The studies described in this report suggest that regulation of the glycosylation of GC-C can act as a means of controlling the ability of cells to respond to the ST peptide. Desensitization studies carried out so far in various members of the receptor guanylyl cyclase family have shown that some of these receptors are down-regulated by rapid dephosphorylation after short treatment with the ligand. For example, the sea urchin sperm receptor guanylyl cyclases are dephosphorylated rapidly upon ligand binding, leading to receptor desensitization [33,34]. Studies carried out with the receptors for natriuretic peptides, GC-A and GC-B, also showed that dephosphorylation is the mechan- ism of desensitization of these receptors. GC-A is phos- phorylated on six serine and threonine residues present in the protein kinase-like domain in the basal state [2]. Mutation of any of these sites to alanine led to a decrease in ANP-mediated activation and simultaneous mutations in all the sites resulted in a complete loss of ANP-mediated activation [38]. Recent studies have indicated that GC-A is possibly dephosphorylated by two phosphatases, a micro- cystin inhibited phosphatase and another phosphatase that is activated by magnesium and manganese [39]. Activation of GC-B by CNP also leads to desensitization of GC-B after brief treatment with ligand, and here again, this is accom- panied by a decrease in the phosphate content of GC-B [40]. Mutational analysis has shown that there are five serine/ threonine phosphorylation sites in GC-B and mutation of all these sites together led to a loss of CNP-dependent activity [41]. The sites for phosphorylation in GC-A or GC-B are not conserved in GC-C. In our initial studies, we had investi- gated the phosphorylation status of GC-C in Caco2 cells, and found that while there is a basal level of phosphory- lation in the receptor on serine residues, there is no alteration in the phosphorylation status on ligand addition (unpublished observations). Given the contrasting rates of desensitization observed between GC-A and GC-C, with inactivation of GC-A occurring in a few minutes following Fig. 5. Desensitization of Caco2 cells to ST peptide requires GC-C internalization. Caco2 cells were treated with 100 n M ST for 9 h at 4 °C to inhibit internalization. Another set was incubated with or without ST at 37 °C and membranes were prepared. (A) Twenty micrograms of membrane was incubated with MgGTP (4 : 1 m M ) in the presence or absence of ST for 10 min cGMP was measured by RIA. Values represent mean ± SEM of duplicate determinations with each experiment performed at least twice. (B) GC-C was immunoprecipi- tated using CTD antibody and the immunoprecipitate was subjected to Western blot analysis using GC-C:4D7 antibody. Lane 1, control cells incubated at 4 °C; lane 2, cells incubated with ST at 4 °C; lane 3, cells incubated at 37 °C; lane 4, cells incubated with ST at 37 °C. (C) Control and desensitized cells (ST treated for 9 h) were surface bio- tinylated and membrane protein prepared. GC-C was immunopre- cipitated from equal amounts of solubilized membrane protein with CTD antibody, and immunoprecipitates analyzed by Western blot analysis using streptavidin–peroxidase conjugate. 3854 Y. Ghanekar et al. (Eur. J. Biochem. 270) Ó FEBS 2003 ligand exposure, while that of GC-C takes many hours, it was likely that distinct regulatory mechanisms are operative in the two receptors, as indeed is the case, and shown in the studies described here. Until date, the role of glycosylation in either GC-A or GC-B signaling has not been studied, but may be worthwhile to pursue now, in light of the observations described here, given the possible similarity in the overall structure of the extracellular domains of the receptors [37]. Using [ 125 I]ANP binding assays, GC-A has also been reported to undergo ligand-mediated internalization with a t 1/2 of 8 min in HEK293 cells [3]. Forty to fifty per cent of the internalized receptor is recycled back to the surface and the rest is directed to the degradation pathway. GC-B and NPR-C, the clearance receptor for atrial natriuretic factor, also undergo ligand-mediated internalization and are recy- cled back to the surface in PC12 cells [42]. GC-C has been shown to be internalized and recycled in T84 cells following ST treatment, but those experiments were conducted with periods of ST treatment for 3 h or less, during which time we observe only a slight desensitization in either T84 or Caco2 cells [43]. Moreover, earlier studies monitored ST binding to monolayers of cells in culture, and not ligand- stimulatable activity of surface-localized GC-C. As shown in the studies described here, the recycling of the 145 kDa form of GC-C does not appear to occur on long-term treatment of cells with ST. As the 130 kDa form of GC-C is still on the surface after prolonged ST treatment, it is not clear at this time whether the 130 kDa form alone is recycled, or not internalized at all. GC-C down-regulation apparently occurs by differential expression of the two glycosylated forms of GC-C, and a strong correlation between the presence of the 145 kDa form and ST-stimulatabilty is seen, indicating that the 145 kDa form is the ligand activated form of GC-C. Our earlier studies have shown that GC-C desensitization is modulated in a cell specific manner [25]. GC-C desensitiza- tion is observed only in cells that endogenously express GC-C, such as T84 and Caco2 but not in HEK293 cells stably transfected with GC-C, HEK293GC-C cells. Inter- estingly, there is no loss of the 145 kDa form in HEK293GC-C cells on ST treatment, providing an explan- ation for the continuous ability of these cells to respond to ST, even on prolonged prior exposure [25]. The absence of GC-C desensitization (which is correlated with removal of the 145 kDa form of GC-C from cells) in HEK293-GC-C cells could be due to lack of a cellular factor that is present in T84 and Caco2 cells, that selectively allows the degradation of the 145 kDa form. Recently a PSD-95, Disc-large, ZO-1 (PDZ) domain protein which interacts with GC-C was identified in a yeast two hybrid screen [44]. This protein named Ôintestine and kidney-enriched PDZ proteinÕ (IKEPP) interacts with GC-C through one of its PDZ domains. In the presence of IKEPP, the EC 50 of GC-C for ST increased 10-fold, suggesting that IKEPP regulates ligand-mediated activation of GC-C. Interestingly, IKEPP is expressed in T84 and Caco2 cells but not in HEK293 cells, and therefore could be a possible candidate protein involved in selective down-regulation of GC-C [44]. Our studies carried out by treating cells with ST at 4 °C suggest that down-regulation is brought about by selective internalization of the 145 kDa form through endocytosis and subsequent degradation. The mechanisms involved in this process are not yet identified. It is possible that the activation of the cyclase domain of the 145 kDa form upon ligand binding leads to a conformational change, exposing a signal that promotes internalization and exposure of a ubiquitination signal, leading to selective degradation of the 145 kDa form. Alternatively the sugar residues present on the 145 kDa form could act as a signal for internalization and/or degradation. Indeed, glycosylation-based recogni- tion motifs are involved in internalization as well as degradation of proteins. N-Linked glycosylation of Edg-1, which is a G-protein-coupled receptor, is essential for targeting the receptor to caveolin-rich domains in the plasma membrane [45]. Glycosylation of the b-adrenergic receptor is also important for its internalization and a single nucleotide polymorphism which leads to mutation of serine 49 to glycine leads to loss of glycosylation and enhances internalization of the b-adrenergic receptor [46]. Recently, glycosylation-based recognition motifs have also been recognized in substrates for the endoplasmic reticulum- associated degradation (ERAD) pathway. One of the proteins associated with the E3 ubiquitin ligase complex, Fbx2, specifically binds N-glycosylated substrates through mannose residues on the glycocalyx [47]. Although glyco- sylation-based recognition signals have not yet been iden- tified for degradation of cell surface receptors, the possibility of such a mechanism operating at the plasma membrane cannot be ruled out, and the two forms of GC-C are perhaps targeted to different endocytic routes based on glycosylation sorting motifs at the plasma membrane. Alternatively, glycosylation-dependent sorting of the two forms could also take place intracellularly after they are endocytosed and not at the plasma membrane. Surface localized receptors such as EGF receptor are carried to the endoplasmic reticulum before being targeted to the degra- dation pathways [48] and it is possible that GC-C is also carried to the endoplasmic reticulum where the 145 kDa form is targeted to the degradation pathway and the 130 kDa form can be recycled back to the surface. Therefore in this study, we have described a novel means of regulation of a member of the guanylyl cyclase receptor family, by controlling the amounts of differentially glycosy- lated forms of GC-C in a cell. Given the fact that the 130 kDa form of the receptor is unresponsive to the ST peptide, even though it can bind the ligand with an affinity similar to the hyperglycosylated form, and remains present on the surface of cells even after desensitization, one can suggest that the 130 kDa form of GC-C can act as a ÔsinkÕ for its ligands, when present on the plasma membrane of intestinal cells. This may partly account for the differential responsiveness of various regions of the intestine to the guanylin/uroguanylin family of peptides [6], and also regulate GC-C signaling in extraintestinal tissues where GC-C and its ligands are expressed. Acknowledgements This work was funded by the Department of Biotechnology, Govern- ment of India. YG is supported by the Indian Council of Medical Research, and AC by the Department of Atomic Energy, Government of India. We would like to thank Ms. Vani Iyer for the purification and radioiodination of ST peptide. Ó FEBS 2003 Glycosylation of GC-C and desensitization (Eur. J. Biochem. 270) 3855 References 1. Lucas, K.A., Pitari, G.M., Kazerounian, S., Ruiz-Stewart, I., Park, J., Schulz, S., Chepenik, K.P. & Waldman, S.A. (2000) Guanylyl cyclases and signaling by cyclic GMP. Pharmacol. Rev. 52, 375–414. 2. Potter, L.R. & Garbers, D.L. (1992) Dephosphorylation of the guanylyl cyclase-A receptor causes desensitization. J. Biol. Chem. 267, 14531–14534. 3. Pandey, K.N. (2002) Intracellular trafficking and metabolic turn- over of ligand-bound guanylyl cyclase/atrial natriuretic peptide receptor-A into subcellular compartments. Mol. Cell Biochem. 230, 61–72. 4. Nandi, A., Bhandari, R. & Visweswariah, S.S. (1997) Epitope conservation and immunohistochemical localization of the gua- nylin/stable toxin peptide receptor, guanylyl cyclase C. J. Cell Biochem. 66, 500–511. 5. Krause, W.J., Cullingford, G.L., Freeman, R.H., Eber, S.L., Richardson,K.C.,Fok,K.F.,Currie,M.G.&Forte,L.R.(1994) Distribution of heat-stable enterotoxin/guanylin receptors in the intestinal tract of man and other mammals. J. Anat. 184, 407–417. 6. Qian,X.,Prabhakar,S.,Nandi,A.,Visweswariah,S.S.&Goy, M.F. (2000) Expression of GC-C, a receptor-guanylate cyclase, and its endogenous ligands uroguanylin and guanylin along the rostrocaudal axis of the intestine. Endocrinology 141, 3210–3224. 7. Scheving, L.A. & Russell, W.E. (1996) Guanylyl cyclase C is up-regulated by nonparenchymal cells and hepatocytes in regenerating rat liver. Cancer Res. 56, 5186–5191. 8. Jaleel, M., London, R.M., Eber, S.L., Forte, L.R. & Visweswar- iah, S.S. (2002) Expression of the receptor guanylyl cyclase C and its ligands in reproductive tissues of the rat: a potential role for a novel signaling pathway in the epididymis. Biol. Reprod. 67, 1975–1980. 9. Forte, L.R., Thorne, P.K., Eber, S.L., Krause, W.J., Freeman, R.H., Francis, S.H. & Corbin, J.D. (1992) Stimulation of intestinal Cl – transport by heat-stable enterotoxin: activation of cAMP- dependent protein kinase by cGMP. Am. J. Physiol. 263, C607– C615. 10. Chao, A.C., de Sauvage, F.J., Dong, Y.J., Wagner, J.A., Goeddel, D.V. & Gardner, P. (1994) Activation of intestinal CFTR Cl – channel by heat-stable enterotoxin and guanylin via cAMP- dependent protein kinase. EMBO J. 13, 1065–1072. 11. Pitari, G.M., Di Guglielmo, M.D., Park, J., Schulz, S. & Wald- man, S.A. (2001) Guanylyl cyclase C agonists regulate progression through the cell cycle of human colon carcinoma cells. Proc. Natl Acad. Sci. USA 98, 7846–7851. 12. Shailubhai, K., Yu, H.H., Karunanandaa, K., Wang, J.Y., Eber, S.L.,Wang,Y.,Joo,N.S.,Kim,H.D.,Miedema,B.W.,Abbas, S.Z., Boddupalli, S.S., Currie, M.G. & Forte, L.R. (2000) Uroguanylin treatment suppresses polyp formation in the Apc (Min/+) mouse and induces apoptosis in human colon adenocarcinoma cells via cyclic GMP. Cancer Res. 60, 5151– 5157. 13. Pitari,G.M.,Zingman,L.V.,Hodgson,D.M.,Alekseev,A.E., Kazerounian, S., Bienengraeber, M., Hajnoczky, G., Terzic, A. & Waldman, S.A. (2003) Bacterial enterotoxins are associated with resistance to colon cancer. Proc.NatlAcad.Sci.USA.100, 2695–2699. 14. Waldman, S.A., Barber, M., Pearlman, J., Park, J., George, R. & Parkinson, S.J. (1998) Heterogeneity of guanylyl cyclase C expressed by human colorectal cancer cell lines in vitro. Cancer Epidemiol. Biomarkers Prev. 7, 505–514. 15. Dharmsathaphorn, K., McRoberts, J.A., Mandel, K.G., Tisdale, L.D. & Masui, H. (1984) A human colonic tumor cell line that maintains vectorial electrolyte transport. Am.J.Physiol.246, G204–G208. 16. Pinto, M., Robine-Leon, S., Appay, M.D., Kedinger, M., Trai- dou, N., Dussaulx, E., Lacroix, B., Simon-Assman, P., Haffen, K., fogh, J. & Zweibaum, A. (1983) Enterocyte-like differentiation and polarization of the human colon carcinoma cell line Caco-2 in culture. Biol. Cell. 47, 323–350. 17. Chantret, I., Barbat, A., Dussaulx, E., Brattain, M.G. & Zwei- baum, A. (1988) Epithelial polarity, villin expression, and enterocytic differentiation of cultured human colon carcinoma cells: a survey of twenty cell lines. Cancer Res. 48, 1936–1942. 18. Mann, E.A., Cohen, M.B. & Giannella, R.A. (1993) Comparison of receptors for Escherichia coli heat-stable enterotoxin: novel receptor present in IEC-6 cells. Am. J. Physiol. 264, G172–G178. 19. Cohen, M.B., Jensen, N.J., Hawkins, J.A., Mann, E.A., Thomp- son, M.R., Lentze, M.J. & Giannella, R.A. (1993) Receptors for Escherichia coli heat stable enterotoxin in human intestine and in a human intestinal cell line (Caco-2). J. Cell Physiol. 156, 138–144. 20. Cohen, M.B., Hawkins, J.A. & Witte, D.P. (1998) Guanylin mRNA expression in human intestine and colorectal adenocarci- noma. Lab. Invest. 78, 101–108. 21. Rudolph, J.A., Hawkins, J.A. & Cohen, M.B. (2002) Proguanylin secretion and the role of negative-feedback inhibition in a villous epithelial cell line. Am. J. Physiol. Gastrointest. Liver Physiol. 283, G695–G702. 22. Gill, R.K., Saksena, S., Syed, I.A., Tyagi, S., Alrefai, W.A., Malakooti,J.,Ramaswamy,K.&Dudeja,P.K.(2002)Regulation of NHE3 by nitric oxide in Caco-2 cells. Am. J Physiol. Gastro- intest. Liver Physiol. 283, G747–G756. 23. Saksena, S., Gill, R.K., Syed, I.A., Tyagi, S., Alrefai, W.A., Ramaswamy, K. & Dudeja, P.K. (2002) Modulation of Cl – /OH – exchange activity in Caco-2 cells by nitric oxide. Am.J.Physiol. Gastrointest. Liver Physiol. 283, G626–G633. 24. Bakre, M.M. & Visweswariah, S.S. (1997) Dual regulation of heat- stable enterotoxin-mediated cGMP accumulation in T84 cells by receptor desensitization and increased phosphodiesterase activity. FEBS Lett. 408, 345–349. 25. Bakre, M.M., Ghanekar, Y. & Visweswariah, S.S. (2000) Homologous desensitization of the human guanylate cyclase C receptor: cell-specific regulation of catalytic activity. Eur. J. Biochem. 267, 179–187. 26. Dwarakanath, P., Visweswariah, S.S., Subrahmanyam, Y.V., Shanthi, G., Jagannatha, H.M. & Balganesh, T.S. (1989) Cloning and hyperexpression of a gene encoding the heat-stable toxin of Escherichia coli. Gene 81, 219–226. 27. Fleet, J.C., Wang, L., Vitek, O., Craig, B.A. & Edenberg, H.J. (2003) Gene expression profiling of Caco-2 BBe cells suggests a role for specific signaling pathways during intestinal differentia- tion. Physiol. Genomics. 13, 57–68. 28. Visweswariah, S.S., Shanthi, G. & Balganesh, T.S. (1992) Inter- action of heat-stable enterotoxins with human colonic (T84) cells: modulation of the activation of guanylyl cyclase. Microb. Pathog. 12, 209–218. 29. Zor, T. & Selinger, Z. (1996) Linearization of the Bradford protein assay increases its sensitivity: theoretical and experimental studies. Anal. Biochem. 236, 302–308. 30. Visweswariah, S.S., Ramachandran, V., Ramamohan, S., Das, G. & Ramachandran, J. (1994) Characterization and partial puri- fication of the human receptor for the heat-stable enterotoxin. Eur. J. Biochem. 219, 727–736. 31. Sindice, A., Basoglu, C., Cerci, A., Hirsch, J.R., Potthast, R., Kuhn, M., Ghanekar, Y., Visweswariah, S.S. & Schlatter, E. (2002) Guanylin, uroguanylin, and heat-stable euterotoxin acti- vate guanylate cyclase C and/or a pertussis toxin-sensitive G protein in human proximal tubule cells. J. Biol. Chem. 277, 17758– 17764. 32. Bhandari, R., Srinivasan, N., Mahaboobi, M., Ghanekar, Y., Suguna, K. & Visweswariah, S.S. (2001) Functional inactivation 3856 Y. Ghanekar et al. (Eur. J. Biochem. 270) Ó FEBS 2003 of the human guanylyl cyclase C receptor: modeling and mutation of the protein kinase-like domain. Biochemistry 40, 9196–9206. 33. Bentley, J.K., Tubb, D.J. & Garbers, D.L. (1986) Receptor- mediated activation of spermatozoan guanylate cyclase. J. Biol. Chem. 261, 14859–14862. 34. Bentley, J.K., Shimomura, H. & Garbers, D.L. (1986) Retention of a functional resact receptor in isolated sperm plasma mem- branes. Cell 45, 281–288. 35. Scheving, L.A., Russell, W.E. & Chong, K.M. (1996) Structure, glycosylation, and localization of rat intestinal guanylyl cyclase C: modulation by fasting. Am. J. Physiol. 271, G959–G968. 36. Scheving, L.A. & Russell, W.E. (2001) Insulin and heregulin-beta1 upregulate guanylyl cyclase C expression in rat hepatocytes: reversal by phosphodiesterase-3 inhibition. Cell Signal. 13,665– 672. 37. van den Akker, F. (2001) Structural insights into the ligand binding domains of membrane bound guanylyl cyclases and natriuretic peptide receptors. J. Mol. Biol. 311, 923–937. 38. Potter, L.R. & Hunter, T. (1998) Phosphorylation of the kinase homology domain is essential for activation of the A-type natriuretic peptide receptor. Mol. Cell Biol. 18, 2164–2172. 39. Bryan, P.M. & Potter, L.R. (2002) The atrial natriuretic peptide receptor (NPR-A/GC-A) is dephosphorylated by distinct micro- cystin-sensitive and magnesium-dependent protein phosphatases. J. Biol. Chem. 277, 16041–16047. 40. Potter, L.R. (1998) Phosphorylation-dependent regulation of the guanylyl cyclase-linked natriuretic peptide receptor B: dephos- phorylation is a mechanism of desensitization. Biochemistry 37, 2422–2429. 41. Potter, L.R. & Hunter, T. (1998) Identification and characteriza- tion of the major phosphorylation sites of the B-type natriuretic peptide receptor. J. Biol. Chem. 273, 15533–15539. 42. Rathinavelu, A. & Isom, G.E. (1991) Differential internalization and processing of atrial-natriuretic-factor B and C receptor in PC12 cells. Biochem. J. 276, 493–497. 43. Urbanski, R., Carrithers, S.L. & Waldman, S.A. (1995) Inter- nalization of E. coli ST mediated by guanylyl cyclase C in T84 human colon carcinoma cells. Biochim. Biophys. Acta 1245, 29–36. 44. Scott, R.O., Thelin, W.R. & Milgram, S.L. (2002) A novel PDZ. protein regulates the activity of guanylyl cyclase C, the heat-stable enterotoxin receptor. J. Biol. Chem. 277, 22934–22941. 45. Kohno, T., Wada, A. & Igarashi, Y. (2002) N-Glycans of sphin- gosine 1-phosphate receptor Edg-1 regulate ligand-induced receptor internalization. FASEB J. 16, 983–992. 46. Rathz,D.A.,Brown,K.M.,Kramer,L.A.&Liggett,S.B.(2002) Amino acid 49 polymorphisms of the human beta1-adrenergic receptor affect agonist-promoted trafficking. J. Cardiovasc. Phar- macol. 39, 155–160. 47. Yoshida, Y., Chiba, T., Tokunaga, F., Kawasaki, H., Iwai, K., Suzuki, T., Ito, Y., Matsuoka, K., Yoshida, M., Tanaka, K. & Tai, T. (2002) E3 ubiquitin ligase that recognizes sugar chains. Nature 418, 438–442. 48. Haj, F.G., Verveer, P.J., Squire, A., Neel, B.G. & Bastiaens, P.I. (2002) Imaging sites of receptor dephosphorylation by PTP1B on the surface of the endoplasmic reticulum. Science 295, 1708–1711. Ó FEBS 2003 Glycosylation of GC-C and desensitization (Eur. J. Biochem. 270) 3857 . Cellular refractoriness to the heat-stable enterotoxin peptide is associated with alterations in levels of the differentially glycosylated forms of guanylyl. colonic cell lines to ST peptides induces cellular refractoriness to the ST peptide, in terms of intra- cellular cGMP accumulation. We have investigated the mechanism