From the Center for Basic Research in Digestive Diseases, Department of Biochemistry and Molecular Biology, Mayo Clinic and Foundation, Rochester, Minnesota 55905
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ABSTRACT |
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An understanding of the molecular basis of
hormonal activation of receptors provides important insights for drug
design. Toward this end, intrinsic photoaffinity labeling is a powerful
tool to directly identify the ligand-binding domain. We have developed a new radioiodinatable agonist ligand of the secretin receptor that
incorporates a photolabile
p-benzoyl-L-phenylalanine (Bpa) into the
position of Leu22 and have utilized this to identify the
adjacent receptor domain. The rat
[Tyr10,Bpa22]secretin-27 probe was a fully
efficacious agonist, with a potency to stimulate cAMP accumulation by
Chinese hamster ovary SecR cells similar to that of natural secretin
(EC50 = 68 ± 22 pM analogue and 95 ± 25 pM secretin). It bound specifically and with high affinity (Ki = 5.0 ± 1.1 nM) and
covalently labeled the Mr = 57,000-62,000
secretin receptor. Cyanogen bromide cleavage of the receptor yielded a
major labeled fragment of apparent Mr = 19,000 that shifted to Mr = 9,000 after
deglycosylation. This was most consistent with either of two
glycosylated domains within the amino-terminal tail of the receptor.
Immunoprecipitation with antibody directed to epitope tags incorporated
into each of the candidate domains established that the fragment at the
amino terminus of the receptor was the site of labeling. This was
further localized to the amino-terminal 30 residues of the receptor by
additional proteolysis of this fragment with endoproteinase Lys-C. This
provides the first direct demonstration of a contact between a
secretin-like agonist and its receptor and will contribute a useful
constraint to the modeling of this interaction.
The secretin receptor is prototypic of a recently recognized
family (Class II) of guanine nucleotide-binding protein (G
protein)1-coupled receptors
(1). Members of this family are believed to have the
seven-transmembrane segment topology typical of the superfamily, but
they share <12% homology with the extensively studied Class I
receptors in the rhodopsin/ In this work, we attempt to establish an initial constraint that will
contribute to the development of a model for the interaction of
secretin with its receptor. We do this through photoaffinity labeling.
This has the theoretical advantage of directly probing the domain
adjacent to the photolabile residue within the probe after it binds to
the receptor. Using this approach, we have successfully identified two
binding contacts between photolabile analogues of cholecystokinin
and its receptor (11, 12).
In this work, we have developed an analogue of secretin that
incorporates a site for radioiodination and a photolabile residue intrinsic to the pharmacophore for establishment of a covalent bond to
a domain of the receptor adjacent to it as it resides in its binding
site. We have characterized this as a fully efficacious and potent
agonist, likely to reside in the natural secretin-binding site within
the secretin receptor. We have characterized its high affinity,
saturable, and specific binding to the receptor, and we have
demonstrated that it efficiently covalently labels the secretin
receptor in a single distinct domain. This domain was identified as the
amino-terminal 30 residues of the amino-terminal tail of the secretin
receptor. Although it is too early to know how generalizable such a
contact may be within the secretin receptor family, this is likely
since themes for the impact of mutagenesis for various members of this
family have been generally consistent.
Materials--
Wheat germ agglutinin-agarose was from EY
Laboratories, Inc. (San Mateo, CA). Endoproteinase Lys-C and
anti-hemagglutinin (HA) epitope monoclonal antibody were from
Boehringer Mannheim. Iodoacetic acid was from Pierce. Endoglycosidase F
was prepared in our laboratory, as we have reported (13). Other
reagents were analytical grade.
Receptor Preparations--
Chinese hamster ovary (CHO) cell
lines were used as source of receptors for this study. The CHO-SecR
cell line stably expressing the wild-type rat secretin receptor has
been previously established and characterized (14). Two new cell lines
were established for this report. These express secretin receptor
mutants in which the HA epitope (with sequence YPYDVPDYA) was inserted
after residue 36 (SecR-HA37) and after residue 78 (SecR-HA79) of the
wild-type rat secretin receptor. Constructs were prepared by polymerase chain reaction mutagenesis (15) of the wild-type rat secretin receptor
cDNA in the pcDNA3 vector (Invitrogen, Carlsbad, CA) and had
their identities checked by direct dideoxynucleotide chain termination
DNA sequencing (16). These cell lines were established in a similar
manner to the CHO-SecR cell line (14), transfecting non-receptor-bearing CHO-K1 cells (American Type Culture Collection, Rockville, MD), enriching the population of cells by
fluorescence-activated cell sorting, and selecting clonal populations
of cells by a series of limiting dilutions. The CHO-SecR-HA37 and
CHO-SecR-HA79 cell lines were selected based on their expression of a
high density of the relevant secretin receptor constructs and having
clear cAMP responses to secretin stimulation. These cell lines
had their binding and signaling characteristics fully characterized.
Cell lines were cultured at 37 °C in an environment containing 5%
CO2 on Falcon tissue culture plastic ware in Ham's F-12 medium supplemented with 5% Fetal Clone-2 (Hyclone Laboratories, Logan, UT). Cells were passaged twice a week and were lifted
mechanically prior to membrane preparation. Enriched plasma membranes
were prepared from these cell lines, as we previously described
(17).
Peptides--
Rat secretin-27, rat
[Tyr10]secretin-27, and rat
[Tyr10,Bpa22]secretin-27 were synthesized
using solid-phase manual techniques, as we previously reported (12,
14). The t-butoxycarbonylbenzoylphenylalanine residue was
synthesized as per Kauer et al. (18) and was incorporated as
an intact blocked residue into the appropriate position during synthesis. Design of the rat
[Tyr10,Bpa22]secretin-27 probe provided a
site for oxidative radioiodination that is known to be accommodated
without interfering with secretin activity (14) and a site for
cross-linking at a photolabile Bpa incorporated into position 22, where
there is a Phe residue in chicken secretin and where a
para-nitrophenylalanine has been previously successfully
incorporated (14). Peptides were purified to homogeneity by
reversed-phase HPLC. The identities of the peptides were assured by
amino acid analysis and Edman degradation sequencing. The
Tyr10-containing peptides were radioiodinated oxidatively
with Na125I, exposing it to the solid-phase oxidant
N-chlorobenzenesulfonamide (IODO-BEAD, Pierce) for 15 s
and purifying the product by reversed-phase HPLC to yield specific
radioactivities of 2,000 Ci/mmol. The peptide corresponding to the
hemagglutinin epitope (YPYDVPDYA) that was incorporated into the
epitope-tagged secretin receptor constructs was also synthesized using
solid-phase techniques and purified by reversed-phase HPLC (19).
Biological Activity Assay--
The agonist activity of rat
[Tyr10, Bpa22]secretin-27 was studied using
an assay for cAMP in lysates from CHO-SecR cells stimulated with
secretin and secretin analogues that was performed with reagents provided by Diagnostic Products Corp. (Los Angeles, CA). The assay was
performed as we have previously described (20). The same assay was
utilized to determine the signaling characteristics of the two cell
lines established to express HA epitope-tagged secretin receptors. In
brief, cells were stimulated at 37 °C for 30 min, and the reaction
was stopped by adding ice-cold perchloric acid. After adjusting the pH
to 6 with KHCO3, cell lysates were cleared by
centrifugation at 3,000 rpm for 10 min, and the supernatants were used
in the assay. Radioactivity was quantified by scintillation counting in
a Beckman LS6000. All assays were performed in duplicate and repeated
in at least three independent experiments.
Receptor Binding Studies--
Binding of rat
[Tyr10,Bpa22]secretin-27 to secretin
receptors was characterized in a standard assay using cells or
membranes from the CHO-SecR cell line as the source of receptor (7,
14). Membranes (1-10 µg) or 106 cells were incubated
with a constant amount of rat
[125I-Tyr10]secretin-27 (3-5 pM)
and increasing concentrations of nonradioactive secretin analogue (0-1
µM) for 1 h at room temperature in
Krebs-Ringer-HEPES (KRH) medium (25 mM HEPES, pH 7.4, 104 mM NaCl, 5 mM KCl, 1 mM KH2PO4, 1.2 mM MgSO4, 2 mM CaCl2, 1 mM phenylmethylsulfonyl
fluoride, and 0.01% soybean trypsin inhibitor containing 0.2% bovine
serum albumin). Bound and free radioligands were separated using a
Skatron cell harvester with glass-fiber filter mats that had been
soaked in 0.3% Polybrene, and bound radioactivity was quantified in a Photoaffinity Labeling of the Secretin Receptor--
For
covalent labeling, receptor-bearing membranes from the secretin
receptor-bearing cells containing ~100 µg of protein were incubated
with rat
[125I-Tyr10,Bpa22]secretin-27
(0.1-1 nM) in KRH medium in the absence or presence of
concentrations of nonradiolabeled secretin ranging up to 1 µM. Incubations were performed for 1 h at room
temperature. The incubation mixture was frozen and exposed to
photolysis in a Rayonet photochemical reactor (Southern New England
Ultraviolet, Hamden, CT) equipped with 3500-Å lamps for 30 min at
4 °C. Membranes were then pelleted, washed, and exposed to reduction
and alkylation. Reduction was accomplished by suspension of the
membranes in KRH medium containing 5 mM dithiothreitol in a
nitrogen environment at room temperature for 1 h. This was
followed by alkylation with 2 mM iodoacetic acid under the
same conditions for another 1 h. Membrane proteins were then
either directly applied to a 10% SDS-polyacrylamide gel for
electrophoresis (21) or solubilized with 1% Nonidet P-40 in KRH
medium, prior to wheat germ agglutinin-agarose affinity chromatography
and subsequent SDS-polyacrylamide gel electrophoresis.
Deglycosylation--
The affinity-labeled secretin receptor and
relevant receptor fragments were deglycosylated with endoglycosidase F
using techniques previously described (17).
Chemical and Enzymatic Cleavage of the Secretin
Receptor--
Gel-purified, affinity-labeled native and deglycosylated
secretin receptors were digested with cyanogen bromide in 70% formic acid according to the procedure previously described (12).
Endoproteinase Lys-C digestion (20 µg/ml) was performed at 37 °C
for 24 h in 50 mM Tris-HCl, pH 8.0, 10 mM
EDTA, and 0.1% SDS. Receptor fragments were resolved on 10% NuPAGE
gels (Novex, San Diego, CA) with MES running buffer. The apparent
molecular masses of radiolabeled receptor fragments were determined by
interpolation on a plot of the mobility of MultimarkTM
protein standards (Novex) versus the log values of their
apparent masses.
Segment Identification--
The radiolabeled fragments of the
affinity-labeled, epitope-tagged secretin receptors resulting from
cyanogen bromide digestion were unambiguously identified by
immunoprecipitation. For this, fragments were solubilized for 15 min at
room temperature in 200 µl of 0.5% Nonidet P-40 in 50 mM
Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM
MgCl2, 1 mM EDTA, 1 mM
dithiothreitol, 0.1% SDS, 0.2% bovine serum albumin, 1 mM
phenylmethylsulfonyl fluoride, and 0.01% soybean trypsin inhibitor.
Samples were divided in half, with one tube containing competing HA
peptide (25 µM) and all tubes having anti-HA monoclonal
antibody (2 µg). After 2 h of incubation at room temperature with gentle shaking, heat-inactivated, fixed Staphylococcus
aureus (10 µl) cells were added to bind antigen-antibody
complexes. After an additional hour, the adsorbent was pelleted by
centrifugation at 6,000 rpm for 2 min and washed with 1 ml of
solubilization buffer without bovine serum albumin. Proteins bound to
the washed pellet were eluted in SDS-containing sample buffer and
separated by NuPAGE gel electrophoresis.
Statistical Analysis--
All observations were repeated at
least three times in independent experiments and are expressed as the
means ± S.E. Binding curves were analyzed using the LIGAND
program of Munson and Rodbard (22) and were plotted using the nonlinear
regression analysis routine for radioligand binding in the Prism
software package (GraphPAD Software for Science, San Diego, CA).
Probe Characterization--
The rat
[Tyr10,Bpa22]secretin-27 analogue of secretin
was synthesized, purified to homogeneity, and characterized by amino
acid analysis and Edman degradation sequencing (Fig.
1). It was a fully efficacious and potent
agonist at the secretin receptor, as demonstrated by its ability to
stimulate cAMP accumulation in receptor-bearing CHO-SecR cells in a
concentration-dependent manner (Fig.
2). The analogue stimulated cAMP with an
EC50 of 68 ± 22 pM, not statistically different from natural secretin (95 ± 25 pM).
Consistent with this, the analogue bound to the secretin receptor
saturably, specifically, and with high affinity (Fig. 2). Analysis of
the binding data with this analogue best fit a single site with a
Ki of 5.0 ± 1.1 nM. This also was
not different from natural secretin.
Photoaffinity Labeling of the Secretin Receptor--
The
photolabile secretin analogue covalently labeled a plasma membrane
protein from the CHO-SecR cells that migrated on an SDS-polyacrylamide
gel at apparent Mr = 57,000-62,000 (Fig.
3). This labeling was saturable and
competed in a concentration-dependent manner with unlabeled
secretin. Densitometric analysis of three similar experiments is also
shown in Fig. 3. The IC50 for this competition was 6.5 ± 2.4 nM, in the range expected from the binding affinity
given the large amount of radioligand used in these experiments. As
previously reported for the secretin receptor (3), deglycosylation with
endoglycosidase F shifted the migration of the labeled band to
Mr = 42,000 (Fig. 3).
Active Site Identification--
Cyanogen bromide cleavage of the
affinity-labeled secretin receptor was used as a first indication of
the receptor domain being covalently labeled. Shown in Fig.
4 is a graphical representation of the
sites of cyanogen bromide cleavage of this receptor and the
characteristics of the expected fragments. Protein cores of these
fragments range in mass from 1 to 11 kDa, with three of the fragments
also containing potential sites of N-linked glycosylation. Also shown in Fig. 4 is a typical autoradiograph of a 10% NuPAGE gel
used to separate the products of cyanogen bromide cleavage of the
labeled native and deglycosylated receptor. This consistently demonstrated a specifically labeled band migrating at
Mr ~ 19,000 that shifted to
Mr ~ 9,000 after deglycosylation. Given that
the ligand probe has a mass of 3,341 Da, the core protein of the
labeled fragment should be in the 5,700-Da range. With this and the
requisite need for glycosylation, the only candidates represent the
first and third fragments, both within the amino-terminal tail of the secretin receptor. One candidate fragment is at the end of this domain,
and the other is adjacent to the first transmembrane domain. These have
characteristics that are too similar for them to be distinguished by
this simple manipulation.
To determine which of these domains included the site of covalent
attachment, two secretin receptor constructs were prepared that
incorporated HA epitope tags within each of these cyanogen bromide
fragments. Each of these constructs was expressed stably in a CHO cell
line. These were characterized to demonstrate normal secretin binding
and signaling characteristics (Fig. 5).
Each of these epitope-tagged receptor-bearing cell lines was
effectively affinity-labeled with the rat
[125I-Tyr10,Bpa22]secretin-27
probe (Fig. 6), with the labeled
epitope-tagged secretin receptor migrating at a slightly higher
apparent Mr than the wild-type receptor (14).
Both intact epitope-tagged receptor constructs were well recognized by
the anti-HA monoclonal antibody, as demonstrated by their saturable
immunoprecipitation in the absence and presence of competing HA peptide
(Fig. 6). However, after cyanogen bromide cleavage, only the
epitope-tagged first fragment was radioactive, containing the site of
affinity labeling with the radioiodinated probe (Fig. 6). This
immunoprecipitation was also saturable with excess HA peptide (Fig. 6).
This provides definitive identification of the amino-terminal end of
the amino-terminal tail of the secretin receptor as the site of
covalent attachment through Bpa22.
To further localize the site of covalent labeling of the receptor with
this probe, this cyanogen bromide fragment was further cleaved with
endoproteinase Lys-C. As shown in Fig. 7,
this proteolytic enzyme cleaves at the carboxyl-terminal side of Lys
residues, dividing the first cyanogen bromide fragment of the receptor
into a non-glycosylated fragment with a mass of 3,425 Da, a
glycosylated fragment with core protein of 1,808 Da, and two tiny
fragments. A typical autoradiograph of a NuPAGE gel used to separate
various fragments of the affinity-labeled secretin receptor is shown in Fig. 7. As expected, the labeled cyanogen bromide fragment of this
receptor migrated at Mr ~ 19,000 and shifted
to Mr = 9,000 after deglycosylation with
endoglycosidase F. Endoproteinase Lys-C digestion of each of these
fragments shifted the labeled fragment to migrate at
Mr ~ 6,000, with similar migration of both the
native and deglycosylated fragments. Given the mass of the attached
probe and the absence of effect of deglycosylation on electrophoretic migration of the labeled products of endoproteinase Lys-C digestion, the data support the covalent attachment of this probe to the secretin
receptor through the amino-terminal portion of the first fragment.
Further evidence for the identity of the site of labeling as the
amino-terminal domain of the first fragment came from the endoproteinase Lys-C digestion of the intact receptor, again yielding a
labeled band of this same size (Fig. 7).
The superfamily of G protein-coupled receptors is extensive and
diverse, binding and being activated by ligands as different as
photons, odorants, biogenic amines, small peptides, and large glycoproteins. Our understanding of the molecular basis of ligand binding is most advanced for small molecules, such as the biogenic amines, where the binding domain is intramembranous (23, 24). Binding
of peptide ligands appear to be more complex, with determinants closer
to the external face of the bilayer and within external loop domains.
The examples of true rational design of drugs acting at peptide hormone
receptors are limited, and almost all of these are antagonists.
Successful mimicking of natural peptide agonists with small
non-peptidyl compounds is only beginning to be successful (25) and only
by screening large libraries of compounds. The development of such
drugs using structure-based rational design will be markedly advanced
by a better understanding of the molecular basis of ligand binding and
receptor activation.
In this work, we focus on the molecular basis of agonist binding for a
recently recognized group of G protein-coupled receptors, the secretin
receptor family (Class II). Members of this family were first
identified in 1991 (26-28) and were noted to be structurally quite
distinct from the extensively studied rhodopsin/ Indeed, mutagenesis studies by our laboratory (5, 7) and others (6, 8,
9) have suggested that the amino-terminal domain of the secretin
receptor is critical for ligand binding and activation of these
receptors (5-9). This theme has also been consistent for several other
receptors in the secretin receptor family, including the
vasoactive intestinal polypeptide receptor (5, 9), the calcitonin
receptor (34), the parathyroid hormone receptor (35), and the pituitary
adenylate cyclase-activating polypeptide receptor (36, 37).
Because of the indirect nature of most mutagenesis studies, in which a
function is lost or impeded, and the paucity of examples of successful
unambiguous gain-of-function mutagenesis experiments, we have used the
complementary approach of direct photoaffinity labeling of the
ligand-binding domain of the receptor. We hoped that this would provide
an unambiguous constraint to use in the initial modeling of the
secretin-binding domain. This approach is dependent on the spatial
proximity between a photolabile group incorporated into a ligand as it
resides in its binding site and a portion of the receptor molecule. It
is also critical that the photolabile group has appropriate chemical
reactivity to form a covalent bond with the receptor. We chose a
benzoylphenylalanine residue to incorporate into a secretin analogue
because of the favorable characteristics and substantial experience
with this residue (12, 18, 38-40). Furthermore, it was a reasonable
substitute for the Phe residue present in position 22 in chicken
secretin and for the para-nitrophenylalanine residue that
was previously well tolerated in the same position in another secretin
analogue probe (14). The efficiency of covalent attachment of the
benzophenone precursor probe to the secretin receptor was much greater
than that of the nitroaryl probe used previously (14). This increased efficiency was critical to facilitate the mapping of the receptor domain that was labeled.
It is noteworthy that the intrinsic photoaffinity labeling approach
identified the amino-terminal tail of the secretin receptor as the site
of covalent attachment to the probe. This is consistent with the
previous secretin receptor mutagenesis studies (5-9). It is also the
domain labeled in analogous affinity labeling approaches to map the
binding domains of the parathyroid hormone receptor (41, 42) and the
calcitonin receptor (43). Of particular interest, this work
demonstrates proximity between the carboxyl-terminal region of secretin
(and residue 22 in particular) and the amino-terminal end of the
amino-terminal tail of the secretin receptor. This is quite similar to
the very recent localization of the region of the parathyroid hormone
receptor interacting with a photolabile residue in position 23 of a
36-residue analogue of parathyroid hormone (42). This is distinct from
the covalent labeling of a domain of the amino-terminal tail of the
parathyroid hormone receptor that is adjacent to the first
transmembrane domain when using a probe with the site of cross-linking
in position 13 of the probe (41). The domain adjacent to the first
transmembrane domain was also identified as important for secretin
action by mutagenesis (7), although it has not yet been directly
identified in affinity labeling experiments.
The molecular contacts in the amino-terminal tail of the Class II G
protein-coupled receptors that have been gleaned from photoaffinity
labeling studies will be much more meaningful once the disulfide bonds
in this domain are also mapped. In the presence of such constraints and
the extensive insights into the conformation of peptides representing
natural ligands for these receptors (44-46), molecular modeling will
be of great interest and should substantially improve opportunities for
rational structure-based design of drugs acting at this important group
of receptors.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-adrenergic receptor family, and they
lack the signature sequences of this family (2, 3). Secretin family
receptors have long amino-terminal domains incorporating six highly
conserved Cys residues, believed to contribute to disulfide bonds that
help define the family (3, 4). Indeed, this complex domain has been
suggested to play a key role in agonist binding, as suggested by
receptor mutagenesis studies (5-9). Other extracellular loop domains
have also been implicated in complementary roles for agonist binding
and receptor activation (4, 5, 7, 10). Natural ligands for this family
of receptors are all peptides longer than 27 residues, with
structure-activity series suggesting the presence of diffuse
pharmacophoric domains (3). Although this large diffuse pharmacophore
nicely complements the multiple domains predicted to be outside the
membrane bilayer, there is no working model to predict how the two
molecules might interact.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-spectrometer. Nonspecific binding was determined in the presence of
1 µM secretin and represented <20% of total binding.
The same assay was also utilized to characterize the binding activity
of the two cell lines established to express HA epitope-tagged secretin receptors.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Fig. 1.
Chemical characterization of synthetic rat
[Tyr10, Bpa22]secretin-27. Shown is
the A220 nm absorbance profile of the products
of synthesis of this secretin receptor probe, separated by
reversed-phase HPLC. The material eluting in the marked peak was found
to have the expected composition by amino acid analysis and the
appropriate sequence by cycles of Edman degradation.
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Fig. 2.
Binding and biological activity
characteristics of rat
[Tyr10,Bpa22]secretin-27. The left
panel illustrates the abilities of increasing concentrations of
secretin (Sec) and the photolabile secretin analogue to
compete for the binding of rat
[125I-Tyr10]secretin-27 to CHO-SecR cell
membranes. Values are expressed as the means ± S.E. of at least
three independent experiments, with data presented as percentages of
maximal saturable binding. The right panel illustrates the
abilities of increasing concentrations of these peptides to stimulate
cAMP accumulation in CHO-SecR cells. Values are expressed as the
means ± S.E. of at least three independent experiments performed
in duplicate, with data normalized relative to the maximal response to
natural secretin.
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Fig. 3.
Photoaffinity labeling of the secretin
receptor on CHO-SecR cells with rat
[125I-Tyr10,Bpa22]secretin-27.
Shown is a typical autoradiograph of an SDS-polyacrylamide gel used to
separate products of the labeling of receptor-bearing membranes in the
presence of increasing amounts of competing unlabeled secretin
(left panel). The saturably labeled band migrated at
Mr = 57,000-62,000, as previously observed for
the secretin receptor (14). Shown also is the densitometric
quantitation of receptor labeling in the presence of increasing
concentrations of secretin (middle panel). Results reflect
the means ± S.E. of data from three similar competition labeling
experiments. After deglycosylation with endoglycosidase F (Endo
F), the labeled band migrated in the expected position of
Mr = 42,000 (right panel).
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Fig. 4.
Cyanogen bromide cleavage of the
photoaffinity-labeled secretin receptor. Shown on the
left is a diagram of the predicted sites of cyanogen bromide
cleavage of the rat secretin receptor, along with the masses of the
protein cores of the fragments and the consensus sites for possible
glycosylation of the receptor. Shown on the right is a
typical autoradiograph of a 10% NuPAGE gel used to separate the
products of cyanogen bromide cleavage of the native and deglycosylated
secretin receptor that had been labeled with rat
[125I-Tyr10,Bpa22]secretin-27.
Cleavage of the native receptor yielded a labeled fragment migrating at
apparent Mr = 19,000. This shifted to
Mr = 9,000 after deglycosylation. These results
are representative of five similar experiments. The first and third
fragments best match the apparent migration of the labeled fragments
observed, given the mass of the covalently attached probe (3,341 Da)
and the presence of glycosylation.
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Fig. 5.
Characterization of the cell lines expressing
epitope-tagged secretin receptors. The CHO-SecR-HA37 and
CHO-SecR-HA79 cell lines were characterized for secretin binding and
secretin-stimulated cAMP accumulation following the methods described
in the legend Fig. 2. The solid lines represent data for the
epitope-tagged constructs, and the dashed lines represent
data from control experiments with the wild-type receptor. Values are
expressed as the means ± S.E. of at least three independent
experiments.
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Fig. 6.
Immunoprecipitation of cyanogen bromide
fragments of secretin receptor constructs incorporating epitope tags
within the first and third fragments. Shown are the sequences of
the key HA epitope-tagged secretin receptor fragments and
autoradiographs of 10% NuPAGE gels used to separate immunoprecipitated
proteins or peptides. Each immunoprecipitation was attempted with a
similar amount of antibody in the absence and presence of competing HA
peptide. The left panel demonstrates that after
photoaffinity labeling of these receptor constructs and solubilization,
they were both immunoprecipitated by the anti-HA monoclonal antibody
(Ab), with this effectively competed by excess HA peptide.
The right panel shows that only the epitope-tagged first
fragment was radioactive, reflecting the presence of covalently
attached rat
[125I-Tyr10,Bpa22]secretin-27.
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Fig. 7.
Further proteolytic mapping of the site of
photoaffinity labeling of the secretin receptor. Shown are
theoretical sites of endoproteinase Lys-C cleavage of the first
cyanogen bromide fragment of the secretin receptor, the expected masses
of resulting fragments, and a typical autoradiograph of a 10% NuPAGE
gel used to separate various fragments of the affinity-labeled
wild-type secretin receptor. The lanes on the gel represent cyanogen
bromide cleavage of the native and deglycosylated secretin receptor,
the products of endoproteinase Lys-C digestion of each of those bands,
and the product of endoproteinase Lys-C digestion of the intact
secretin receptor. Each of the conditions including endoproteinase
Lys-C digestion resulted in the migration of the labeled band at
Mr ~ 6,000. This is consistent with this
fragment representing the region of the receptor between residues 1 and
30, covalently bound to the probe (3,341 Da). Endo F,
endoglycosidase F.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-adrenergic receptor
family (Class I). The secretin family receptors have <12% sequence
homology to the rhodopsin family and are missing all the signature
sequences of that family. Although the predicted seven-transmembrane
segment topology was felt to be intact in the secretin receptor family,
substantial differences are even predicted to be present in the helical
packing motifs (29). A prominent feature of this family is a moderately
long and complex amino-terminal tail region, incorporating a series of
conserved Cys residues that are predicted to contribute to critical
disulfide bonds (3, 4, 30-33).
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ACKNOWLEDGEMENTS |
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We acknowledge the excellent technical assistance of E. Holicky and the excellent secretarial support of S. Erickson.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant DK46577 and the Fiterman Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
These authors contributed equally to this work.
§ To whom correspondence and reprint requests should be addressed: Center for Basic Research in Digestive Diseases, Guggenheim 17, Mayo Clinic, Rochester, MN 55905. Tel.: 507-284-0680; Fax: 507-284-0762; E-mail: miller{at}mayo.edu.
The abbreviations used are: G protein, guanine nucleotide-binding protein; HA, hemagglutinin; CHO, Chinese hamster ovary; Bpa, p-benzoyl-L-phenylalanine; HPLC, high pressure liquid chromatography; MES, 4-morpholineethanesulfonic acid.
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REFERENCES |
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