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J Biol Chem, Vol. 273, Issue 44, 28779-28784, October 30, 1998
,From the Division of Gastroenterology and GRASP Digestive Disease Center, Tupper Research Institute, New England Medical Center, Tufts University School of Medicine, Boston, Massachusetts 02111
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ABSTRACT |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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The enteroendocrine hormone, gastrin, exerts
trophic effects on the gastric mucosa through the CCK-B/gastrin
receptor (CCK-BR). To varying degrees in different species, excess
circulating gastrin leads to proliferation of enterochromaffin-like
cells and to the development of gastric carcinoid tumors. The African
rodent, Mastomys natalensis, is distinguished from other
mammals by its propensity toward CCK-BR-mediated growth even in the
absence of hypergastrinemia. Here, we report that the
Mastomys CCK-BR, when expressed in COS-7 cells, differs
from the respective human, canine, and rat receptor homologs by its
ability to trigger ligand-independent (i.e., constitutive) inositol phosphate formation. To define the molecular basis of this
observation, a series of Mastomys-human chimeric receptors was investigated. Functional characterization of these constructs revealed that a limited segment of the Mastomys CCK-BR,
transmembrane domain VI through the C-terminal end, is sufficient to
confer constitutive activity to the human protein. Mutagenesis studies within this CCK-BR region defined a combination of three
Mastomys amino acids that, when introduced into the human
receptor, together conferred a level of ligand-independent signaling
comparable with the Mastomys CCK-BR. Complementing prior
observations that single point mutations can lead to ligand-independent
signaling, our findings suggest that multiple naturally occurring amino
acid polymorphisms and/or mutations may together result in an enhanced basal level of receptor activity.
The cholecystokinin-B/gastrin receptor
(CCK-BR)1 is a seven
transmembrane domain G protein-coupled receptor that is widely expressed in the gastrointestinal tract and in the central nervous system (1). Stimulation of the CCK-BR by either of the endogenous peptides, CCK-8 or gastrin, triggers phospholipase C-mediated conversion of membrane phospholipids to inositol phosphates and diacylglycerol, which in turn initiates a series of downstream signaling events (1).
In the CNS, the CCK-BR is postulated to modulate anxiety as well as the
perception of pain (2, 3). In the stomach, it is well established that
activation of this receptor subtype stimulates acid secretion as well
as mucosal growth (1). Recent studies in which CCK-BR-deficient mice
were generated by targeted gene disruption revealed that absence of the
CCK-BR results in thinning of the gastric mucosa and a reduced density
of both gastric enterochromaffin-like (ECL) and parietal cells (4, 5).
Conversely, receptor overactivity in rodents, induced by
hypergastrinemia, leads to gastric mucosal hypertrophy marked by
increased synthesis of DNA, RNA, and protein (6). Long-term stimulation
of the CCK-BR by elevated circulating gastrin may ultimately result in
the development of ECL cell carcinoid tumors (7).
Among mammals, there is a broad spectrum of susceptibility to
gastrin-induced proliferation and tumor formation, with the highest
sensitivity observed in the African rodent, Mastomys
natalensis. During its normal course of aging, Mastomys
may develop gastric carcinoid tumors even with normal circulating
gastrin levels. In this animal, hypergastrinemia potentiates the
enhanced rate of gastric ECL cell tumor development (7). The increased
susceptibility of Mastomys to ECL cell growth, even in the
absence of elevated circulating gastrin, led us to speculate that this
receptor homolog was functionally different from the CCK-BR in other
species. There is ample precedent that species-dependent
differences in the amino acid sequence of the CCK-BR can lead to
alterations in the pharmacologic properties of the receptor. We have
previously shown that interspecies polymorphisms markedly alter CCK-BR
interactions with synthetic ligands, i.e., a single amino
acid difference between the canine and human CCK-BRs results in a
reversal of the affinity rank order for non-peptide antagonists (8). In
a subsequent study, we demonstrated that single and double amino acid
differences between the mouse, human, and dog receptors led to marked
alterations in drug-induced inositol phosphate formation, the primary
second messenger generated in response to activation of the CCK-BR
(9).
In this study, recombinant CCK-BRs from different species were
expressed in COS-7 cells and functionally compared. Only the Mastomys receptor had a significant level of
ligand-independent (i.e., constitutive) signaling activity.
To understand the molecular basis of this finding, we utilized a series
of human/Mastomys CCK-BR chimeric receptors to map the
limited domain that confers ligand-independent signaling. Point
mutations were then introduced within this region of the protein to
identify residues that underlie the species-dependent
constitutive activity.
Materials--
Restriction endonucleases and T4 DNA ligase were
purchased from New England Biolabs Inc. Iodinated cholecystokinin
octapeptide (125I CCK-8; specific activity, 2200 Ci/mmol)
and [3H]myoinositol (specific activity 40-60
Ci/mmol) were obtained from NEN Life Science Products. Unlabeled CCK-8
was purchased from Peninsula Laboratories (Belmont, CA).
Construction of Chimeric Receptor cDNAs--
The
Mastomys CCK-BR cDNA (generously provided by Dr. T. Chiba) was subcloned into the expression vector pcDNA I
(Invitrogen) and sequenced. Two differences in translated amino acid
sequence from the originally published version (10) were noted:
317GPGLASANQA326 (old)
versus 317GPGPGPRPNQA327
(revised) and 359C (old) versus 360R
(revised). Of note, the 320PGPRP revision results in the
introduction of an additional amino acid, thus increasing the numbering
by "1" of residues C-terminal to proline 324. The revised
Mastomys cDNA has been submitted to GenBank. Naturally
occurring PstI sites, in the same relative position of the
human (11) and the Mastomys (10) CCK-BR (corresponding to
human cDNA nucleotides 607-612), were used to generate the PstI chimeras. Oligonucleotide-directed mutagenesis was used
to introduce XhoI and MluI sites (corresponding
to human cDNA nucleotides 910-915 and 986-992) into the
respective receptor cDNAs from each species (12). The
XhoI and MluI chimeric receptor cDNAs were constructed by replacing segments of the human CCK-BR cDNA with the
corresponding Mastomys sequence. Mutant receptors, in which 1-3 amino acids were replaced, were generated using
oligonucleotide-directed mutagenesis (12). All chimeric and mutant
receptor cDNA constructs were confirmed using the Applied
Biosystems 373 DNA sequencer.
Cell Culture and Transfection--
COS-7 cells were grown in
Dulbecco's modified Eagle's medium supplemented with 10% fetal calf
serum and gentamicin (100 µg/ml). The cells were maintained at
37 °C in a 5% CO2 atmosphere. Utilizing the
DEAE-dextran method (13), cells (106/10-cm dish) were
transiently transfected with 5 µg of either the expression vector,
pcDNA I, or the relevant CCK-BR cDNA construct. In control
experiments (see Fig. 4), the amount of transfected DNA was adjusted
(1-5 µg/10-cm dish) to obtain comparable receptor expression levels
of the human and Mastomys CCK-BR.
Radioligand Binding Experiments--
24 h after transfection,
cells were trypsinized and divided into 24-well plates (5 × 103 cells/well). The following day, competition binding
experiments were performed in Hank's balanced salt solution
supplemented with 25 mM HEPES, pH 7.3, 0.2% bovine serum
albumin, and 0.15 mM phenylmethylsulfonyl fluoride. 20 pM of 125I-CCK-8 (NEN Life Science Products)
was used as the radioligand in the absence or presence of increasing
concentrations of unlabeled CCK-8 (0-0.3 µM). After
incubation for 80 min at 37 °C, cells were washed three times with
Hank's balanced salt solution and hydrolyzed in 1 N NaOH.
Bound radioactivity was quantified, and receptor expression levels were
calculated using computerized nonlinear curve fitting (MacLigand). In
all experiments, the human wild-type CCK-BR was included as a
control.
Measurement of Inositol Phosphate Formation--
Transfected
COS-7 cells were cultured overnight in serum-free medium containing 3 µCi of [3H]myoinositol/ml. Ligand-independent and
agonist (0.3 µM CCK-8)-stimulated [3H]inositol phosphate production were assessed at
37 °C in the presence of 10 mM LiCl. After incubation of
the cells for either 0.5 or 1 h (see text), inositol metabolites
were extracted with methanol/chloroform, and the upper phase was
analyzed for inositol phosphates by strong anion exchange
chromatography. The production of inositol phosphates was expressed as
a fraction of the total cellular tritium content. To determine the
receptor-mediated component of IP production, all measurements were
corrected by the amount of IP formation in COS-7 cells expressing
pcDNA I (the expression vector) alone.
Second messenger signaling by COS-7 cells expressing the human
(11, 14, 15), canine (13), rat (16), or Mastomys (10) CCK-B
receptors was assessed in the presence and in the absence of
cholecystokinin octapeptide (CCK-8). With agonist stimulation, each
receptor led to comparable levels of IP production (Fig. 1A). In contrast, only the
Mastomys protein triggered ligand-independent IP formation
that was significantly higher than the level observed in cells
transfected with the expression vector, pcDNA I (Fig. 1B).
INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References
RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References
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Fig. 1.
Ligand-independent second messenger signaling
distinguishes the Mastomys CCK-BR from other species
homologs. [3H]Inositol phosphate production was
assessed in COS-7 cells expressing the human, canine, rat, or
Mastomys CCK-BR. IP production (y axis) is expressed as a
fraction of the total tritium incorporated after an overnight exposure
of transfected cells to tritiated myoinositol. Asterisk
denotes values that are significantly different from cells transfected
with the expression vector, pcDNA I, alone (p < 0.05). A, all species homologs of the CCK-BR trigger
comparable levels of IP production when stimulated with a saturating
concentration of CCK-8 (0.3 µM). B, in the
absence of agonist ("basal" activity), only the Mastomys
CCK-BR triggers significant production of IP.
Increased expression of a constitutively active receptor will result in a parallel increase in both agonist-induced and ligand-independent second messenger signaling (17). Consistent with this expectation, a positive correlation was observed between CCK-8-stimulated and ligand-independent signaling in COS-7 cells expressing the Mastomys CCK-BR (Fig. 2). In contrast, signaling by the human CCK-BR in the absence of ligand was always close to zero regardless of the level of CCK-8-induced IP formation. These observations confirm that the Mastomys CCK-BR is constitutively active, whereas the human homolog is not.
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A comparison of IP production after 0.5 and 1 h provided additional support that only the Mastomys CCK-BR is constitutively active. As shown in Fig. 3A, a time-dependent increase in basal IP production is observed in cells expressing the Mastomys CCK-BR, whereas this is not the case in cells expressing the human receptor. As a control, the maximal level of CCK-8-induced signaling was also compared at 0.5- and 1-h incubation times. Consistent with expectation, agonist-induced IP production by either the Mastomys or the human CCK-BR increased to a similar extent independent of the respective basal level of signaling (Fig. 3B).
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It has been recently established that constitutive activity of G protein-coupled receptors can be attenuated by a novel class of ligands, inverse agonists (18, 19). The availability of such a compound for the CCK-BR, L-740,093 (R enantiomer), enabled us to provide additional confirmation of constitutive signaling by the Mastomys CCK-BR. L-740,093 R attenuated ligand-independent IP production by the Mastomys receptor but had no effect on the basal activity of the human CCK-BR (Fig. 4A). Half-maximal inhibition of ligand-independent signaling by L-740,093 R in cells expressing the Mastomys receptor was achieved at a concentration of 1.73 nM (Fig. 4B).
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The marked difference in ligand-independent second messenger signaling by the Mastomys and human CCK-BR homologs was observed despite 92% amino acid identity between the two proteins. In an attempt to define a minimal domain of the Mastomys receptor, which conferred ligand-independent activity, a series of chimeric receptors was constructed with varying N-terminal portions of human sequence linked to complementary C-terminal portions of the Mastomys protein (PstI, XhoI, and MluI chimeras, see Fig. 5). When functionally assessed in COS-7 cells, each of these chimeric receptors had a level of constitutive activity approaching that of the Mastomys wild-type CCK-BR.
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Constitutive activity of the MluI chimera suggested that transfer of a small segment of the Mastomys receptor was sufficient to confer constitutive activity to the human protein. Sequence comparison between the Mastomys and human receptors within this limited domain, which spans transmembrane domain VI through the C terminus (see Fig. 5), revealed four candidate amino acids that could potentially confer constitutive signaling activity (Fig. 6A). Many previously reported examples of constitutively active G protein-coupled receptors are the result of single amino acid substitutions (20-22). Based on this precedent, a series of four mutant human CCK-BRs was generated. In each receptor, one of the four human amino acids was replaced with the corresponding Mastomys residue. None of the single point mutations in the human CCK-BR was sufficient to significantly increase ligand-independent signaling relative to the human wild-type value (data not shown). The absence of ligand-independent signaling with single amino acid substitutions suggests that a combination of several Mastomys residues is required to confer constitutive signaling activity.
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To determine which of the four candidate Mastomys amino
acids contributed to constitutive activity, an alternative strategy was
utilized. With the cDNA encoding the human/Mastomys MluI
chimera as a template for mutagenesis, each residue unique to the
Mastomys receptor (Leu344, Ile353,
Asp407, Pro423) was sequentially
replaced with its corresponding human homolog (Val340,
Val349, Glu403, Ala419). A second
series of four mutant human receptors was thus generated, each
including a different combination of three Mastomys amino acids (Fig. 6B). A decrease in constitutive signaling with
replacement of the respective Mastomys amino acid indicated
that the corresponding residue participated in ligand-independent
signaling. Mutants 1, 2, and 3 had a significantly lower level of
constitutive activity than the Mastomys wild-type receptor,
suggesting that each of the Mastomys amino acids
(Leu344, Ile353, Asp407), which had
been substituted with the corresponding human residue, contributed to
ligand-independent signaling (Fig. 6B). In contrast, triple
mutant 4 maintained constitutive activity comparable with the level of
the Mastomys protein, despite substitution of
Pro423 by an alanine residue. The latter finding suggests
that the residue at position 423 played no appreciable role in
conferring constitutive activity to the Mastomys CCK-BR.
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DISCUSSION |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Comparison of the recombinant canine, rat, Mastomys, and human CCK-BRs expressed in COS-7 cells revealed that only the Mastomys receptor triggered ligand-independent IP formation. Constitutive signaling activity of the Mastomys protein was confirmed by three additional observations: (i) the level of ligand-independent signaling correlated with respective levels of CCK-8-induced signaling, (ii) ligand-independent [3H]inositol phosphate formation increased with extended incubation time, and (iii) ligand-independent signaling could be inhibited in a concentration-dependent manner by the inverse agonist, L-740,093 R. Of note, none of these criteria applied to the human CCK-BR, which was tested in parallel. Together, these findings establish that the Mastomys receptor is constitutively active as a consequence of interspecies variability in the CCK-BR.
Since the Mastomys cDNA utilized in this study was isolated from an enterochromaffin-like cell carcinoid (10), it was important to consider whether the corresponding protein resulted from a somatic mutation in the tumor. This possibility is unlikely based on sequence identity between the cDNA used in our experiments and CCK-BR cDNAs isolated from a number of normal Mastomys tissues, including isolated gastric parietal cells and cerebral cortical tissue (7).
The observation that species-dependent polymorphisms can result in constitutive activation of a G protein-coupled receptor suggests an important mechanism by which ligand-independent signaling can occur in nature. As such, our observation complements two previous reports which revealed that constitutive activity may be a distinguishing feature of certain G protein-coupled receptor subtypes (23) or splice variants (24). Study of both human and rat dopamine receptors indicated that the D1B subtype is constitutively active, whereas the D1A subtype is not (23). In the mouse, it has been found that alternative splicing of the prostaglandin E3 receptor results in a constitutively active isoform of this receptor (24). For both the D1 and the mouse prostaglandin E3 receptor, ligand-independent signaling is a property of naturally occurring receptor variants within a given species rather than a species-dependent phenomenon as observed with the Mastomys CCK-BR.
The vast majority of naturally occurring constitutively active
receptors reported in the literature can be attributed to single amino
acid mutations. These point mutations have been identified in
extracellular (25), transmembrane domain (21, 26-28), and intracytoplasmic segments (20, 22, 29, 30) of the corresponding G
protein-coupled receptors. In contrast to this precedent, it is of note
that three amino acids act in concert to confer constitutive activity
to the Mastomys CCK-BR. Two of these residues
(Leu344, Ile353) are located in transmembrane
domain VI of the Mastomys CCK-BR, consistent with reports
indicating a role of TMD VI in receptor activation. Studies with the
photoreceptor rhodopsin, another member of the seven transmembrane
domain receptor superfamily, suggest that movement of TMD VI is
required for G protein activation (31). In addition, rotation of TMD VI
has been demonstrated with either ligand or mutation-induced activation
of the 
2 adrenergic receptor (32, 33). Complementing
these prior observations, the occurrence of activating polymorphisms in
TMD VI of the CCK-BR further illustrates that this portion of the
protein may be an important regulator of receptor-mediated
signaling.
The third activating polymorphism in the Mastomys CCK-BR (Asp407 versus Glu403 in the human receptor) is found in the carboxyl terminus. This region of G protein-coupled receptors has been hypothesized to play a role in constraining the protein through palmitoylated cysteine residues that are believed to be anchored in the cell membrane. Truncations of the carboxyl terminus can result in constitutive activity as shown for the PGE3 receptor (24) and the thyrotropin-releasing hormone receptor (34). Alternately, deletion or truncation of the carboxyl terminus may leave the signaling properties of a G protein-coupled receptor unchanged (35). The variable consequences of carboxyl-terminal truncations may indicate that this domain may act in concert with other regions of the receptor to define the basal level of signaling. Consistent with this possibility, our results suggest that a combination of transmembrane domain VI and carboxyl terminus amino acids together confer constitutive activity to the Mastomys CCK-BR.
Several naturally occurring constitutively active G protein-coupled receptors, each the result of single amino acid mutations, cause alterations of physiologic function both in animals as well as in humans. Constitutively active melanocyte-stimulating hormone receptor isoforms result in darkening of fur and skin color in mice (26). Activating mutations in the luteinizing hormone receptor lead to precocious puberty (21), whereas constitutively active parathyroid hormone receptors result in Jansen-type metaphyseal chondrodysplasia (29). Constitutively active mutants of the thyroid-stimulating hormone receptor have been identified in patients with thyroid adenomas (20).
In contrast to constitutively active receptors that arise from single
point mutations (discussed above), ligand-independent signaling by the
Mastomys CCK-BR results from a triple amino acid alteration.
The observation that multiple naturally occurring residue changes can
lead to constitutive receptor activity introduces a new consideration
in the search for receptor abnormalities underlying physiologic
alterations. In the case of the Mastomys CCK-BR, the in vivo consequences of receptor overactivity remain to be
determined. Based on precedent with constitutively active G
protein-coupled receptors resulting in tumor formation (20, 36), it is
intriguing to speculate that the ligand-independent signaling by the
Mastomys CCK-BR may in part explain the increased
susceptibility of this species to the development of ECL cell
hyperplasia as well as ECL cell-derived carcinoid tumors.
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ACKNOWLEDGEMENTS |
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We thank Wyeth-Lederle for providing L- 740,093 R, Dr. T. Chiba for providing the Mastomys CCK-BR cDNA, and Dr. S. A. Wank for providing the rat CCK-BR cDNA. We are grateful to Drs. M. Bläker, Y. Ren, A. Leiter, and A. Kane for critical reading of the manuscript and to B. Desai, C. Chen, and L. Sundararajan for technical assistance.
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FOOTNOTES |
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* This work was supported by NIDDK, National Institutes of Health, Grant DK46767, an American Digestive Health Foundation/American Gastroenterological Association Industry Research Scholar Award (to M. B.), and the GRASP Center (NIDDK Grant P30 DK34928).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.
Supported by the Deutsche Forschungsgemeinschaft (Scha
739/1-1).
§ To whom correspondence should be addressed: The Division of Gastroenterology and GRASP Digestive Disease Center, Tupper Research Institute, New England Medical Center, Tufts University School of Medicine, 750 Washington St., Boston, MA 02111. Tel.: 617-636-5875; Fax: 617-636-4207; E-mail: alan.kopin{at}es.nemc.org.
The abbreviations used are: CCK-BR, cholecystokinin-B/gastrin receptor; ECL, enterochromaffin-like; IP, inositol phosphate; TMD, transmembrane domain.
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REFERENCES |
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References
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10 independent experiments.
Asterisk denotes a significant increase (0.5 h
versus 1 h) in IP production (p < 0.01). A, basal IP production in COS-7 cells expressing the
Mastomys CCK-BR significantly increases between 0.5 and
1 h. In contrast, in cells expressing the human CCK-BR, no
significant increase (n.s.) in IP production is observed.
B, when stimulated with a saturating concentration of CCK-8
(0.3 µM), cells expressing either the Mastomys
or the human CCK-BR show a similar increase in IP production. Of note,
after either 0.5 or 1 h, ligand-independent IP production
expressed as a fraction of the CCK-8-induced maximum [(basal IP
production/CCK-8-stimulated IP production) × 100] approximates 13%
for the Mastomys CCK-BR versus 0% for the human
CCK-BR.
) in the
cartoon of the CCK-BR; their position in the human receptor is noted.
N, amino terminus; C, carboxyl terminus. B, substitution of
three human amino acids with the corresponding Mastomys
residues (human mutant 4) confers constitutive activity to the human
CCK-BR. The four amino acids in the MluI chimera, which are
unique to the Mastomys receptor (344L,
353I, 407D, 423P), were
sequentially substituted with the corresponding human residue
(340V, 349V, 403E,
419A). For each of the resulting human triple mutants 1-4
(human CCK-BR with three Mastomys amino acid substitutions),
basal IP production was measured and expressed as a percentage of the
CCK-8-induced maximum. Human mutants 1, 2, and 3 had constitutive
activity significantly lower than the level of the Mastomys
wild-type receptor, suggesting that each of the Mastomys
amino acids (344L, 353I, 407D),
which had been substituted with the corresponding human homolog,
contributes to constitutive receptor activity. In contrast, triple
mutant 4 maintained constitutive activity comparable with the level of
the Mastomys protein despite substitution of
Pro423 by alanine, suggesting that the Mastomys
residue that had been replaced plays no appreciable role in
ligand-independent signaling. Data represent the means ± S.E. of