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 cell
Trang 1Cellular 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 4C 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)
Trang 2Several 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 Vmaxof the guanylyl cyclase catalytic
activity of GC-C with no change in the S0.5of 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 PlusTM
Western blotting detection reagent were obtained from
Amersham Biosciences, UK 125Iodine 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, STY72F, 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)1penicillin and
270 mgÆL)1streptomycin 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)7MSTh in DMEM/F12 for
9 h Monolayers were then washed and re-stimulated with fresh STh (10)7M) in the presence or absence of 500 lM
isobutylmethyl xanthine (IBMX) for 30 min in serum-free medium for 15 min Cell monolayers were washed, and cells lysed in 0.1Mcitric acid or 0.1MHCl 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/Pi, 10 mM sodium phos-phate buffer, pH 7.2, 0.9% sodium chloride) and scraped into homogenization buffer (50 mM Hepes, pH 7.5,
100 mM NaCl, 5 mM EDTA, 1 mM dithiothreitol,
5 lgÆmL)1soybean trypsin inhibitor, 5 lgÆmL)1leupeptin, and 5 lgÆmL)1aprotinin) The cell lysate was sonicated and centrifuged at 10 000 g for 1 h at 4C The pellet obtained was resuspended in buffer containing 50 mM Hepes,
pH 7.5, 5 lgÆmL)1 soybean trypsin inhibitor, 5 lgÆmL)1 leupeptin, 5 lgÆmL)1aprotinin and 100 lMsodium 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)7M ST in assay buffer consisting of 60 mM Tris-Cl,
pH 7.6, 500 l IBMX, and a GTP regeneration system
Trang 3consisting of 10 lg creatine kinase and 7.5 mM creatine
phosphate The assay was initiated by adding 4 mMMgCl2
and 1 mMGTP as substrate and incubated at 37C for 5–10
min The reaction was terminated by addition of 400 lL of
50 mM 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 mMmanganese and 1 mM
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 37C with
4 mMMgCl2and 1 mMGTP as substrate
Receptor binding analysis
STY72Fwas iodinated using Na125I as described earlier [30]
and was available in the laboratory Membrane protein
(100–200 lg) was incubated with increasing concentrations
of 125I-labeled STY72Ffor 1 h at 37C in binding buffer
(50 mMHepes, pH 7.5, 4 mMMgCl2, 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 usingGRAPHPAD PRISM(San Diego, CA, USA)
Immunodetection of GC-C
Western blot analysis was performed with 50 lg of
mem-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/Pi
and fixed in NaCl/Picontaining 4% paraformaldehyde for
20–30 min Cells were washed and incubated with 2%
bovine serum albumin and 0.1% Triton X-100 in NaCl/Pi
for 1 h at room temperature to block nonspecific sites and
permeabilize cells Cells were then incubated overnight with
5 lgÆmL)1GC-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)1in immunoprecipitation buffer
(20 m Tris-Cl pH 7.5, 100 m NaCl, 2 m EDTA, 1%
Triton X-100, 5 lgÆmL)1 soybean trypsin inhibitor,
5 lgÆmL)1 aprotinin, 5 lgÆmL)1 leupeptin, and 100 lM
sodium orthovanadate) for 1 h at 4C 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 mMsodium phosphate buffer, pH 7.2 and then boiled in 50 mMsodium phosphate buffer with 0.1% SDS and 50 mM2-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 37C 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 37C was per-formed for 6 h and samples were then subjected to SDS gel electrophoresis and Western blot analysis as described above Surface biotinylation of Caco2 cells
Cells were washed with NaCl/Pi(pH 8.0) containing 1 mM
CaCl2and 0.5 mMMgCl2 (NaCl/Pi-CM), and then incu-bated in NaCl/Pi-CM containing 500 lgÆmL)1 sulfo-NHS-biotin (Sigma) for 30 min at room temperature Excess biotin was quenched by incubation with 50 mM Tris-Cl,
pH 7.5, for 5 min Cells were briefly washed with NaCl/Pi 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)7M
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
Trang 4ST 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 Mr 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)7M ST for 18 h, monolayers were washed and restimulated with
10)7M 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)7M ST
for the indicated times At the end of incubation, cells were washed and
restimulated with 10)7M 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)7M ST for 15 min in the presence of IBMX Values represent
mean ± SEM of duplicate determinations with each experiment
performed at least twice.
Trang 5the 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)7M 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 of125I-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, and cG MP synthesized was monitored by RIA 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)1GC-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.
Trang 6the 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 degradainternaliza-tion 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.
Trang 7fragment 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 4C, 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 4C 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 4C
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.
Trang 8ligand 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 [125I]ANP binding assays, GC-A has also been
reported to undergo ligand-mediated internalization with a
t1/2of 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 intestine and kidney-enriched PDZ protein
(IKEPP) interacts with GC-C through one of its PDZ
domains In the presence of IKEPP, the EC50of 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 4C
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 internalizarecogni-tion 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 sink 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 YGis 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.
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