(Received for publication, October 29, 1996, and in revised form, January 15, 1997)
From the CURE/Digestive Diseases Research Center, Cholecystokinin (CCK)-A and CCK-B receptors are
highly homologous members of the seven transmembrane domain
G-protein-coupled receptor superfamily. Genes of both receptors contain
five exons and share a similar exon-intron organization. To determine
the structural basis of CCK-A receptor (CCK-AR) functionally coupled to
Gs, a series of chimeric mutants were constructed by
replacing exons of human CCK-B receptor (CCK-BR), from the second to
the fifth (last) exon, with human CCK-AR counterparts. Binding and signal transduction properties of wild-type and chimeric receptors were
examined in stably transfected HEK-293 cells. Chimeric receptors that
maintained high affinity binding to CCK exhibited
dose-dependent increases in intracellular calcium
mobilization similar to both wild-type receptors. However, only the
wild-type CCK-AR and chimeric mutants containing the second exon of
CCK-AR were able to mediate significantly greater increases in
intracellular cAMP content and adenylyl cyclase activity compared with
wild-type CCK-BR. A CCK-BR mutant was further constructed by replacing
five amino acids, Gly-Leu-Ser-Arg-(Arg)-Leu, in the first intracellular
loop with the corresponding five CCK-AR specific amino acids,
Ile-Arg-Asn-Lys-(Arg)-Met. The resultant receptor maintained high
affinity binding to both CCK and gastrin and dose-dependent
calcium responses similar to wild-type CCK-BR. However, this first
intracellular loop mutant also gained positive cAMP responses to both
sulfated CCK-8 and gastrin-17 with EC50 values of 8.5 ± 1 nM and 23 ± 7 nM, respectively. These data suggest that the first intracellular loop of CCK-AR is
essential for coupling to Gs and activation of adenylyl
cyclase signal transduction cascade.
Peptides in the cholecystokinin (CCK)1
family have a variety of biological functions in the central and
peripheral nervous systems as well as in the gastrointestinal tract
(1). Most of these activities are mediated by two distinct membrane
receptors that were initially defined by their binding specificity to
CCK and gastrin. Molecular cloning of CCK-AR and CCK-BR cDNAs
(2-6) and genes (7-10) identified that CCK-AR and CCK-BR are
structurally homologous members of the seven transmembrane domain
G-protein-coupled receptor superfamily. A comparison based on
previously described gene structures of CCK-BR (7-8) and CCK-AR
(9-10), as well as our own genomic DNA PCR analysis of human CCK-AR,
indicate that both receptor subtypes contain five exons and share very
similar exon-intron organization.
The molecular basis of CCK receptor interactions with peptide agonists
and non-peptide antagonists has been examined in cells transfected with
the two CCK receptor subtypes (11-14). Previous mutagenesis studies of
interaction between CCK ligand and receptors demonstrated that a Val to
Leu substitution in the sixth transmembrane domain of the canine
gastrin/CCK-B receptor reverses the rank-affinity order of two
non-peptide antagonists L-364,718 and L-365,260 (11). In the rat
CCK-AR, six amino acids in the seventh transmembrane domain may
contribute to high affinity binding to CCK-AR preferring antagonist
L-364,718 (12). Furthermore, at least eight amino acid residues
scattered throughout all seven transmembrane domains of CCK-BR are
crucial for binding of the CCK-BR preferring antagonist L-365,260 (13).
Recently, a sequence of five amino acids in the second extracellular
loop of rat CCK-BR has been identified to be important for gastrin
selectivity (14). These studies mainly described the interaction
between CCK ligand and receptors but did not address the specific
receptor domains involved in G-protein coupling.
Although both receptors appear to stimulate intracellular calcium
mobilization through Gq coupling (15), CCK-AR also exhibits selective stimulation of cAMP through Gs coupling (16). To
gain further insight into structure-function relationships of CCK
receptors in intracellular signaling and, particularly, to define
CCK-AR sequences essential for Gs coupling, we constructed
a series of CCK receptor mutants and examined their in vitro
binding and biological functions. Our strategy of mutagenesis was as
follows. First, we exchanged exons between the CCK-BR and CCK-AR,
utilizing their homologous splice sites to construct a series of
functional CCK receptor chimeras. Based on the preliminary findings
that the second exon, which encodes from the end of the N-terminal
extracellular domain to the beginning of the third transmembrane
domain, of CCK-AR was associated with positive cAMP responses in these
mutants, we further replaced CCK-BR with the entire second exon, or a
segment encoding the first intracellular loop of CCK-AR, to generate
domain-specific mutants. The present study describes the changes of
binding and second messenger signaling properties of these chimeric
mutants in comparison with wild-type CCK receptors in stably
transfected human embryonic kidney fibroblast cells (HEK-293). Human
CCK-BR mutants containing the CCK-AR second exon, or more specifically, the first intracellular loop, exhibited significantly increased cAMP
responses to both CCK and gastrin compared with the transfected wild-type CCK-BR in HEK-293 cells. These data suggest that critical residues located in the first intracellular loop of CCK-AR are required
for functional Gs coupling with the receptor.
Human CCK-AR
cDNA was a gift from Dr. S. A. Wank, National Institutes of Health
(Bethesda, MD). Human CCK-BR cDNA was isolated from a human fetal
brain cDNA library (Clontech, Palo Alto, CA) as described
previously (17). Chimeric CCK receptors containing different
combinations of exons from CCK-BR and CCK-AR were constructed by
overlap extension PCR according to the procedure described by Horton
et al. (18). Primers were designed based on the conserved junction regions between CCK-AR and CCK-BR, and Pfu DNA
polymerase was used in all PCR reactions (Stratagene, La Jolla, CA).
For each CCK receptor mutant, first PCR was performed with wild-type N-
or C-terminal primers with their respective junction primers using
appropriate receptor template to generate two intended PCR fragments;
and in second PCR, these two fragments were annealed and amplified by
adding the appropriate wild-type N- and C-terminal primers only. All
wild-type and mutant receptors were cloned into pCR-Script SK(+)
(Stratagene), and their DNA sequences were confirmed (Sequenase 2.0, Amersham Corp.) before subcloned into a mammalian expression vector
pcDNA3 (Invitrogen, San Diego, CA).
HEK-293 cells (ATCC CRL 1573) were maintained in
Dulbecco's modified Eagle medium/nutrient mixture F-12 (DMEM/F-12,
1:1) supplemented with 10% fetal bovine serum at 37 °C in a
humidified atmosphere of 5% CO2. Cells grown to 60-70%
confluence in 100-mm dishes were incubated with 5-10 µg of plasmid
DNA in lipofectamine mixture for 4-6 h in serum-free medium (Opti-MEM,
Life Technologies Inc., Gaithersburg, MD). Cells were cultured for an
additional 48 h in serum containing DMEM/F-12 medium before adding
G418 (500 µg/ml Geneticin, Life Technologies Inc.). G418-resistant
colonies were first screened by radiolabeled CCK binding, and positive
cells were subsequently isolated from a single cell by limited
dilution. From each transfection experiment, at least 10 positive
clones were developed and 1-2 representative cell lines were further characterized.
Stable cell lines that
express transfected CCK receptors were cultured in
poly-D-lysine coated 24-well dishes. All binding experiments were performed in 1 × Hanks' balanced salt solution supplemented with 20 mM HEPES, pH 7.4, 0.1% bacitracin,
0.2% bovine serum albumin. Competition of radioligand binding was
performed with 40 pM of 125I-labeled
Bolton-Hunter-CCK-8 (2000 Ci/mmol, Amersham Corp.) in the presence of
unlabeled peptide at the indicated concentrations. At the end of the
incubation period, cells were washed twice with 2 ml of ice-cold PBS
and solubilized with 1 ml of 1% Triton X-100 in PBS. Bound and free
radioactivity were counted, and values were subjected to data
analysis.
Cells were cultured
for 48 h on 20-mm glass coverslips precoated with
poly-D-lysine. Cells were pre-incubated with 5 µM Ca2+ indicator dye, Fura-2/AM (Molecular
Probes, Eugene, OR), for 30 min at 37 °C. Coverslips were then
mounted in a perfusion chamber with 0.9 ml of Hanks' balanced salt
solution containing 20 mM Hepes, pH 7.4. To each disc, 0.1 ml CCK-8 s was added to produce final concentrations from 1 pM to 1 µM. A video imaging workstation consisting of a Zeiss 100TV inverted microscope with a × 40 objective and a computerized videomicroscopy system (Attofluor Digital
Imaging System, Atto Instruments, Rockville, MD).
Ca2+-dependent fluorescent signals were
obtained by exciting Fura-2 at 340 and 380 nm. The indicator was
calibrated before the measurements with saturated and
Ca2+-free solutions, and accuracy was controlled with
standard solutions of known Ca2+ concentrations. Basal
calcium concentrations were recorded before the addition of peptides,
and the difference between the first predominant peak (after
stimulation) and the basal values ( Cells
were grown to densities of 2 × 106 cells/well in
six-well plates or of 5 × 106 cells in 100-mm dishes.
For intracellular cAMP measurement, cells were rinsed with serum-free
DMEM/F-12 medium and incubated with 10 pM to 1 µM CCK-8 s in the presence of 1 mM IBMX for
15 min at 37 °C. The treatment was stopped by the addition of 65%
ice-cold ethanol, and cell extracts were harvested. The cell extracts
were centrifuged at 2000 × g for 15 min at 4 °C,
and the supernatants were collected and concentrated. The extract
concentrates were dissolved in assay buffer (106 cells/100
µl) and measured for cAMP content from 10- to 2000-fold dilution. A
non-acylation protocol was performed with an RIA kit (Amersham
Corp.).
Adenylyl cyclase activity was measured using a modification of the
method described by Bockaert et al. (19). Cells grown on
100-mm plates to 80-90% confluence were rinsed and scraped off the
plates in ice-cold PBS. After centrifugation, the cell pellet was
resuspended in 0.5 ml homogenization buffer (20 mM HEPES,
pH 7.8, 1 mM EDTA, 27% sucrose), and homogenates were
prepared using 10 strokes in tight-fitting Dounce homogenizer. 10-µl
aliquots of homogenates (1 µg/µl) were added to a reaction mixture
containing 25 mM Tris-Cl, pH 8.0, 2.68 mM
MgCl2, 1 mM EDTA, 1 mM cAMP, 1 mM IBMX, 100 µM ATP, 25 µM GTP,
20 µM creatine phosphate, 400 units/ml creatine kinase,
400 units/ml myokinase, 0.2% bovine serum albumin, 1 × 104 cpm [3H]-cAMP, and 2×106 cpm
[ Dose response curves and kinetic binding data
were analyzed using GraphPad Prism nonlinear regression software
programs (GraphPad, San Diego, CA). Significance was determined using
the Student's unpaired t test with p < 0.05 (Statistix, NH Analytical, Roseville, MN). When more than two
groups were compared, significance was determined by one-way analysis
of variance followed by Tukey-Kramer posttest comparisons.
Human CCK-AR and CCK-BR genes contain five exons
and share similar exon-intron organization (Fig.
1A). A sequence homology analysis indicates
that the highest homology exists between the exons of two CCK receptor
subtypes. To determine receptor domains that are critical for G-protein
coupling and signaling, a series of six chimeric receptors were
constructed by substituting exons of CCK-BR with their CCK-AR
counterparts except a CCK-B receptor mutant, BAICL-1, which
was constructed by replacing five residues in the first intracellular
loop of the CCK-BR,
Gly80-Leu81-Ser82-Arg83-(Arg)-Leu85, with the corresponding amino acids of the CCK-AR,
Ile-Arg-Asn-Lys-(Arg)-Met (Fig. 1B). All six receptor
mutants were transfected into HEK-293 cells and selected based on
positive binding with radiolabeled CCK-8 s. Representative cell lines
derived from stable clones were further characterized. Wild-type CCK-AR
and CCK-BR or nontransfected HEK-293 cells were used as controls.
Scatchard analysis indicated that the
wild-type and chimeric receptors showed high affinity binding to
CCK-8 s with Kd values between 0.1-10
nM based on a single site model (Table I). Bmax for most receptors ranged from 2-6 × 105 sites/cell, except for wild-type CCK-BR which was
expressed at a significantly higher level (1.6 ± 0.3 × 106 sites/cell), and for
CCK-B1-2A3-5, which was expressed at a much
lower level (4 ± 1 × 104 sites/cell from three
separate transfection experiments) (Table I). Nontransfected HEK-293
cells exhibited no significant binding to radiolabeled CCK-8 s and
thus precluded the estimation of endogenous CCK receptors. Competitive
binding was studied to determine agonist affinity and specificity to
wild-type and chimeric receptors (Table I). Binding of radiolabeled
CCK-8 s to wild-type CCK-AR could be displaced competitively by
CCK-8 s but not by gastrin-17, whereas binding to wild-type CCK-BR was
displaced by both CCK-8 s and gastrin-17 with high affinities. Three
of the chimeric receptors, CCK-B1-4A5,
B1-3A4-5, and BAICL-1 retained
high affinity binding to both CCK-8 s and gastrin-17 and thus mimicked wild-type CCK-BR binding. However, two mutants with CCK-AR exon 2 replacement, CCK-B1A2-5 and
B1A2B3-5, lost high affinity
binding to gastrin-17 and thus resembled wild-type CCK-AR (Table
I).
Binding properties of human CCK wild-type and chimeric receptors
expressed in HEK-293 cells
125I-labeled Bolton-Hunter-CCK-8 s was used as radioligand to
perform binding and competition with increasing concentrations of CCK-8
s or gastrin-17. Values of IC50 are presented in nM
as the mean ± S.E. from three to six experiments.
Bmax was calculated by Scatchard analysis of
representative clones of wild-type (WT) and chimeric receptors (numbers
in subscript indicate specific exons contributed by CCK-BR or CCK-AR).
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
Construction of CCK Chimeric Receptors
[Ca2+]i) was calculated to represent values
for intracellular calcium increase. Twelve or more cells were selected
for imaging at each measurement, and at least three experiments were
performed for each receptor.
-32P]ATP, plus the given concentration of CCK in a
total volume of 50 µl. After 10 min at 32 °C, the reactions were
stopped by the addition of 100 µl of stop solution (40 mM
ATP, 10 mM cAMP, and 1% SDS), and labeled cAMP was
purified by sequential column chromatography over Dowex and Alumina
columns. All determinations were carried out in triplicate, and the
amount of [
-32P]cAMP synthesized was corrected for
overall recovery by comparing with the yield of [3H]cAMP.
Overall recoveries were typically 70-75%.
Characterization of CCK Wild-type and Chimeric Receptors Expressed
in HEK-293 Cells
Fig. 1.
Strategy to construct human CCK receptor
chimeric mutants. A, schematic representation of human
CCK-AR and CCK-BR genes. Organization of human CCK-BR gene structure is
adopted from Song et al. (7) and Miyake (8) and human CCK-AR
from Miller et al. (10) and Wank (20) (V. Wu, unpublished
data). Upper and lower numbers
indicate estimated base pairs in each exon and intron, respectively. An apparent size discrepancy in intron 1 of human CCK-BR
(7, 8) and similarly in intron 2 of human CCK-AR (10, 20) were
observed. Note that intron 1 for human CCK-BR was reported to be 1177 bp (7) or > 10 kilobases (8), and intron 2 for human CCK-AR was
estimated to be 650 bp (10) or 2800 bp (20) and 3200 bp by our own
genomic PCR analysis (data not shown). B, human chimeric
receptor mutants generated by exon replacement. Numbers in subscript of
each receptor indicate the contribution of specific exons from CCK-BR
and CCK-AR. Mutant CCK-BAICL-1 was generated from wild-type
CCK-BR by substitution of the five unique first intracellular loop
amino acids of CCK-AR. CCK-BR components were represented by
solid shading and CCK-AR components by light
shading with arrowheads indicating exon-intron splice
sites.
[View Larger Version of this Image (30K GIF file)]
Receptor
Kd,
nM
IC50,
nM
Bmax (No. × 105
sites/cell)
CCK-8 s
Gastrin-17
B1-5
(WT)
1.1 ± 0.2
3.2 ± 0.3
16.0
± 0.3
B1-4A5
2.5 ± 0.5
6.9
± 0.7
1.3 ± 0.2
B1-3A4-5
0.3
± 0.1
0.7 ± 0.3
2.4 ± 0.3
B1-2A3-5
0.15 ± 0.04
0.4
± 0.1
0.4 ± 0.1
B1A2-5
7.6
± 1.5
>1000
6.1 ± 0.5
A1-5 (WT)
1.3
± 0.2
>1000
4.4 ± 0.8
B1A2B3-5
7.2 ± 0.3
238
± 43
2.1 ± 0.3
BAICL-1
8.2 ± 0.5
17.5
± 2
4.2 ± 0.7
HEK-293
NSBa
NSB
NAb
a
NSB, no significant binding.
b
NA, not applicable.
To demonstrate that all six mutant
receptors were capable of transducing calcium signal in response to
CCK, intracellular calcium mobilizations were studied by single cell
imaging in cell lines expressing wild-type and chimeric CCK receptors.
Differences between the basal and the peak level of Ca2+
([Ca2+]i) in responsive cells were obtained,
and the average increase by a given dose was calculated from at least
12 cells. Typical transient increases of calcium levels were observed
within 1-2 min following stimulation with 1 µM CCK-8 s
in all wild-type and chimeric receptors. Although the maximal increases
of calcium stimulated by CCK-8 s were varied among different cell
lines from 300-900 nM, when normalized by their basal
levels ranging from 10-40 nM, the average increases were
between 30-40-fold (Table II). Further studies using
different concentrations of CCK-8 s confirmed the
dose-dependent effect on calcium mobilization in wild-types
as well as in the chimeric receptors with EC50 values ranging from 0.1 to 6 nM (Fig. 2, Table
II).
|
Intracellular cAMP Accumulation
To determine that any of the
chimeric mutants were capable of transducing cAMP signal as wild-type
CCK-AR, intracellular cAMP was measured in the wild-type and chimeric
receptor cell lines following stimulation with CCK-8 s in the presence
of IBMX. At the maximal dose of 1 µM, CCK-8 s caused
profound increases of cAMP from basal levels of 17 ± 1 pmol to
1.225 ± 0.11 nmol in wild-type CCK-AR, 24 ± 4 pmol to
1.130 ± 0.083 nmol in B1A2-5, 17 ± 2 to 0.328 ± 0.048 nmol in
B1A2B3-5, and 32 ± 5 pmol to
0.578 ± 0.075 nmol in BAICL-1 in 15 min/106 cells (Table II). In contrast, 1 µM
CCK-8 s stimulated only a small but significant increase in wild-type
CCK-BR from 26 ± 3 to 46 ± 12 pmol/15 min/106
cells while it caused no significant changes of cAMP levels in any
other chimeric receptor cell lines (Table II).
Dose-dependent increases in cAMP levels were subsequently
measured in wild-type CCK-AR and in three responsive chimeras. Fig.
3A shows the dose-dependent effect of CCK-8 s on cAMP production in these cell lines.
EC50 values for wild-type CCK-AR, chimeric receptors
CCK-B1A2-5, B1A2B3-5, and BAICL-1
were estimated to be 20 ± 4, 15 ± 5, 140 ± 28, and
8.5 ± 1 nM, respectively.
Chimeric receptors CCK-B1A2B3-5 and BAICL-1 were further examined for their cAMP response to CCK-BR selective agonist gastrin-17. While gastrin-17 produced no significant increases in cAMP levels in wild-type CCK-AR and CCK-BR, it stimulated dose-dependent increases in cAMP in CCK-B1A2B3-5 (EC50, 260 ± 15 nM) and in CCK-BAICL-1 (EC50, 23 ± 7 nM) (Fig. 3B). Chimeric receptor CCK-B1A2-5, which has a similar cAMP response as that of wild-type CCK-AR to CCK, did not respond to gastrin at all (data not shown).
Membrane Adenylyl Cyclase StimulationTo determine if
increased cAMP was mediated directly through Gs, adenylyl
cyclase was measured independently utilizing cell homogenates prepared
from transfected cells. Table II shows the results of the adenylyl
cyclase activity in the absence (basal) or presence of 1 µM CCK-8 s for both wild-type and six of the CCK
receptor chimeras. The responses of the cell homogenates to CCK were
similar to those found in whole cells. Cells expressing the wild-type
CCK-AR responded with a 7-fold increase in cAMP production over basal
(9.9 ± 1.9 nmol/h/mg of protein), while those expressing the
wild-type CCK-BR responded with an insignificant increase in cAMP
production (0.2 ± 0.1 nmol/h/mg of protein). Cells that were
expressing chimeric receptor B1A2-5 had similar responses (7.9 ± 1.7 nmol/h/mg of protein) to those
expressing wild-type CCK-AR. Chimeric receptors,
B1A2B3-5 and
BAICL-1, also gave significant responses to CCK although
the magnitude of the response was only 1.0 ± 0.3 and 1.5 ± 0.1 nmol/h/mg of protein, respectively. In contrast, chimeric receptors
B1-4A5, B1-3A4-5,
B1-2A3-5 had no measurable
CCK-8 s-stimulated adenylyl cyclase activity (Table II).
Dose-dependent effects of CCK-8 s on adenylyl cyclase
activity in the four responsive cell lines are shown in Fig.
4. Nonlinear regression analysis of the wild-type
CCK-AR, and mutants B1A2-5,
B1A2B3-5, and BAICL1
gave EC50 values of 24 ± 3, 29 ± 5, 151 ± 37, and 5 ± 1 nM, respectively.
Simultaneous stimulation of intracellular second messengers such as Ca2+ and cAMP by CCK has been described previously in cells naturally expressing wild-type CCK-AR and in cells transfected with recombinant CCK-AR (21, 22). Stimulation of CCK-BR, on the other hand, leads to intracellular Ca2+ mobilization and MAP kinase activation by a pathway that involves p74raf-1 kinase (23, 24) but does not appear to activate the cAMP pathway (25). It is reasonable to postulate that a critical region in CCK-AR may determine the intracellular signal transduction pathway that leads to cAMP production specifically stimulated by CCK.
CCK-A and CCK-B receptors demonstrate a highly conserved molecular organization with five exons and similar splice junctions, suggesting a common ancestral origin of these genes (7-10). Such structural homology suggests the exchange of functional domains encoded by a specific exon between two CCK receptors subtypes can serve as a starting point to create receptor chimeras. It was assumed that replacing an exon or group of exons in one of the receptor subtypes with that from the other closely related receptor would preserve the overall structure of the molecule. As an initial approach to generate CCK receptor chimeras, we replaced exons of human CCK-BR, from the second to the fifth exon, with human CCK-AR counterparts. Based on the information obtained from these chimeras, we made additional receptor mutants in which only a critical exon or an intracellular loop was replaced (Fig. 1).
Both CCK receptor subtypes share high affinity for binding of sulfated cholecystokinin peptides but only CCK-BR has high affinity to gastrin. This pharmacological selectivity served as a good marker to identify functional chimeric receptors that retained either CCK-AR or CCK-BR binding properties (Table I). Furthermore, since both wild-type receptors are primarily coupled to Gq and transduce calcium signals, integrity of these chimeric receptors was confirmed by their ability to mobilize Ca2+ in response to CCK (Table II, Fig. 2).
In the present study, both wild-type receptors transduced similar intracellular Ca2+ signals when stimulated by CCK-8 s (Fig. 2). However, transfected CCK-AR produced a more profound cAMP response than transfected CCK-BR measured either by cAMP accumulation (60-fold) or by adenylyl cyclase activation (50-fold), even though CCK-BR expressed 4 times more receptors/cell than CCK-AR in HEK-293 (Table I). Thus the HEK-293 cells into which these receptors were introduced possessed the necessary intracellular G-protein and effector components required to activate the calcium and cAMP pathways. In addition, within the same cellular context, a differential coupling of CCK-AR and CCK-BR to Gs was revealed by their markedly different cAMP responses to a common agonist. Such selectivity makes this in vitro model useful since these functional responses mimicked those found in native cell types such as pancreatic acini (16) and gastric enterochromaffin-like cells (26).
There are two possible mechanisms by which CCK-AR could couple to production of cAMP in intact cells. The most direct mechanism is coupling of the receptor to Gs, which activates adenylyl cyclase upon agonist binding to the receptor. A second possible mechanism is indirect and involves activation of phospholipase A2 leading to liberation of arachidonic acid which can then be converted to prostaglandin E2 by prostaglandin synthase. Prostaglandin E2 can then stimulate cell surface prostaglandin receptors leading to the production of cAMP via Gs stimulation of adenylyl cyclase. This latter mechanism has been described by Rozengurt and colleague (27) in Swiss 3T3 cells expressing the gastrin releasing peptide receptor. However, no significant differences in CCK-stimulated cAMP responses were detected in wild-type CCK-AR transfected HEK-293 cells treated with or without 1 mM indomethacin, an inhibitor of prostaglandin synthase, during a 1-h time course study (data not shown). These data, together with the demonstration of CCK-activated adenylyl cyclase in cell homogenates, indicate that the wild-type CCK-AR on HEK-293 cells is coupled to adenylyl cyclase exclusively through interaction with Gs.
Previous studies of other seven transmembrane domain receptors coupled
to Gs indicate that the second intracellular loop of thyrotropin receptor (28) and the third intracellular loop of 2-adrenergic receptor (29, 30) are important for
adenylyl cyclase activation. A recent study showed that a Leu to Ser
mutation at the proximal region of the third loop of human CCK-BR
resulted in a constitutively active mutant with elevated basal turnover of inositol phosphates (31). In two of the chimeric mutants, CCK-B1A2-5 and
B1A2B3-5, which exhibited positive cAMP responses to agonist stimulation, exon 2 of the human CCK-AR was
apparently required for functional Gs coupling. Moreover, a
five amino acid change, Gly80
Ile,
Leu81
Arg, Ser82
Asn,
Arg83
Lys, and Leu85
Met, in the first
intracellular loop was sufficient to confer such "gain-of-function"
phenomenon in CCK-BR. Since CCK-B1A2B3-5 and
BAICL-1 receptor mutants did not attain similar increases
as high as those found in wild-type CCK-AR and in chimeric receptor
B1A2-5, it is likely that other intracellular regions may also contribute to optimal Gs coupling.
However, while gastrin had no effect on cAMP levels in wild-type CCK
receptors, it was able to stimulate cAMP formation in both
CCK-B1A2B3-5 and
BAICL-1 chimeric mutants. Therefore, the gain-of-function of Gs coupling to these mutant receptors is primarily the
result of alteration of intracellular loop structure without affecting the interaction between CCK-BR and its native ligand.
One suggestion for the role of the first intracellular loop affecting G-protein coupling is a Ser to Lys substitution in the melanocyte-stimulating hormone receptor that causes the mutant receptor to be hyperactive in its coupling to Gs (32). A corticotropin releasing factor receptor splice variant has recently been identified in the first intracellular loop by a 29-amino acid insertion (33). This corticotropin releasing factor receptor, referred to as type II, showed markedly diminished coupling efficiency to Gs compared with type I receptor without insert (34). However, a direct functional involvement of the first intracellular loop in cAMP signaling has not been established in serpentine receptors with dual coupling to both Gq and Gs. A data base search shows that other peptide receptors that share the highest amino acid sequence homology in the first intracellular loop with CCK-AR include endothelin and tachykinin receptor families. Like CCK-AR, these receptors are primarily coupled to Gq and activate calcium mobilization, but some of the members are also able to couple to Gs and activate adenylyl cyclase (35). In the present study, our results demonstrate that the first intracellular loop of CCK-AR is essential for G-protein coupling and effector activation. In view of the emerging evidence from x-ray crystallography (36) and from molecular modeling (37), we hypothesize that first intracellular loop may interact with A1 domain in the C-terminal of Gs, which is mostly distinct from those of Gq and Gi.
In conclusion, our strategy to use exon replacement as an initial approach to generate a series of structurally compatible CCK receptor mutants allowed rapid scanning of regions critical for binding and signaling. Identification of a small area within exon 2, which is responsible for CCK-AR subtype-specific binding and cAMP response, revealed an intracellular target for Gs coupling. A CCK-BR mutant containing five amino acid residues from the first intracellular loop of CCK-AR gained significant cAMP responses to both CCK and gastrin through activation of adenylyl cyclase. Future identification of specific amino acid residues in the first intracellular loop and in other potential G-protein binding areas will aid in understanding the interaction of CCK receptors and Gs.
Imaging services were provided by the Imaging/Morphology Core and oligonucleotide and peptide services were provided by the Peptide Biochemistry Core of CURE/Digestive Diseases Research Center. We thank Drs. Nigel Bunnett and Joseph Pisegna for helpful discussions and Mary Ma for technical assistance