Departments of Internal Medicine and Biochemistry/Molecular Biology, Center for Basic Research in Digestive Diseases, Mayo Clinic and Foundation, Rochester, Minnesota 55905
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Differences in the molecular structure or organ distribution of receptors can limit the usefulness of a given species for drug studies. In this work, we have studied cholecystokinin (CCK) receptors in cynomolgus monkey, an animal model useful for preclinical testing. The type A CCK receptor cDNA was cloned and predicted to encode a 428 amino acid peptide that was 98% identical to the human receptor. Only 2 of the 10 residues that were distinct from the human receptor were not present in other cloned CCK receptor species. A Chinese hamster ovary cell line that stably expressed this receptor was developed. The cynomolgus receptor expressed in this environment was functionally indistinguishable from the human receptor, binding CCK with high affinity [inhibition constant (KI) = 1.8 ± 0.5 nM] and exhibiting a potent intracellular calcium signaling response to this hormone (EC50 = 6.6 ± 2.1 pM). Like the human type A CCK receptor, this receptor was expressed prominently in monkey gallbladder and stomach and was expressed in low levels in brain and pancreas. The type B CCK receptor cDNA was cloned from stomach and brain (450 residue receptor that is 96% identical to the human receptor), where it was highly expressed yet was undetectable in gallbladder or pancreas. This work confirms the relevance of the cynomolgus species for preclinical testing of drugs acting on the type A CCK receptor.
G protein-coupled receptor; ligand binding; cDNA cloning; cynomolgus monkey
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
ANIMAL MODELS ARE EXTREMELY important for assessing the safety and efficacy of new drugs. Before potential new therapeutic agents can enter phase one human clinical trials, regulatory agencies require studies in relevant rodent and mammalian animal models. The cynomolgus monkey has become a particularly useful species for preclinical studies in which more evolutionarily divergent animals may not be appropriate. However, while the amino acid sequence of a specific receptor molecule is usually highly conserved across closely related species, modification of even a single residue can have profound implications for ligand binding characteristics and for biological actions (3). Also, the cellular distribution of receptors may differ across species, thereby affecting its utility as an animal model (5, 21).
Because the type A cholecystokinin (CCK) receptor is a potentially important target for drug development (2, 11), and because the neurohormonal control of the exocrine pancreas can be quite different in distinct species (10), we have examined CCK receptors in the nonhuman primate species, cynomolgus monkey. In this work, we have cloned the cDNAs encoding the type A and type B CCK receptors from this species. This allowed us to use ribonuclease protection assays and ultrasensitive reverse transcriptase polymerase chain reactions to examine the organ distribution of each of these receptors in this species. We established a Chinese hamster ovary cell line stably expressing the cynomolgus type A receptor and determined its ligand binding and signaling characteristics.
We found that the cynomolgus type A CCK receptor is 98% conserved relative to the human receptor (31), with only 10 residues that are distinct. Of these, eight are variants that are present in CCK receptors from at least one of the four other species that have been cloned to date [rat (32), guinea pig (7), rabbit (26), and mouse (9)]. This receptor was functionally indistinguishable from the human receptor. Like the human type A CCK receptor, this receptor was expressed prominently in gallbladder and stomach and was expressed in low levels in brain and pancreas.
The cynomolgus type B CCK receptor was also closely related to the human receptor (96% conserved). There were 20 residues that were distinct from those in the human receptor, with 9 of these unique among the type B CCK receptor sequences cloned to date [dog (18), human (25), Mastomys (23), rat (16), rabbit (4), and cow (8)]. The largest variability was in the intracellular third loop domain (13 of the variations). This receptor was highly expressed in the brain and stomach of this species, with no detectable expression in cynomolgus gallbladder or pancreas. The latter is a clear difference from man, where the type B CCK receptor is prominently expressed in pancreatic islets (27).
This work confirms the relevance of the cynomolgus species for preclinical testing of drugs acting on the type A CCK receptor.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
cDNA cloning.
Aliquots of dissected, snap-frozen cynomolgus tissues (brain,
gallbladder, pancreas, and gastric fundus) were kindly provided by Dr.
Elizabeth Sugg at Glaxo-Wellcome Research Laboratories. These were
stored at 80°C before use. Total RNA was extracted from each using
TRIZOL reagent (Life Technologies, Grand Island, NY). First strand cDNA
was produced using 10µg RNA as template with random hexamer primers
and AMV reverse transcriptase (Roche Molecular Biochemicals,
Indianapolis, IN), incubating for 1 h at 42°C, followed by 10 min at 65°C.
Establishment of receptor-bearing cell line. The full-length cynomolgus type A CCK receptor cDNA construct was transfected into CHO-K1 cells using lipofectin (Life Technologies, Grand Island, NY). After 48 h, neomycin-resistant cells were selected with 1 mg/ml G418. Receptor-expressing cells were further selected using fluorescence activated cell sorting after binding a fluorescein-conjugated CCK analog, as we described previously (12). Cells were cultured in Ham's F-12 medium supplemented with 100 U/ml penicillin, 100 U/ml streptomycin, and 5 % fetal clone II (HyClone). They were lifted using trypsin or cell dissociation media and passaged approximately twice per week.
Functional characterization of receptor. CCK receptor binding was studied in the receptor-expressing cells using the previously validated radioligand, 125I-D-Tyr-Gly-[(Nle28,31)CCK-26-33] (24), and previously established conditions (12). Binding to intact cells was performed in 24-well culture dishes with incubation performed for 1 h at 25°C.
Signaling in response to CCK was studied using a fully validated assay for intracellular calcium utilizing fura 2-AM-loaded cells (13).Determination of tissue distribution of receptors. The tissue distribution of mRNA species encoding type A and type B CCK receptors was determined by Northern blotting, ribonuclease protection assays, and reverse transcriptase polymerase chain reactions, in order of increasing sensitivity. 32P-labeled transcripts for the receptors and GAPDH were prepared for the hybridization assays. For ribonuclease protection assays, relevant radiolabeled transcripts were hybridized with 20 µg of total RNA or 2 µg of yeast RNA (negative control) at 45°C overnight. Reactions were then digested with ribonuclease T1 at 37°C for 30 min and precipitated with 95% ethanol, before being separated on 5% acrylamide 8 M urea gels. Products were identified by autoradiography.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
cDNA cloning of cynomolgus CCK receptors.
The cDNA encoding the type A CCK receptor was cloned from cynomolgus
gastric fundus (Fig. 1A).
Translation of this predicted it to be a 428 amino acid protein that
was 98% identical to the human receptor (Fig.
2A). There
were only 10 residues that were different in the cynomolgus receptor
compared with the human receptor, with 8 of these also present in at
least one other cloned type A CCK receptor from another species (Fig.
2A). The region containing the largest variation was the
intracellular third loop (five changes from the human sequence). The
other changes were in the first extracellular loop and the
carboxyl-terminal tail domain. Comparison with the sequences of the
type A CCK receptors from the other species revealed 94% identity with
the rat and mouse receptors, 93% identity with the guinea pig
receptor, and 91% identity with the rabbit receptor.
|
|
Functional characterization of the cynomolgus type A CCK receptor.
Figure 3 illustrates CCK radioligand
binding and CCK-stimulated intracellular calcium responses in the
cynomolgus type A CCK receptor-bearing CHO cell line. CCK bound to this
receptor with high affinity [inhibition constant
(KI) = 1.8 ± 0.5 nM], analogous to
that of the human receptor (31). CCK was also a potent
stimulant of signaling in these cells (EC50 = 6.6 ± 2.1 pM), like its action in human receptor-bearing cells
(31).
|
Tissue distribution of CCK receptors in cynomolgus.
Total RNA extracted from cynomolgus gastric fundus, brain, gallbladder,
and pancreas was high quality, as assessed by its staining on a gel
(not shown) and in ribonuclease protection assay for GAPDH message
expression (Fig. 4). The ribonuclease
protection assay was a clear indication of the higher expressing
tissues. For the type A CCK receptor, this revealed a strong signal in stomach, a weaker signal in gallbladder, and no detectable signal above
background in brain and pancreas (Fig. 4). When the more sensitive
reverse transcriptase polymerase chain reaction assay was applied to
the same tissues, stomach and gallbladder continued to have strong
signals, while brain and pancreas were weakly positive (Fig.
5). For the type B CCK receptor,
ribonuclease protection assay revealed strong signals in stomach and
brain, with no detectable signal above background in gallbladder and
pancreas (Fig. 4). The polymerase chain reaction assay for the type B
receptor confirmed this distribution, without showing any signal in
gallbladder or pancreas (Fig. 5).
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The cynomolgus type A CCK receptor has molecular structure, hormone binding and signaling, and organ distribution similar to the human receptor. These features make it a potentially quite useful animal model for the preclinical testing of drugs acting at this important receptor. Of interest, the type B CCK receptor in the cynomolgus monkey has a key difference, in being below detectability in the pancreas of this species, while being quite prominent in human islets (27).
There has been considerable variation in the pancreatic expression of CCK family receptors in different species (5). The rat and mouse are believed to express only type A CCK receptors, with no type B receptors present in adult pancreas. In these rodent species, CCK is a potent secretagogue for the exocrine pancreas. However, even between these closely related rodent species, there are clear differences in pancreatic responses to selected CCK analogs (9). Higher mammalian species have been shown to express both type A and type B CCK receptors, with a possible predominance of the type B receptors (19, 20, 22), although there continue to be questions related to cellular distribution and physiological relevance of each. In the calf, where type B receptors seem to predominate, these seem to play no role in meal-induced stimulation of exocrine secretion (19, 20). CCK appears to stimulate human exocrine pancreatic secretion via a neuronal type A CCK receptor, rather than through an acinar cell receptor (1, 30).
However, the distribution of CCK receptors in human pancreas has been problematic to study directly. This is likely due to the paucity of available healthy tissue, the high content of proteases and ribonucleases, and the difficulties in dissociating healthy cells from the adult organ. There is general agreement that the type B CCK receptor mRNA is present in considerably higher concentration in the human pancreas than the type A CCK receptor mRNA (33), but the specific cellular localization of each is not clear. A recent report (27) claims that the type B receptor is present on human islet cells. If this receptor were expressed on the human acinar cell, one would expect a strong secretory response to gastrin, but this has been absent in physiological studies (6).
There has also been controversy about the expression of CCK family receptors on pancreatic neoplasms (29, 34). Due to the trophic actions of this hormone (15), expression on healthy pancreatic cells and on pancreatic neoplasms could have substantial implications for long-term agonist therapeutics.
The type A CCK receptor cDNA has been cloned in six different species, rat (32), human (31), guinea pig (7), rabbit (26), and mouse (9) and now also in cynomolgus monkey. Of these, the species most closely related in evolution to the human is the primate. Indeed the cynomolgus receptor is most closely related to the human receptor, being 98% conserved with only 10 different residues. Of these, the major variation was found in intracellular domains, rather than in the extracellular loop and tail domains that have been shown to be important for CCK peptide binding (13, 14, 17, 28). Only one residue was different from the human in any extracellular domain, with that representing a Ser in the first extracellular loop, a variant also described in the guinea pig receptor. All other changes from the human sequence were in predicted intracellular domains. The high affinity binding and potent signaling of CCK at this receptor support the prediction that this high degree of conservation makes it an excellent model to study receptor-active drugs. The relationship of this receptor to the other nonhuman species was in the range of 90 to 94% conservation.
The type B CCK receptor cDNA has been cloned in seven different species, dog (18), human (25), Mastomys (23), rat (16), rabbit (4), and cow (8) and now also in cynomolgus monkey. Of these, the species most closely related in evolution to the human receptor is the primate. The cynomolgus type B CCK receptor is 96% conserved relative to the human receptor, with 20 residues different. Of these, the major variation was found in the intracellular third loop domain. In contrast to the type A receptor, there were multiple variations in domains potentially important for drug action at this receptor, with four changes observed in transmembrane segments one, three, and six, and with changes observed in the amino-terminal tail and third extracellular loop domain. Of these, two of the transmembrane segment changes and both of the extracellular changes were unique to cynomolgus, not occurring in any other species cloned to date. The relationship of this receptor to the other nonhuman species was in the range of 90 to 94% conservation.
As part of this project, because of its clear utility in drug-screening efforts, we established a Chinese hamster ovary cell line that stably expresses the cynomolgus type A CCK receptor. This binds CCK with high affinity and signals in response to hormone binding in a concentration-dependent manner, as does the human receptor expressed in a similar environment (31). This can provide a valuable tool to study the action of potential new drugs in vitro, before initiating the quite expensive in vivo testing required by regulatory agencies before initiating phase one human clinical trials.
![]() |
ACKNOWLEDGEMENTS |
---|
This work was supported by grants from the National Institute of Diabetes and Digestive and Kidney Diseases (DK-32878), the Fiterman Foundation, and the Glaxo-Wellcome Research Institute.
![]() |
FOOTNOTES |
---|
Address for reprint requests and other correspondence: L. J. Miller, Center for Basic Research in Digestive Diseases, Guggenheim 17, Mayo Clinic, Rochester, MN 55905 (E-mail: miller{at}mayo.edu).
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.
Received 13 September 2000; accepted in final form 27 March 2001.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Adler, G,
Nelson DK,
Katschinski M,
and
Beglinger C.
Neurohormonal control of human pancreatic exocrine secretion.
Pancreas
10:
1-13,
1995[ISI][Medline].
2.
Aquino, CJ,
Armour DR,
Berman JM,
Birkemo LS,
Carr RAE,
Croom DK,
Dezube M,
Dougherty RW, Jr,
Ervin GN,
Grizzle MK,
Head JE,
Hirst GC,
James MK,
Johnson MF,
Miller LJ,
Queen KL,
Rimele TJ,
Smith DN,
and
Sugg EE.
Discovery of 1,5-benzodiazepines with peripheral cholecystokinin (CCK-A) receptor agonist activity.1. Optimization of the agonist "Trigger."
J Med Chem
39:
562-569,
1996[ISI][Medline].
3.
Beinborn, M,
Lee Y-M,
McBride EW,
Quinn SM,
and
Kopin AS.
A single amino acid of the cholecystokinin-B/gastrin receptor determines specificity for non-peptide antagonists.
Nature
362:
348-350,
1993[ISI][Medline].
4.
Blandizzi, C,
Song I,
and
Yamada T.
Molecular cloning and structural analysis of the rabbit gastrin/CCKB receptor gene.
Biochem Biophys Res Commun
202:
947-953,
1994[ISI][Medline].
5.
Bourassa, J,
Lainé J,
Kruse ML,
Gagnon MC,
and
Calvo
É, and Morisset J. Ontogeny and species differences in the pancreatic expression and localization of the CCKA receptors.
Biochem Biophys Res Commun
260:
820-828,
1999[ISI][Medline].
6.
Cantor, P,
Petronijevic L,
Pedersen JF,
and
Worning H.
Cholecystokinetic and pancreozymic effect of O-sulfated gastrin compared with nonsulfated gastrin and cholecystokinin.
Gastroenterology
91:
1154-1163,
1986[ISI][Medline].
7.
De Weerth, A,
Pisegna JR,
and
Wank SA.
Guinea pig gallbladder and pancreas possess identical CCK-A receptor subtypes: receptor cloning and expression.
Am J Physiol Gastrointest Liver Physiol
265:
G1116-G1121,
1993
8.
Dufresne, M,
Escrieut C,
Clerc P,
Le Huerou-Luron I,
Prats H,
Bertrand V,
Le Meuth V,
Guilloteau P,
Vaysse N,
and
Fourmy D.
Molecular cloning, developmental expression and pharmacological characterization of the CCKB gastrin receptor in the calf pancreas.
Eur J Pharmacol
297:
165-179,
1996[ISI][Medline].
9.
Ghanekar, D,
Hadac EM,
Holicky EL,
and
Miller LJ.
Differences in partial agonist action at cholecystokinin receptors of mouse and rat are dependent on parameters extrinsic to receptor structure: molecular cloning, expression and functional characterization of the mouse type a cholecystokinin receptor.
J Pharmacol Exp Ther
282:
1206-1212,
1997
10.
Go, VLW,
DiMagno EP,
Gardner JD,
Lebenthal E,
Reber HA,
and
Scheele GA.
The Pancreas. Biology, Pathobiology, and Disease. New York: Raven, 1993.
11.
Gouldson, P,
Legoux P,
Carillon C,
Dumont X,
Le Fur G,
Ferrara P,
and
Shire D.
Essential role of extracellular charged residues of the human CCK1 receptor for interactions with SR 146131, SR 27897 and CCK-8S.
Eur J Pharmacol
389:
115-124,
2000[ISI][Medline].
12.
Hadac, EM,
Ghanekar DV,
Holicky EL,
Pinon DI,
Dougherty RW,
and
Miller LJ.
Relationship between native and recombinant cholecystokinin receptors-role of differential glycosylation.
Pancreas
13:
130-139,
1996[ISI][Medline].
13.
Hadac, EM,
Ji ZS,
Pinon DI,
Henne RM,
Lybrand TP,
and
Miller LJ.
A peptide agonist acts by occupation of a monomeric G protein-coupled receptor: dual sites of covalent attachment to domains near TM1 and TM7 of the same molecule make biologically significant domain-swapped dimerization unlikely.
J Med Chem
42:
2105-2111,
1999[ISI][Medline].
14.
Hadac, EM,
Pinon DI,
Ji Z,
Holicky EL,
Henne R,
Lybrand T,
and
Miller LJ.
Direct identification of a second distinct site of contact between cholecystokinin and its receptor.
J Biol Chem
273:
12988-12993,
1998
15.
Hoshi, H,
and
Logsdon CD.
Both low- and high-affinity CCK receptor states mediate trophic effects on rat pancreatic acinar cells.
Am J Physiol Gastrointest Liver Physiol
265:
G1177-G1181,
1993
16.
Jagerschmidt, A,
Popovici T,
O'Donohue M,
and
Roques BP.
Identification and characterization of various cholecystokinin B receptor mRNA forms in rat brain tissue and partial determination of the cholecystokinin B receptor gene structure.
J Neurochem
63:
1199-1206,
1994[ISI][Medline].
17.
Ji, ZS,
Hadac EM,
Henne RM,
Patel SA,
Lybrand TP,
and
Miller LJ.
Direct identification of a distinct site of interaction between the carboxyl-terminal residue of cholecystokinin and the type A cholecystokinin receptor using photoaffinity labeling.
J Biol Chem
272:
24393-24401,
1997
18.
Kopin, AS,
Lee YM,
McBride EW,
Miller LJ,
Lu M,
Lin HY,
Kolakowski LF,
and
Beinborn M.
Expression cloning and characterization of the canine parietal cell gastrin receptor.
Proc Natl Acad Sci USA
89:
3605-3609,
1992[Abstract].
19.
Le Drean, G,
Huerou-Luron I,
Gestin M,
Desbois C,
Rome V,
Bernard C,
Dufresne M,
Moroder L,
Gully D,
Chayvialle JA,
Fourmy D,
and
Guilloteau P.
Exogenous CCK and gastrin stimulate pancreatic exocrine secretion via CCK-A but also via CCK-B/gastrin receptors in the calf.
Pflügers Arch
438:
86-93,
1999[ISI][Medline].
20.
Le Dréan, G,
Huërou-Luron I,
Gestin M,
Romé V,
Bernard C,
Chayvialle JA,
Fourmy D,
and
Guilloteau P.
Pancreatic secretory response to feeding in the calf: CCK-A receptors, but not CCK-B/gastrin receptors are involved.
Can J Physiol Pharmacol
78:
813-819,
2000[ISI][Medline].
21.
Li, Y,
and
Owyang C.
Endogenous cholecystokinin stimulates pancreatic enzyme secretion via vagal afferent pathway in rats.
Gastroenterology
107:
525-531,
1994[ISI][Medline].
22.
Morisset, J,
Levenez F,
Corring T,
Benrezzak O,
Pelletier G,
and
Calvo E.
Pig pancreatic acinar cells possess predominantly the CCK-B receptor subtype.
Am J Physiol Endocrinol Metab
271:
E397-E402,
1996
23.
Nakata, H,
Matsui T,
Ito M,
Taniguchi T,
Naribayashi Y,
Arima N,
Nakamura A,
Kinoshita Y,
Chihara K,
Hosoda S,
and
Chiba T.
Cloning and characterization of gastrin receptor from ECL carcinoid tumor of Mastomys natalensis.
Biochem Biophys Res Commun
187:
1151-1157,
1992[ISI][Medline].
24.
Pearson, RK,
Powers SP,
Hadac EM,
Gaisano H,
and
Miller LJ.
Establishment of a new short, protease-resistant, affinity labeling reagent for the cholecystokinin receptor.
Biochem Biophys Res Commun
147:
346-353,
1987[ISI][Medline].
25.
Pisegna, JR,
De Weerth A,
Huppi K,
and
Wank SA.
Molecular cloning of the human brain and gastric cholecystokinin receptor: structure, functional expression and chromosomal localization.
Biochem Biophys Res Commun
189:
296-303,
1992[ISI][Medline].
26.
Reuben, M,
Rising L,
Prinz C,
Hersey S,
and
Sachs G.
Cloning and expression of the rabbit gastric CCK-A receptor.
Biochim Biophys Acta
1219:
321-327,
1994[ISI][Medline].
27.
Saillan-Barreau, C,
Dufresne M,
Clerc P,
Sanchez D,
Corominola H,
Moriscot C,
Guy-Crotte O,
Escrieut C,
Vaysse N,
Gomis R,
Tarasova N,
and
Fourmy D.
Evidence for a functional role of the cholecystokinin-B/gastrin receptor in the human fetal and adult pancreas.
Diabetes
48:
2015-2021,
1999[Abstract].
28.
Silvente-Poirot, S,
Escrieut C,
and
Wank SA.
Role of the extracellular domains of the cholecystokinin receptor in agonist binding.
Mol Pharmacol
54:
364-371,
1998
29.
Smith, JP,
Liu G,
Soundararajan V,
McLaughlin PJ,
and
Zagon IS.
Identification and characterization of CCK-B/gastrin receptors in human pancreatic cancer cell lines.
Am J Physiol Regulatory Integrative Comp Physiol
266:
R277-R283,
1994
30.
Soudah, HC,
Lu Y,
Hasler WL,
and
Owyang C.
Cholecystokinin at physiological levels evokes pancreatic enzyme secretion via a cholinergic pathway.
Am J Physiol Gastrointest Liver Physiol
263:
G102-G107,
1992
31.
Ulrich, CD,
Ferber I,
Holicky E,
Hadac EM,
Buell G,
and
Miller LJ.
Molecular cloning and functional expression of the human gallbladder cholecystokinin A receptor.
Biochem Biophys Res Commun
193:
204-211,
1993[ISI][Medline].
32.
Wank, SA,
Harkins R,
Jensen RT,
Shapira H,
De Weerth A,
and
Slattery T.
Purification, molecular cloning, and functional expression of the cholecystokinin receptor from rat pancreas.
Proc Natl Acad Sci USA
89:
3125-3129,
1992[Abstract].
33.
Wank, SA,
Pisegna JR,
and
De Weerth A.
Cholecystokinin receptor family. Molecular cloning, structure, and functional expression in rat, guinea pig, and human.
Ann NY Acad Sci
713:
49-66,
1994[Abstract].
34.
Weinberg, DS,
Ruggeri B,
Barber MT,
Biswas S,
Miknyocki S,
and
Waldman SA.
Cholecystokinin A and B receptors are differentially expressed in normal pancreas and pancreatic adenocarcinoma.
J Clin Invest
100:
597-603,
1997