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INTRODUCTION |
Guanine nucleotide-binding protein (G
protein)1-coupled receptors
represent a remarkable group of structurally homologous membrane proteins that can bind and be activated by widely diverse ligands. The
molecular details of how ligands as structurally dissimilar as photons,
biogenic amines, peptides, and glycoproteins can elicit similar
conformational changes in the cytosolic face of their receptors (where
G protein-coupling occurs) are far from clear. Our best understanding
of this process relates to the smallest ligands that appear to bind
within the confluence of helices within the lipid bilayer (1), where we
have analogous low resolution crystal structures on which to rely (2,
3). As the ligands get larger and more structurally complex, the
binding domains tend to move toward the extracellular face of the
membrane, with extracellular loop and amino-terminal tail domains
becoming more important (1, 4). These are receptor domains for which we have minimal meaningful structural data.
We have been quite interested in the molecular basis of ligand binding
to the type A cholecystokinin (CCK) receptor (5-10). This receptor is
a member of the class I family of G protein-coupled receptors, along
with rhodopsin and the
-adrenergic receptor (11). CCK occurs as a
series of linear peptides, having lengths ranging from 8 to 58 residues
(12). These all share their carboxyl-terminal domain, with the
carboxyl-terminal heptapeptide-amide representing the minimal region
that has full potency and efficacy for stimulating targets of this
hormone. Two molecular approximations have been experimentally
determined for residues within this region of CCK and residues within
the ligand-binding domain of this receptor (8-10). Extension or
structural modification of the amino terminus of CCK-8 has been well
tolerated, without interfering with receptor binding or activation (13,
14). One provocative report has recently suggested that the naturally
occurring amino-terminal extension that is present in CCK-58 can affect
the conformation of the biologically active carboxyl-terminal
octapeptide and can have a positive effect on the action of this
hormone (15).
With these observations in mind, we initiated the current group of
studies. We were particularly interested in the structural details of
how an amino-terminal extension from the CCK pharmacophore might be
positioned relative to the CCK receptor. This work has given us new
insights into the structure and function of the amino-terminal tail of
this receptor. Unlike affinity labeling through photolabile residues
that are positioned within the pharmacophore of CCK that have yielded
only focused sites of covalent attachment to the receptor (8, 9), the
present work with a photolabile benzophenone residue positioned outside
of this domain has resulted in demonstration of the ability to label
either of two sites in distinct regions of the CCK receptor. This
suggests that this position within the receptor-bound ligand may retain
substantial mobility and not be held tightly in a single position
relative to the receptor.
One of the sites of covalent labeling was in a position in the
amino-terminal tail of the CCK receptor that can be eliminated without
having any negative impact on receptor binding or signaling. The other
site of labeling was within the third extracellular loop domain.
However, by truncation of the region of the receptor amino terminus in
which the first contact was present, this photolabile residue no longer
came in contact with or labeled the CCK receptor. These observations
suggest that the amino-terminal domain of the receptor might provide a
protective cover for the peptide-binding domain within the receptor.
Indeed, when comparing the sensitivity of wild type and truncated
receptor constructs to extracellular protease treatment, we found the
latter to be much more amenable to damage by tryptic protease.
The insights coming from the molecular approximations with a
photolabile residue sited in position 24 of a CCK-like ligand, when
combined with our previous ligand binding and cross-linking data
(8-10), also provided us with the opportunity to propose a distinct
topological model for CCK binding to its receptor, having a
counterclockwise helix bundle topology. This refined molecular model of
the ligand-receptor complex is quite distinct from models recently
proposed in the literature based on less direct receptor mutagenesis
studies (16-18). The current model is fully consistent with all
existing experimental data and will continue to spawn experimentally
testable predictions as it is further refined.
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EXPERIMENTAL PROCEDURES |
Materials--
Synthetic CCK-8 was purchased from Peninsula
Laboratories (Belmont, CA). Fura-2AM was from Molecular Probes (Eugene,
OR); wheat germ agglutinin-agarose was from EY Laboratories (San Mateo, CA); cyanogen bromide (CNBr) and phenylisothiocyanate were from Pierce;
endoproteinase Lys-C and staphylococcal V8 protease (SAP) were from
Roche Molecular Biochemicals; L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin was from Worthington; and N-(2-aminoethyl-1)-3-aminopropyl glass beads were form
Sigma. Endoglycosidase F was prepared in our laboratory as reported
(19). Other reagents were analytical grade.
Preparation of CCK Receptor
Probes--
D-Tyr-Gly-[Nle28,31]CCK-(26-33),
previously characterized to represent a CCK-like agonist ligand, was
prepared as described (20). Des-amino-Tyr-Gly-Bpa-Gly-[Nle28,31]CCK-(26-33)
(Bpa24 analogue) and
D-Tyr-Gly-[Nle28,31,Bpa29]CCK-(26-33)
(Bpa29 analogue) are photolabile and radioiodinatable CCK
receptor ligand probes that have been recently described (9, 21). The
Bpa24 analogue probe has not previously been fully
characterized. It was synthesized by solid-phase techniques and
purified to homogeneity by reversed-phase HPLC (Fig. 1), as we have
previously reported for other CCK analogues (8). The identity of the
product was established by mass spectrometry. Each of the ligands was
radioiodinated using the solid-phase oxidant, IODO-BEADS (Pierce), with
Na125I and was purified to yield a specific radioactivity
of 2000 Ci/mmol using reverse-phase HPLC, in manner analogous to that
described previously (6).
Receptor Preparations--
The Chinese hamster ovary cell line
that was previously prepared to express the rat type A CCK receptor
(CHO-CCKR) (22) was used as a source of wild type receptor for this
work. Cells were grown as a monolayer in flasks containing Ham's F-12
medium supplemented with 5% fetal Clone-2 (HyClone Laboratories,
Logan, UT) in a humidified environment containing 5% carbon dioxide. Cells were harvested with protease-free cell dissociation medium or
mechanically for all described uses. Enriched plasma membranes were
prepared by suspending these cells in 0.3 M sucrose
containing 0.01% soybean trypsin inhibitor and 1 mM
phenylmethylsulfonyl fluoride and sonicating in a Sonifier cell
disrupter (Plainview, NY) at setting 7 for 10 s. The sucrose
concentration was then adjusted to 1.3 M, placed in the
bottom of the tube, and overlayered with 0.3 M sucrose
prior to centrifugation at 225,000 × g for 1 h.
The membrane band at the sucrose interface was then harvested, diluted
with iced-cold water, and pelleted by centrifugation at 225,000 × g for 30 min. Membranes were then resuspended in
Krebs-Ringers/HEPES (KRH) medium containing 25 mM HEPES, pH
7.4, 104 mM NaCl, 5 mM KCl, 2 mM
CaCl2, 1.2 mM MgSO4, 1 mM KH2PO4, 0.01% soybean trypsin inhibitor, and 1 mM phenylmethylsulfonyl fluoride and were
stored at
80 °C until ready for use.
A CCK receptor construct in which the Val residue at position 342 in
the third extracellular loop of the receptor is replaced by Met (V342M)
was previously produced and characterized to bind and signal like wild
type CCK receptor (9). This cell line was cultured as described above
for the CHO-CCKR cell line.
An amino-terminally truncated CCK receptor construct was also prepared.
This represented the elimination of receptor residues 1-30. This was
accomplished using oligonucleotide-directed mutagenesis, with the wild
type rat CCK receptor construct used as template (23). The sequence of
the construct was confirmed by direct DNA sequencing (24). A CHO-K1
cell line stably expressing this construct was produced using the same
methodology previously utilized (22).
Functional Characterization of the CCK Receptor Probe--
The
Bpa24 analogue of CCK was characterized to determine its
ability to bind to and to activate the CCK receptor. Binding to the CCK
receptor was characterized in a standard competition-binding assay,
using conditions that have been previously established (22). This
utilized enriched plasma membranes prepared from the CHO-CCKR cells as
source of receptor (5-10 µg per tube) and the CCK-like radioligand,
125I-D-Tyr-Gly-[Nle28,31]CCK-(26-33)
(1-2 pM) (14). Incubations were carried out at 25 °C
for 60 min, conditions adequate to achieve steady-state binding, in the
absence and presence of varied concentrations of the Bpa24
analogue. Rapid separation of bound from free radioligand was accomplished with a Skatron cell harvester (Molecular Devices, Sunnyvale, CA), using receptor-binding filter mats. Bound radioactivity was quantified with a gamma spectrometer. Data were graphed using Prism
software (GraphPad software, San Diego, CA) and were analyzed using the
nonlinear least squares curve fitting program of Munson and Rodbard
(25), LIGAND.
The agonist activity of the probe was studied using a well
characterized assay for stimulation of intracellular calcium
accumulation in CHO-CCKR cells (22). In this assay, ~2 million
receptor-bearing cells were loaded with 5 µM Fura-2AM in
Ham's F-12 medium for 30 min at 37 °C. They were then washed and
stimulated with varied concentrations of the Bpa24 analogue
at 37 °C, with fluorescence quantified in a PerkinElmer Life
Sciences LS50B luminescence spectrometer. Excitation was performed at
340 and 380 nm, and emissions were determined at 520 nm, with calcium
concentration calculated from the ratios (26). The peak intracellular
calcium transient was utilized to determine the agonist concentration
dependence of this biological response.
Photoaffinity Labeling of the CCK Receptor--
Photoaffinity
labeling of the CCK receptor was performed using 75-100 pM
radioiodinated CCK analogue probes and 50-100 µg of membrane. This
was incubated at 25 °C for 60 min in KRH medium in the absence and
presence of competing unlabeled peptide. Nonspecific binding was
determined in the presence of 1 µM CCK. Photolysis was
performed in a Rayonet model RP-100 apparatus equipped with 3500-Å
lamps at 4 °C for 30 min. Bound and free radioligand were separated
by centrifugation. Membrane proteins were then solubilized with 1%
Nonidet P-40 in KRH medium, prior to wheat germ agglutinin-agarose affinity chromatography and subsequent SDS-polyacrylamide gel electrophoresis using the conditions described by Laemmli (27). After
resolution on SDS-polyacrylamide gel electrophoresis, the affinity
labeled receptor was visualized by autoradiography, eluted, and lyophilized.
Identification of the Domains of CCK Receptor
Labeling--
Affinity labeled, lectin-purified receptor and relevant
receptor fragments were deglycosylated with endoglycosidase F using conditions we described previously (19).
The initial level of identification of the domains of labeling was
performed using CNBr cleavage. This highly efficient cleavage method
theoretically results in 17 receptor fragments having a broad range of
expected masses (see Fig. 4). As we described previously (8), this was
applied to the affinity labeled, gel-purified native and deglycosylated
CCK receptor in 70% formic acid. The resultant fragments were then
resolved on 10% NuPAGE gels (Novex, San Diego, CA) with MES running
buffer and were visualized by autoradiography.
Each of the gel-purified, labeled CNBr-cleaved fragments was further
digested with endoproteinase Lys-C and SAP. Lys-C (200 µg/ml)
digestion was performed at 37 °C for 24 h in 50 mM
Tris-HCl, pH 8.0, 10 mM EDTA, and 0.1% SDS. SAP (500 µg/ml) digestion was performed in 0.1 M phosphate buffer,
pH 7.8, containing 0.1% SDS at 25 °C for 24 h. The products of
digestion were also resolved on 10% NuPAGE gels.
Identification of the Sites of CCK Receptor Labeling--
The
gel-purified receptor fragments were desalted using a
Dispo-BiodialyzerTM (The Nest Group Inc., Southborough, MA) at
25 °C for 48 h prior to HPLC purification. For this, the
samples were injected onto a PerkinElmer Life Sciences/Brownlee
microbore C-18 column (number 219855), at a flow rate of 5 µl/min.
The system was run isocratically at 5% solution B for 50 min, followed
by a linear gradient up to 90% solution B over 320 min (solution A,
0.1% trifluoroacetic acid, and solution B, 0.085% trifluoroacetic acid in acetonitrile). A280 nm was
monitored with UV absorbance detector. Eluate from the microbore column
was collected directly onto a polyvinylidene difluoride membrane, and
autoradiography was used to detect the position of the radioactive
product. This position on the filter was excised and subjected to Edman
degradation sequencing using an Applied Biosystems automated instrument.
The purified products of CNBr digestion of the CCK receptor that were
labeled with the Bpa24 analogue were also manually
sequenced using Edman degradation chemistry, quantifying radioactivity
released in each cycle. For this, purified fragments were coupled to
N-(2-aminoethyl-1)-3-aminopropyl glass beads through the
sulfhydryl side chain of a Cys residue. This was accomplished by
derivatizing the amino groups on the beads with
m-maleimidobenzoyl-N-hydroxysuccinimide ester at
pH 7.0, quenching remaining amino reactivity with Tris, and then adding
a purified, labeled receptor fragment. Cycles of Edman degradation were
repeated manually in a manner that has been described previously in
detail (8).
Characterization of Amino-terminally Truncated CCK Receptor
Construct--
Existing literature (28) has suggested that truncation
of the first 37 residues of the amino-terminal tail of the CCK receptor does not affect CCK binding or biological action at the CCK receptor. We performed binding and biological activity studies with the cell line
expressing our truncated receptor construct (eliminating residues
1-30), using the methodology described above. This construct was also
used as a target of affinity labeling using the Bpa24 and
Bpa29 probes described above.
Protease Resistance of CCK Receptor Constructs--
Truncated
and wild type CCK receptor constructs were studied to determine their
abilities to resist attack by proteases from outside the cell. Trypsin
was used since there are seven tryptic cleavage sites within this
receptor that are theoretically exposed to the extracellular milieu.
Intact receptor-bearing cells were lifted from their culture dishes
using protease-free cell dissociation medium, washed in KRH medium, and
suspended in this medium in the presence or absence of 0.05% trypsin.
This was allowed to react for 20 min at 37 °C. At that time, the
cells were washed with KRH medium and standard CCK radioligand-binding
assays were performed (as described above).
Molecular Modeling--
Three-dimensional models were generated
for the type A CCK receptor using the low resolution rhodopsin
structure (3) as a template. Both clockwise and counterclockwise
seven-helix bundles were generated, and extracellular and cytosolic
loops were added as described previously (29). The ligand,
D-Tyr-Gly-[Nle28,31,Bpa29]CCK-(26-33),
was docked manually in the receptor models, using available
photoaffinity labeling and other ligand binding data as constraints to
facilitate exact placement and orientation. The solution phase
conformation reported for CCK-(26-33) (30) was initially adopted for
the ligand, although this conformation was permitted to relax in
subsequent energy minimization calculations. All manual
three-dimensional model building was performed with the interactive
molecular graphics program, PSSHOW (31). Docked ligand-receptor
complexes were refined with limited energy minimization and low
temperature molecular dynamics calculations using the AMBER 5.0 suite
of programs (32).
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RESULTS |
Characterization of the Amino-terminal CCK Receptor Probe--
The
synthetic Bpa24 analogue of CCK was purified to homogeneity
(Fig. 1) and was analyzed by mass
spectrometry to ensure its identity. This was oxidatively
radioiodinated and purified by reversed-phase HPLC to yield a specific
radioactivity of 2000 Ci/mmol. The probe bound to the CCK
receptor-bearing membranes from CHO-CCKR cells saturably, specifically,
and with high affinity (Ki = 8.9 ± 1.1 nM) (Fig. 2). Nonspecific
binding, as determined in the presence of 1 µM CCK,
represented less than 10% of total binding. This CCK analogue
stimulated an increase in intracellular calcium concentrations in
Fura-2AM-loaded CHO-CCKR cells in a concentration-dependent
manner (Fig. 2). It was similarly efficacious to natural CCK and was
quite potent (EC50 = 0.5 ± 0.04 nM).

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Fig. 1.
Preparation of photoreactive
Bpa24 analogue of CCK. The probe,
desamino-Tyr-Gly-Bpa-Gly-[Nle28,31]CCK-(26-33), was
synthesized and purified to homogeneity, as demonstrated by this
reversed-phase HPLC elution profile.
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Fig. 2.
Binding and biological activity of the
photoreactive Bpa24 analogue of CCK. The probe,
desamino-Tyr-Gly-Bpa-Gly-[Nle28,31]CCK-(26-33),
displaced CCK radioligand binding to CCK receptor-bearing
membranes from CHO-CCKR cells (left) and stimulated an
increase in intracellular calcium in Fura-2AM-loaded CHO-CCKR cells
(right) in a concentration-dependent manner.
Data are expressed as means ± S.E. of values from three
independent experiments. Inset is a typical intracellular
calcium transient response to 100 nM peptide over time in
these cells.
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Photoaffinity Labeling of the CCK Receptor--
The photolabile
CCK probe covalently labeled the CCK receptor, with the product
migrating at Mr = 85,000-95,000 on a 10%
SDS-polyacrylamide gel (Fig. 3). The
labeling of this band was inhibited in a
concentration-dependent manner by competition with
unlabeled CCK, and labeling was absent in nonreceptor bearing CHO cell
membranes. As previously reported for the CCK receptor, deglycosylation
with endoglycosidase F shifted the migration of the labeled band to
Mr = 42,000, the position of migration of the
receptor core protein. Fig. 3 also shows the densitometric quantitation
of the receptor labeling in the presence of increasing concentrations
of CCK, supporting the expected saturability and high affinity
interaction between ligand and receptor.

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Fig. 3.
Photoaffinity labeling of the CCK receptor
with
desamino-Tyr-Gly-Bpa-Gly-[Nle28,31]CCK-(26-33).
Shown on the left is a typical autoradiograph of an
SDS-polyacrylamide gel used to separate products of the labeling of
receptor-bearing membranes in the absence and presence of increasing
amounts of competing unlabeled CCK. The labeled band migrated at the
expected position of Mr = 85,000-95,000 that
shifted to Mr = 42,000 after deglycosylation
with endoglycosidase F. Absence of significant labeling of
nonreceptor-bearing CHO cell membranes is also shown. Shown on the
right is the densitometric analysis of the receptor labeling
in the presence of increasing concentrations of competing CCK in four
independent experiments (means ± S.E.).
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Identification of the Domains of Receptor Labeling--
CNBr
cleavage of the affinity labeled CCK receptor was used as a first
indication of the receptor domains being covalently labeled by the
Bpa24 analogue of CCK. Fig. 4
shows a typical autoradiograph of a NuPAGE gel used to separate the
products of CNBr cleavage of the affinity labeled native and
deglycosylated CCK receptor. Two distinct mixtures of molecular weight
markers were used, due to substantial differences in the migration of
the lower mass markers (migration positions noted). This cleavage of
the intact receptor reproducibly generated labeled fragments migrating
in the range of Mr = 4,000-7,000 (the smaller
fragment) and Mr = 25,000 (the larger fragment).
In addition, as seen in this figure, larger labeled bands representing
products of partial digestion were also sometimes observed. The smaller fragment was always predominant, representing 75 ± 2% of the
labeling.

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Fig. 4.
CNBr cleavage of the photoaffinity labeled
CCK receptor. The left panel represents a diagram of
the rat CCK receptor structure that includes theoretical sites of
cleavage by CNBr, with the masses noted for the generated fragments.
The first 15 residues of the amino terminus of the rat receptor are
shown in parentheses, since this region is not present in
the type A CCK receptor cloned from human or other species. The
numbering scheme used, therefore, reflects the more highly conserved
domains. A typical autoradiograph of a NuPAGE gel used to separate the
products of CNBr cleavage of affinity labeled native and deglycosylated
CCK receptor is shown on the right. CNBr digestion of
affinity labeled CCK receptor molecules generated two distinct
fragments, migrating at apparent positions of Mr = 4,000-7,000 and Mr = 25,000, the former being
predominant. Fragment 4 best matches the Mr = 25,000 fragment, as evidenced by its migration at apparent
Mr = 8,500 after deglycosylation. Fragments 7, 9, 11, and 14 are potential candidates for the
Mr = 4,000-7,000 fragment, based on apparent
size and absence of glycosylation. Two mixtures of molecular weight
markers, MultimarkTM standards (Invitrogen, Carlsbad, CA) and
KaleidoscopeTM polypeptide standards (Bio-Rad), were used to indicate
better the apparent mass of the smaller fragment.
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The cleavage of the deglycosylated CCK receptor always produced only
two labeled bands, migrating at Mr = 4,000-7,000 and Mr = 8,500 (Fig. 4). The former
was shown to come from the band migrating in the same position from the
digestion of the native receptor, thus representing a nonglycosylated
fragment. The latter was shown to come from deglycosylation of the
glycosylated larger products of CNBr digestion of the intact receptor.
The specific covalent labeling of multiple bands was different from our
previous experience with affinity labeling this receptor through
photolabile residues sited within the pharmacophoric domain of the
ligand in which only a single site of labeling was observed (8, 9).
Fig. 4 also shows the structure of the rat CCK receptor with
theoretical sites of cleavage by CNBr and the characteristics of the
expected fragments. The fragment extending from residue 10 to 72 best
matches the Mr = 25,000 fragment, as supported
by its shift in migration after deglycosylation and the sum of the masses of that fragment and the covalently attached probe (1,506 Da).
Fragments 7, 9, 11, and 14 were all potential candidates for
representing the smaller labeled fragment, based on topology, apparent
size, and absence of glycosylation.
The identity of the Mr = 25,000 fragment was
further defined by enzymatic cleavage of the purified fragment. The
receptor fragment extending from residues 10 to 72 contains sites of
N-linked glycosylation and two lysine residues that can be
cleaved by endoproteinase Lys-C (Fig. 5).
One Lys is very close to the carboxyl terminus of this fragment, and
the other is positioned effectively to separate a nonglycosylated
fragment with a mass of 3,771 Da from a glycosylated fragment with core
protein of 3,045 Da. The gel-purified Mr = 25,000 product of CNBr cleavage was further digested with
endoproteinase Lys-C and deglycosylated with endoglycosidase F. Fig. 5
shows a typical autoradiograph of a NuPAGE gel used to separate the products of these treatments. Lys-C cleavage of this fragment yielded a
band migrating at approximate Mr = 21,500 that
shifted to approximate Mr = 5,000 after
deglycosylation. Considering the mass of the covalently attached probe
(1,506 Da), the amino-terminal part of the amino-terminal receptor
fragment, extending from residues 10 to 37, best fit the data. This
region of the CCK receptor is known not to be critical for CCK binding,
based on the ability to truncate the first 37 residues of the
amino-terminal tail of the receptor, while maintaining normal function
(28). For this reason, no further specific localization of this site of
labeling was pursued.

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Fig. 5.
Proteolysis and deglycosylation of the
purified Mr = 25,000 CNBr
fragment. Shown on the left is a diagram of fragment 4 with endoproteinase Lys-C cleavage sites. Shown on the right
is a typical autoradiograph of a NuPAGE gel used to separate the
products of digestion and deglycosylation of this fragment. Lys-C
digestion yielded a band migrating at approximate
Mr = 21,500 that shifted to apparent
Mr = 5,000 after deglycosylation. This localized
one site of labeling to the region between Asn10 and
Lys37, based on the mass of this fragment and that of the
covalently attached probe (1,506 Da) and its glycosylation
status.
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Identification of the smaller labeled fragment
(Mr = 4,000-7,000) was more of a challenge,
since migration on a gel may not precisely reflect mass (particularly
for small peptides, as demonstrated by the differential migration of
the molecular weight standards in the two mixtures). Focus on the
absolute mass of a single standard marker in a previous report (21)
resulted in consideration of too few candidate fragments. Of the
potential candidates for the current labeling (fragments 7, 9, 11, and
14), fragments 7, 9, and 14 contain Lys, Asp, or Glu residues that can
be cleaved by the specific proteases, Lys-C or SAP. Therefore, these
enzymatic reactions were first utilized to gain preliminary insight
into the identity of this fragment. Fig.
6 shows a diagram of these CNBr
fragments, with theoretical cleavage sites by these proteases. As
hoped, both Lys-C and SAP treatment resulted in shifts in the migration
of the labeled band on a gel (Fig. 6). Only fragments 7 and 14 contain
residues that can be cleaved by both of these enzymes, suggesting that
they were the most suitable candidates to represent this smaller
labeled fragment. This fragment was definitively identified as CNBr
fragment 14 by direct Edman degradation sequencing, in which the first
11 cycles matched the expected sequence
(Pro-Ile-Phe-Ser-Ala-Asn-Ala-Trp-Arg-Ala-Tyr).

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Fig. 6.
Proteolysis of the purified
Mr = 4,000-7,000 CNBr fragment.
Shown on the left is a diagram of fragments 7, 9, 11, and 14 with sites of cleavage by endoproteinase Lys-C and SAP. Shown on the
right is a typical autoradiograph of a NuPAGE gel used to
separate the products of proteolysis of the labeled fragment. Treatment
with both Lys-C and SAP resulted in shifts in the electrophoretic
migration of the labeled band, suggesting that fragments 7 and 14 are
the most suitable candidates to represent this smaller labeled
fragment.
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The site of covalent attachment of this probe to this fragment was
further characterized by radiochemical sequencing of the labeled
products of CNBr cleavage of wild type and V342M receptor constructs.
Although no radioactivity above background was observed through 15 cycles for the labeled CNBr fragment of the wild type receptor, a peak
was consistently observed in cycle 3 for this fragment of the V342M
construct in four independent experiments (Fig.
7). This corresponds with covalent
labeling of Glu345 in the third extracellular loop of the
receptor.

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Fig. 7.
Edman degradation sequencing of the affinity
labeled V342M mutant CCK receptor fragment. The affinity labeled
V342M mutant CCK receptor was cleaved with CNBr and purified to
homogeneity before being subjected to manual Edman degradation
sequencing. Cycle three, corresponding to Glu345, was
significantly above background (p < 0.05). Data
represent means ± S.E. of values from four independent
experiments. a.a., amino acid.
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Characterization and Affinity Labeling of the Amino-terminally
Truncated CCK Receptor Construct--
Fig.
8 shows that the amino-terminally
truncated CCK receptor construct recognizes and responds to CCK in a
similar manner to the wild type receptor. This confirms the high
affinity binding of CCK previously reported for another truncated CCK
receptor construct in which residues 1-37 were eliminated (28). It
extends the previous observations by adding insight into normal
signaling by such a construct.

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Fig. 8.
Characterization of the amino-terminally
truncated CCK receptor. Shown are data for CCK binding
(left) and biological activity (right) at the
truncated CCK receptor. This receptor construct bound CCK and signaled
in a concentration-dependent manner, similar to wild type
(WT) receptor. Data represent means ± S.E. of values
from three independent experiments. Inset is a typical
intracellular calcium transient response to 10 nM CCK in
cells expressing the truncated receptor.
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Fig. 9 shows the attempt to affinity
label the truncated CCK receptor construct with the Bpa24
analogue of CCK. Of note, despite high affinity binding, this probe did
not covalently label this receptor construct. Even prolonged exposure
of the gel for autoradiography failed to demonstrate a labeled receptor
band. This means that both sites of covalent attachment were impacted
by the absence of the receptor amino-terminal domain. As a control,
another analogue of CCK that incorporated the same photolabile residue
within the pharmacophoric domain was also used in an analogous series
of affinity labeling experiments. This probe, the Bpa29
analogue of CCK (9), efficiently labeled this receptor construct.

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Fig. 9.
Affinity labeling of the truncated CCK
receptor with Bpa24 and Bpa29 probes.
Shown is a typical autoradiograph of an SDS-polyacrylamide gel used to
separate products of the labeling of the truncated receptor-bearing
membranes using Bpa24 and Bpa29 probes in the
absence and presence of competing unlabeled CCK (1 µM)
(representative of three similar experiments). The Bpa24
probe failed to label covalently the truncated receptor, whereas the
Bpa29 probe efficiently labeled this preparation.
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Protease Resistance of CCK Receptor Constructs--
Exposure to
trypsin of cells expressing similar densities of wild type and
truncated CCK receptor constructs that had similar CCK-binding
capacities resulted in substantial differences. Despite having multiple
sites in the extracellular domain for digestion with this protease,
only the truncated construct was negatively affected by this treatment,
with its binding markedly inhibited (Fig.
10). The glycosylated amino terminus
was able to provide a protective cover for the peptide-binding domain
of the fully intact wild type CCK receptor.

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Fig. 10.
Protease resistance of CCK receptor
constructs. Shown are CCK competition-binding curves for CHO cell
lines expressing wild type (WT) and truncated CCK receptor
constructs that had been treated with trypsin. Cells were exposed to
trypsin digestion for 20 min at 37 °C, followed by extensive washing
and standard CCK radioligand binding assay. The binding capacity of the
truncated receptor was significantly negatively impacted by trypsin
treatment, whereas the wild type receptor was resistant to this
treatment. Values are expressed as the means ± S.E. of data from
three independent experiments.
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Molecular Modeling--
Based on results from our earlier
photoaffinity labeling studies (8-10), we initially positioned the
peptide ligand such that a photolabile residue (Bpa) at position 29 would be well situated to form covalent cross-links with receptor
residues His347 and Leu348 in the third
extracellular loop, whereas a photolabile residue at position 33 would
form cross-links with Trp39 in the amino terminus of the
receptor. Since structure-activity relationship data for CCK peptides
indicate that residues positioned as an extension from the amino
terminus of the pharmacophoric domain (residues Tyr24 and
Gly25) have no impact on receptor binding (5, 20, 33), we
oriented the peptide ligand so that its amino terminus made no specific contacts with the receptor but was instead exposed to solvent. Given
that the amino terminus and extracellular loops of the receptor likely
possess some conformational flexibility, then it is quite plausible
that a photoaffinity label substituted in the position of
Tyr24 (shown in Fig. 11)
would alkylate the amino terminus and possibly the third extracellular
loop of the receptor.

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Fig. 11.
Molecular model of peptide-receptor
complex. Shown are stereoscopic pairs of images of the agonist
peptide ligand,
D-Tyr-Gly-[Nle28,31,Bpa29]CCK-(26-33),
bound to the CCK receptor, as seen from the side and top. The CCK
receptor backbone is shown in cyan, and the peptide ligand
backbone is highlighted in yellow. Key peptide residue side
chains are displayed and labeled, including Tyr24,
Tyr27(SO4), Bpa29, and
Phe33. Sites of receptor photoaffinity labeling are
displayed in red, and a putative interaction between ligand
residue, Tyr27(SO4), and receptor residue,
Arg197, is illustrated in white.
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The experimental results reported here are completely consistent with
our models for the CCK peptide-receptor complex. The specific
photoaffinity labeling patterns observed in this study (i.e.
labeling of Glu345 in the third extracellular loop plus
residue(s) in the amino terminus), together with results from previous
ligand binding and photoaffinity labeling studies, allow us for the
first time to propose a specific helix bundle topology for the CCK
receptor. If we assume that the bound conformation of the CCK peptide
ligand is not dramatically different from the solution conformation for CCK-(26-33) (30), then a counterclockwise helix bundle arrangement for
the CCK receptor best accommodates the available ligand binding data
and the full set of covalent cross-links we observe in our photoaffinity labeling experiments.
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DISCUSSION |
G protein-coupled receptors can provide pockets for binding small
ligands within the confluence of their seven intramembranous helical
segments and for binding large glycoprotein ligands in specialized
structural domains configured within large amino-terminal domains (1,
4). These receptors can also bind and be activated by peptide ligands,
such as CCK. The emerging themes for such binding suggest critical
contributions by extracellular loop and amino-terminal tail domains
that are near the external face of the membrane, as well as including
examples of portions of ligands dipping down into the confluence of helices.
Our current understanding of the molecular basis of CCK binding is
based on receptor mutagenesis and affinity labeling studies. Both
support the importance for peptide binding of regions just outside of
the first transmembrane segment and in the extracellular loop domains
(8, 9, 16). There has, however, been substantial controversy related to
the details of CCK peptide docking to these domains. Very distinct
models of the peptide-occupied receptor have been proposed (8, 16, 17).
Nonpeptidyl ligand binding to this receptor seems to occur deeper in
the membrane within the confluence of helices (34).
Established structure-activity relationships for CCK have localized the
pharmacophoric domain to the carboxyl-terminal heptapeptide-amide (12,
13). Almost every residue within this domain makes an important
contribution to binding and activity. In contrast, almost any
modification to the amino terminus of CCK-8 that has been attempted has
been well tolerated, without modifying receptor binding or signaling.
It was this feature that encouraged our positioning of the amino
terminus of CCK-8 above the ligand-binding domain of the CCK receptor
in our evolving model, such that an extension would not make contact
with the regions of this receptor that are known to be important for
function (8, 10). It is noteworthy that another molecular model has
placed the amino terminus of CCK much closer to the membrane and
directed toward the other side of the helical bundle (16, 17). Such a
model is inconsistent with the residue-residue approximations that have
been directly established by photoaffinity labeling studies (8,
10).
To date, there has been the successful photoaffinity labeling of
distinct spatially approximated residues within the CCK receptor through two positions within the pharmacophoric domain of CCK (8-10).
This has been accomplished with two different photoreactive moieties in
position 33 of CCK (8, 10) and with a benzophenone moiety in position
29 of CCK (9). The position 33 photoprobes covalently labeled receptor
residue Trp39 just above the first transmembrane segment.
The position 29 photoprobe established a covalent bond with receptor
residues His347 and Leu348 just above the
seventh transmembrane segment. It is noteworthy that both of these
represented focused contacts with a single receptor domain, as might be
expected from the high affinity tight interaction between native
agonist ligand and the ligand-binding domain of the receptor.
In contrast, in the present work, the Bpa residue in position 24 of CCK
established covalent bonds to either of two distinct domains of the CCK
receptor. This probe was fully characterized as a high affinity ligand
that had full efficacy relative to natural CCK. While one of the
covalently labeled receptor domains was within a region that is known
to be important, the third extracellular loop, it is notable that this
contact was lost when the amino terminus of the receptor was shortened
by truncation. The second labeled domain was a portion of the
amino-terminal tail of the CCK receptor that can be eliminated by
truncation, without any detrimental effects on CCK binding or
agonist-stimulated signaling. This contact is therefore not critical
for ligand affinity or function.
These spatial approximations, however, provide the basis to postulate
that the docked peptide is tucked under the protective cover of the
amino terminus of the CCK receptor. This function was further supported
by the demonstration that this domain protected the receptor from
extracellular proteolytic attack. Eliminating this region of the
receptor by truncation had no detrimental effect on ligand binding
affinity or agonist-stimulated signaling, but resulted in a more labile
receptor that was much more sensitive to proteolysis. The glycosylation
of the amino-terminal domain of the CCK receptor is probably most
responsible for this resistance to proteolysis. This is a recognized
function of the glycosylation of membrane proteins (35, 36). Another
established function for membrane protein glycosylation relates
to assisting in solubility and folding during biosynthesis that is also
likely relevant to the CCK receptor.
Although position 24 in the CCK ligands is external to the established
pharmacophore, cross-linking data generated by substitution of a
photolabile residue at this position provides extremely useful constraint data for three-dimensional model building studies. The
results reported here provide clear evidence that our earlier decision
to position the amino terminus of the ligand facing away from receptor
binding site (8, 10) was appropriate. The photoaffinity labeling data
for the ligand amino terminus, combined with ligand binding studies and
cross-linking data for positions within the peptide pharmacophore, also
enable us for the first time to propose a distinct topological model
for the peptide ligand-CCK receptor complex. Based on the full set of
photoaffinity labeling and ligand binding data now available, it
appears likely that the CCK receptor possesses a counterclockwise helix
bundle topology. More definitive biophysical data will be needed to
ultimately verify this prediction.
With this refinement in our working model, the tyrosine-sulfate residue
in position 27 of CCK that has been shown to be so important in
structure-activity studies (37) is now in direct contact with
Arg197. These residues, therefore, represent potential
partners for charge-charge interaction. Indeed, Arg197 is
one of only three basic residues in the extracellular domain of the CCK
receptor that has been reported to have a marked negative impact on CCK
binding when replaced with an Ala residue (38).
Thus, using an analogue of CCK with a photolabile Bpa moiety in
position 24, we have learned much about the binding domain for this
hormone within its receptor. While the pharmacophoric domain of the
peptide is held in constant approximation with the regions of the CCK
receptor close to the membrane, the amino-terminal extension from the
pharmacophore likely moves further away from the bilayer and is
overlaid by a region of the receptor amino terminus that has no direct
effect on binding affinity or on biological activity. Instead, this
region of the receptor serves a protective function over the more
critical domain below. The distinct positions of covalent labeling with
this probe also provide additional detail to refine the molecular model
of the docked peptide agonist. This evolving model now best supports a
counterclockwise helical bundle topology for the receptor and residue
approximations that are fully consistent with all existing experimental data.