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Department of Zoology, The University of Hong Kong (R.T.-K.P., S.S.-M.N., B.K.-C.C.), and the Department of Biochemistry, Chinese University of Hong Kong (C.H.-K.C.), Hong Kong, Special Administrative Region, Peoples Republic of China; and the Center for Basic Research in Digestive Diseases, Mayo Clinic and Foundation (M.H.H., L.J.M.), Rochester, Minnesota 55905
Address all correspondence and requests for reprints to: Dr. B. K. C. Chow, Department of Zoology, University of Hong Kong, Pokfulam Road, Hong Kong, Special Administrative Region, Peoples Republic of China. E-mail: bkcc{at}hkusua.hku.hk
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Secretin elicits its biological effects by interacting with specific
cell surface receptors. Recently, rat (17) and human (18, 19, 20) secretin
receptors have been characterized. These receptors belong to a distinct
family of guanine nucleotide-binding protein (G
protein-coupled)-coupled receptors, including pituitary adenylate
cyclase-activating polypeptide (PACAP type 1) vasoactive intestinal
peptide-1 (VIP1), VIP2, GH-releasing hormone (GHRH), glucagon, glucagon
like peptide-1, and gastric inhibitory polypeptide receptors. The human
secretin receptor (hSR) is 440 amino acids in size and contains a
putative hydrophobic leader peptide (22 amino acids), a large
hydrophilic ectodomain (122 amino acids), 7 transmembrane regions with
3 exoloops and 3 cytoplasmic loops (254 amino acids), and a hydrophilic
cytoplasmic tail (42 amino acids). There are also 5 putative
N-linked glycosylation consensus sequences at positions 72,
100, 106, 128, and 291 (Fig. 1
). The
glycoprotein nature of rat secretin receptor has been demonstrated
using wheat-germ agglutinin affinity chromatography (21) and
cross-linking studies in rat gastric gland (22) and pancreatic acini
(23).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Inhibitor treatment of stably transfected cells
The hSR complementary DNA (cDNA) was previously cloned and
functionally characterized in our laboratory (18). The full-length cDNA
(1.7 kb) encoding the human secretin receptor was subcloned into an
expression vector pRc-CMV (Stratagene, La Jolla, CA).
Chinese hamster ovary (CHO) cells and COS-7 cells were kept in MEM
supplemented with 10% FBS and antibiotics (100 U/ml penicillin and 100
µg/ml streptomycin). A stable cell line was generated by transfecting
CHO cells using Lipofectamine (Life Technologies, Inc.).
Two days after transfection, the cells were subjected to G418 selection
at 500 µg/ml for 2 weeks. For inhibitor treatment, 0.2 million cells
were seeded onto 6-well plates (Costar, Cambridge, MA)
supplemented with 1.7 µg/ml swainsonine, 200 µg/ml
deoxymannojirimycin, 200 µg/ml castanospermine, or 1 µg/ml
tunicamycin. The cells were incubated with the inhibitors for 30 or
60 h before being stimulated with 0.5 nM secretin. In
this experiment, the culture medium was changed every 20 h. In
control experiments, the cells incubated with the inhibitors were
stimulated with 10 µM forskolin. Conditions for peptide
stimulation and cAMP extraction were described previously (18). For
RT-PCR, RNA was extracted from the cells incubated with or without
glycosylation inhibitors for 60 h. Total RNA was isolated by the
guanidium thiocyanate/phenol extraction method (30). Residue genomic
DNA was digested with 1 U deoxyribonuclease I in the buffer provided by
the manufacturer (Pharmacia Biotech, Piscataway, NJ) for
15 min at 37 C. The messenger RNA (mRNA) was extracted using the
PolyATtract mRNA isolation system (Promega Corp., Madison,
WI) and was used as the template for first strand cDNA. The RT reaction
contained 10 mM dithiothreitol, 0.33 mM dNTPs,
0.2 µg random hexamer, 20 U RNasin (Promega Corp.), and
200 U Moloney murine leukemia virus reverse transcriptase in buffer
provided by the manufacturer (Life Technologies, Inc.).
The reaction was carried out at 37 C for 2 h. A standard PCR was
performed using a pair of hSR-specific primers: hSRF, 5'-ACACAGAGGG
CACGGGCAGG CGGACGTCGG; and hSRR, 5'-TGCAGGACCA GCATCATCTG AGAGGGAATT
CGC. The PCR contained 50 pmol of each primer, 200 µM
dNTPs, and 2.5 U Taq DNA polymerase (Life Technologies, Inc.) in the buffer provided by the manufacturer.
The reaction conditions were 1 min each at 94, 55, and 72 C for 30
cycles, and the PCR products were analyzed by a 1% ethidium
bromide-stained agarose gel.
Construction of hSR mutants by site-directed mutagenesis
The 1.7-kb full-length hSR cDNA was subcloned into the plasmid
vector pAlter-1 (Promega Corp.). Single stranded template
for site-directed mutagenesis was produced using the R408 helper phage.
Mutants were generated using an oligonucleotide-directed method
according to the manufacturer’s procedure (Promega Corp.). Mutations were designed at positions according to the
N-linked glycosylation consensus sequences (Asn-X-Ser/Thr)
by replacing Asn with Leu. The mismatch oligonucleotides used for Asn
mutants were: Asn72Leu, 5'-GGGATGTGGG ACCTCATAAG TGCTGG;
Asn100Leu, 5'-CTCACCAGCA GACTTGGTTC CTTGTTC;
Asn106Leu, 5'-TCCTTGTTCC GACTCTGCAC ACAGGAT;
Asn128Leu, 5'-GGCGTTAATG CGCTCGACTC TTCCAAC; and
Asn291Leu, 5'-GACATCAATG CCCTCGCATC CATCTGG. The Ser
mutants were constructed by replacing Ser with Ala. The mismatch
oligonucleotides used were: Ser74Ala, 5'-TGGGACAACA
TAGCATGCTG GCCCTCT; and Ser130Ala, 5'-AATGTGAACG ACGCTTCCAA
CGAGAAG. The mutants were confirmed by DNA sequence analysis using a T7
DNA sequencing kit (Pharmacia Biotech). The mutant
receptor cDNAs were subcloned into the vector pRc-CMV
(Stratagene) for expression studies.
Functional analysis of the glycosylation mutants by cAMP
assay
cAMP assay was performed as described previously (18). Briefly,
1 million cells seeded overnight were transfected with the wild-type
and mutant hSR cDNA constructs in 6-well plates (Costar)
using Lipofectamine according to the manufacturer’s protocol. Peptide
stimulation and cAMP assays were performed 3 days after transfection as
described above. The basal and maximally stimulated cAMP levels were
typically, on the average, 4.4 and 23.1 pmol/well, respectively. To
control for the transfection efficiency in different transfection
experiments, the cells were cotransfected with 1 µg pGL2-Control
luciferase reporter plasmid (Promega Corp.). Luciferase
assays were performed according to the manufacturer’s protocol
(Promega Corp.), and luciferase reporter activities were
quantified using a luminometer (LUMAT LB 9507, EG&G Berthold, Bad
Wildbad, Germany).
Measurement of the extracellular acidification rate
Extracellular acidification rates were measured using the
Cytosensor microphysiometer (Molecular Devices, Menlo
Park, CA). Transfected CHO cells were seeded into sterile cell capsule
cups (Molecular Devices) at a density of 0.6 million
cells/capsule in growth medium for 24 h. The internal size of the
capsule was 50 µm high and 6 mm in diameter as defined by a spacer
and a capsule insert (Molecular Devices). The cells were
trapped between two microporus polycarbonate membranes. The assembled
capsule cups were loaded into the Cytosensor chambers. The chambers
were perfused with the running medium (bicarbonate-free MEM
supplemented with 1 mg/ml BSA). The extracellular acidification rates
were monitored at 90-sec intervals. A stable basal acidification rate
was established before peptide stimulation. The acidification rate
response upon peptide stimulation was expressed as a percentage of the
basal rate.
Competitive binding assays
The Kd and binding capacity values of the wild-type
and mutant receptors (Table 2) were determined by the homologous
competitive binding approach using PRISM version 2.0 computer software
(GraphPad Software, Inc., San Diego, CA). For the
competitive binding experiments, the wild-type and mutant human
secretin receptors were expressed transiently in CHO cells using a
protocol described previously (18). In brief, CHO cells (0.5 million)
were seeded into 6-well plates (Costar) and transfected
with 1.5 µg expression plasmid using the Lipofectamine reagent
(Life Technologies, Inc.). After 48 h,
[125I]human secretin (100,000 cpm) was incubated with the
transfected CHO cells expressing the wild-type or mutant secretin
receptor in the presence of increasing concentrations (picomolar to
micromolar concentrations) of cold peptide for 1 h at 22 C. Human
secretin was labeled using 125I according to the method
described by Chang and Chey (31). The specific activity of the labeled
peptide was 463 cpm/fmol. Peptide was diluted from a stock solution
(100 µM) in the binding buffer [50 mM
Tris-HCl (pH 7.4), 200 mM sucrose, 5 mM
MgCl2, 10 mg/ml BSA, 0.1 mM
phenylmethylsulfonylfluoride, and 1 mg/ml bacitracin]. After the
incubation, cells were washed twice with ice-cold binding buffer and
lysed with 1 M NaOH, and radioactivity was measured in a
-counter. Nonspecific binding was determined in the presence of 1
µM human secretin. Total specific binding was, on the
average, 25–30% (25,000–30,000 cpm) of the total radioactivity
added.
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Statistical analysis
All observations were repeated six times, and values are
expressed as the mean ± SE. Statistical analyses were
carried out using Student’s t test, and differences were
considered significant at P < 0.01.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-mannosidase. Castanospermine is a plant alkaloid that inhibits
-glucosidases. Deoxymannojirimycin is a mannose analog that
specifically inhibits Golgi -mannosidases IA/IB. Tunicamycin is an
antibiotic that blocks N-linked glycosylation by preventing
the transfer of N-acetyl-glucosamine residue to dolichol
phosphate, thus inhibiting the synthesis of all N-linked
glycoproteins (Fig. 2). The cells
expressing the hSR, as indicated by RT-PCR (Fig. 3C, positive control lane), cAMP, and
acidification rate response (Fig. 4),
were cultured for 30 or 60 h in the presence of various
inhibitors. The cells were then stimulated with secretin (0.5
nM), and the accumulation of intracellular cAMP was
measured. Tunicamycin led to a 87% reduction or a total loss of cAMP
response after 30 or 60 h of inhibitor treatment, respectively
(Fig. 3, A and B, lane a). Besides tunicamycin, castanospermine (Fig. 3, A and B, lane b) also had a significant inhibitory effect on cAMP
production in the cells; the responses were reduced by 58% and 78%
after 30 and 60 h of treatment, respectively. In contrast, the
effect of deoxymannojirimycin or swainsonine was less pronounced and
statistically insignificant (Fig. 3, lane c and d). Functional analysis
of inhibitor-treated cells using forskolin indicated that these
inhibitors do not affect the cAMP signal transduction system (Fig. 3D).
Hence, the effects of tunicamycin and castanospermine are probably at
the receptor level. In addition, a semiquantitative RT-PCR was
performed using mRNA extracted from an equal number of cells (1
million) treated with various inhibitors for 60 h. Our results
indicated that inhibitor treatment does not have an adverse effect on
the transcription of hSR in the transfected cells. However, we cannot
exclude the possibility that these inhibitors may have altered the
translation and/or presentation of the receptor to the cell surface,
although the morphologies of the cells were unchanged during the study.
Taken together, our data suggested that N-linked
glycosylation is essential to the functional expression of hSR, and
glycosylation of the receptor is probably of the high mannose type.
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) indicated that the
Cytosensor bioassay is a more sensitive assay; the ED50
values of the wild-type receptor in these two assay systems are 0.25
and 0.11 nM, respectively. In both experiments, the
transfection efficiencies were monitored by cotransfection with the
luciferase pGL-2 control vector (data not shown). In the negative
control experiments, the vector alone-transfected cells showed neither
secretin-dependent cAMP nor acidification responses (data not
shown).
Mutation at position 100, 106, or 291 did not alter ED50
values or maximal responses in the two assays (Fig. 5), indicating either that glycosylation
at these sites does not affect receptor function or these sites are not
glycosylated. However, the Asn72Leu mutation significantly
increased the ED50 values (3.1 and 0.51 nM for
the cAMP and Cytosensor measurements, respectively; see Table 1) and reduced the maximal responses
(50.1% and 35.2%, respectively; Table 1). To show that these effects
are related to the changes in receptor glycosylation, a second mutant
(Ser74Ala) corresponding to the same glycosylation
consensus was constructed and analyzed. By cAMP assays, this mutant was
also found to be defective, there was a 6-fold increase in the
ED50 value (1.5 nM) and a 50% reduction in the
maximal response (Fig. 6 and Table 2). In fact, there was no significant
difference in the cAMP responses between these two mutants, further
suggesting that they have the same defect. The diminished activities of
the mutants could be due to a reduction in receptor presentation to the
cell surface, binding affinity, or G protein coupling. We next
investigate the binding affinities of the wild-type and mutant
receptors using competitive binding assays (Fig. 6 and Table 2).
Consistent with the cAMP and Cytosensor studies, the
Asn72Leu and Ser74Ala mutants were also
defective in binding to the radiolabeled human secretin. The
Kd values were increased (from 0.24 to 2.2 and 1.0
nM, respectively), whereas the binding capacities were
reduced (from 606 to 104 and 107 fmol/106 cells,
respectively). Taken together, all of these results strongly indicate
that this site (position 72–74) is a true glycosylation signal, and
glycosylation at this site is required for the interaction of the
peptide with the receptor.
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), but no
significant change in ED50 values (Table 1). When the
mutant was subjected to competitive binding study, there were no
significant differences between the mutant and the wild-type receptors
in the Kd and binding capacity values. The reason for the
discrepancy between our functional and binding data is unclear at the
moment. It is possible that the augmented cAMP and Cytosensor responses
reflect differences in the levels of receptor expression. Future
experimentation is needed to further investigate this observation. In
contrast, a second mutation of the same glycosylation consensus
sequence (Ser130Ala) led to reduced maximal responses and
increased ED50 and Kd values in the cAMP and
competitive binding assays (Fig. 6 and Table 2). Our results suggest
that the mutation at this position from serine to alanine has a
negative effect on the binding of the ligand with the receptor.
Immunostaining of the wild-type and mutant receptors
The variation in the functional activities of the mutant receptors
may be caused by the changes in membrane trafficking or ligand binding
(24, 25). To distinguish these possibilities, we tagged the
c-Myc epitope to the C-termini of the wild-type and mutant
receptors for immunostaining studies. In the control cAMP assays, the
wild-type and the c-Myc-tagged receptor were found to have
similar responses, indicating that the presence of a c-Myc tail
does not alter receptor function (data not shown). By immunostaining of
the c-Myc tail with the 9E10 mouse monoclonal antibody, the
location of the receptors within the cells was detected. Confocal laser
microscopy clearly showed that both the wild-type and mutant receptors
were delivered and expressed on the cell surface (Fig. 7, A and B), as the predominant
fluorescent signals were detected as a ring-like structure at the
plasma membrane.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine whether glycosylation is necessary for hSR function, we
have used various glycosylation inhibitors to alter the
N-linked glycosylation pattern in the transfected cells.
There are three main types of N-linked glycosylation
patterns: the complex, the hybrid, and the high mannose type. The
glycosylation pattern of a protein can be studied by using inhibitors
that can specifically interfere with the key enzymes or act as
substrate analogs in the glycosylation pathway. Tunicamycin inhibits
all kinds of N-linked glycosylation. Castanospermine
prevents the formation of high mannose oligosaccharides by the
inhibition of glucosidases I and II in the endoplasmic reticulum.
Swainsonine alters the glycosylation pattern of a protein to the hybrid
type by competitive inhibition of the enzyme -mannosidase I.
Deoxymannojirimycin is a mannose analog that can prevent hybrid chain
formation by inhibiting the removal of mannose residues from high
mannose glycans in the Golgi apparatus. Thus, by including these
inhibitors in the culture medium, the glycosylation pattern of hSR
expressed on the cell surface can be altered, and the effects of these
inhibitors on hSR function can then be examined. Our studies showed
that N-linked glycosylation, especially the formation of the
pentasaccharide core and the high mannose side chain, is essential to
hSR function. Our data are consistent with the findings in human VIP1
receptor (40). It was suggested that the folding process of receptor
proteins had been completed after high mannose-type
N-glycosylation, thus allowing the matured receptors to
leave the endoplasmic reticulum (41).
To extend our study on the effect of glycosylation on hSR function, five Asn to Leu mutants and subsequently two Ser to Ala mutants corresponding to the consensus N-linked glycosylation sequence (Asn-X-Ser/Thr, where X can be any amino acid except proline) (42, 43, 44) were constructed. The Asn72Leu and Ser74Ala mutants consistently caused drastic impairment of receptor functions from ligand interaction, to cAMP production, to changes in extracellular acidification rates. Evidently, in addition to secretin receptors (18, 19, 20), the glycosylation consensus at this position is conserved among all members within this receptor family, including human, rat, mouse, and bovine PACAP type 1 receptors (45, 46, 47, 48), human and rat VIP1 receptors (49, 50), and human VIP2 receptor (51). A similar mutagenesis approach in human VIP1 receptor also indicated the functional importance of this glycosylation site. Couvineau et al. (40) found that N-glycosylation at either Asn64 (not conserved in hSR) or Asn69 (corresponding position in hSR is Asn72) is necessary and sufficient to ensure presentation of functional receptors to the cell surface. Taken together, this motif is probably a true glycosylation site for this subfamily of receptors, and glycosylation at this position is essential to the interaction of this family of receptors with their peptide ligands. However, at present, we do not know whether the apparent contribution of the carbohydrate side chain at this position to biological activity is due to its direct involvement in ligand binding. It is equally possible that the effect is secondary, and there is no direct contact between the carbohydrate moieties and human secretin during receptor activation. In other words, the lack of glycosylation at this position could have significantly changed the overall tertiary conformation of the receptor and, hence, altered its interaction with secretin.
One interesting but controversial finding of the present study was that the Asn to Leu mutation at position 128 enhanced receptor function in cAMP and Cytosensor assays. The maximal responses of this mutant in both measurements are significantly higher than those of the wild-type receptor. This enhancement effect is probably due to a postreceptor mechanism, as there is no change in ligand affinity as indicated by the Kd value in the competitive binding studies. By sequence comparison with other family members, this site is conserved with only the rat secretin receptor, suggesting that this effect is secretin receptor specific. In fact, the Asn128Leu mutant is of special interest because it represents the first example in this family of receptors where mutation of a single residue could lead to enhanced receptor function. In the second Ser130Ala mutant corresponding to the same N-glycosylation site, significant decreases in the maximal responses were observed in both cAMP and binding assays. The current work suggests that mutation of amino acid residue at position 128 or 130 may affect G protein coupling or presentation of critical regions of the extracellular portion of this receptor. This can have either a positive or a negative impact, with the elimination of a distinct site of glycosylation either augmenting or reducing the response to secretin stimulation. The present findings open new avenues suggesting that hSR function could be manipulated in either direction through site-specific mutagenesis.
Another aspect of the study was to investigate the functional impact of individual carbohydrate side-chains on cell surface targeting. The wild-type and mutant receptors were visualized by fluorescence laser confocal microscopy. In this study, the fluorescent signal was observed as a ring-like structure on the surface of the cells expressing the wild-type and mutant receptors, indicating that the receptors are properly addressed to the cell surface. In addition, although the morphology and the fluorescent signals of individual cells are highly variable, it was found that average fluorescent signals on the cells expressing the mutant and wild-type receptors were similar. Taken together, our data suggest that mutation of individual N-glycosylation sites of the hSR have little effect on the delivery of the receptor to the plasma membrane. This is consistent with the observation in human VIP1 receptor, in which N-glycosylation at either Asn58 or Asn69 is sufficient to ensure correct delivery to the cell surface (40). It was suggested that transfer of N-linked glycans from the oligosaccharide-lipid donor to the receptor in the endoplasmic reticulum is needed for subsequent transport of the receptor out of the endoplasmic reticulum (40, 52). Data from this experiment support the idea that functional changes in the mutants (position 72, 74, 128, and 130) are unlikely to be due to changes in the maturation or the delivery of the receptors to the cell surface, but are related to changes in the structure of the receptor. These conformational changes may lead to the reduction in binding affinities (mutants Asn72Leu, Ser74Ala, and Ser130Ala) or augmentation of signal transduction mechanisms (mutant Asn128Leu).
In summary, the present study supports the idea that N-glycosylation of peptidergic receptors is required to maintain high affinity binding states, as demonstrated in receptors for VIP1 (40), calcitonin (53), somatostatin (54), and cholecystokinin (55). Our study demonstrates for the first time that N-glycosylation, particularly at positions 72–74, is essential to hSR function. In addition, mutations at position 72, 74, 128, or 130 may affect receptor function by changing the structure of the receptor, rather than by changing the process of receptor delivery to the cell surface.
Received March 22, 1999.
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
, mannose; 
,
N-acetylglucosamine; PP, pyrophosphoryl bridge; Dol,
dolichol.
) and
acidification rate (
, Wild-type receptor; 
, mutant
receptors.
