Antibodies raised against the extracellular tail of the CCKB/gastrin receptor inhibit gastrin-stimulated signalling (original) (raw)
Related papers
Expression of CCKB/gastrin receptor isoforms in gastro‐intestinal tumour cells
International Journal of Cancer, 1998
Anti-serum raised against the human cholecystokinin B (CCKB)/gastrin receptor was used in Western blotting to differentiate the cellular locations of receptor isoforms expressed by human gastro-intestinal (GI) tumour cell lines. Using anti-serum directed against the amino-terminal extracellular tail of the CCKB/gastrin receptor, 8/9 cell lines were shown to express immunoreactive proteins of either m.w. 70 or 40 kDa, or both. Both isoforms were found to be associated with intracellular, non-nuclear membranes, whereas only the 70 kDa protein was expressed in the plasma membrane. Receptor expression was related to gastrin production and secretion. Both progastrin and glycine-extended gastrin-17 were produced and secreted by the tumour cell lines; however, carboxy amidated gastrin was not detected by radioimmunoassay. A CCKB/gastrin receptor transfectant NIH3T3 cell line did not produce detectable gastrin and showed exclusive expression of the 70 kDa receptor on the plasma membrane. One cell line had Ͻ50 pg/ml cell-associated progastrin and no detectable receptor form. Cell lines expressing 50-150 pg/ml had both 40 and 70 kDa receptor forms. Those expressing Ͼ150 pg/ml progastrin had only the 40 kDa isoform, which was shown to be exclusively expressed on intracellular, non-nuclear membranes, in one of the cell lines. Of the 4 cell lines exclusively expressing the lower m.w. receptor, 3 had gastrin present within the cell, which was not secreted. Thus, if cell-associated gastrin induces a proliferative effect, it may be by an intracrine pathway. Our study has identified the presence of CCKB/gastrin receptor isoforms in different cellular locations and may help toward understanding the complex autocrine and intracrine pathways mediated by gastrin peptides. Int.
FEBS Letters, 2004
and N-carboxymethylgastrin (G17-Gly) have been shown to stimulate the growth of colon cancer cells both in vivo and in vitro. The identity of the receptor mediating these e¡ects is controversial. A recent study demonstrated the presence of a low a⁄nity binding site for G17 and G17-Gly on the DLD-1 human colon cancer cell line. The goal of the current study was to further investigate the role of this receptor in mediating the growth-promoting e¡ects of gastrin peptides. Binding of [Leu 15 ]G17 and [Leu 15 ]G17-Gly to DLD-1 cell membranes in competition with [ 3 H]G17-Gly was examined. Binding of [ 3 H]cholecystokinin-8 (CCK8) to DLD-1 cell membranes was also assessed. Whole cell binding experiments were carried out using [ 125 I-Tyr 12 ,Leu 15 ]G17-Gly. In addition, the ability of [Leu 15 ]G17 and [Leu 15 ]G17-Gly to stimulate cell growth, as determined by cell counting, was tested. [Leu 15 ]G17 and [Leu 15 ]G17-Gly competed with [ 3 H]G17-Gly at both a high and a low a⁄nity site on DLD-1 membranes. The IC 50 values for [Leu 15 ]G17 were 6.0U U10 38 M and 6.9U U10 36 M while those for [Leu 15 ]G17-Gly were 3.2U U10 39 M and 4.9U U10 36 M. [ 3 H]CCK8 did not bind to either site. [Leu 15 ]G17-Gly also competed with [ 125 I-Tyr 12 ,Leu 15 ]G17-Gly at both a high and a low a⁄nity site on DLD-1 cells with similar a⁄nities as observed with membranes. [Leu 15 ]G17 and [Leu 15 ]G17-Gly signi¢cantly stimulated the growth of DLD-1 cells in a dose-dependent and biphasic manner. The binding pro¢les of the peptides tested suggest that these sites are di¡erent from previously identi¢ed wild-type and mutant CCK 1 or CCK 2 receptors. ß
European Journal of Pharmacology: Molecular Pharmacology, 1995
We have shown that gastrin and cholecystokinin octapeptide (CCK-8) are differently coupled to G protein (GTP-binding protein) through type B cholecystokinin receptors in guinea-pig brain membranes and Jurkat cells. Indeed, the gastrin-13 binding affinity is strongly reduced by stable guanyl nucleotides, whereas CCK-8 binding is only slightly affected. In order to determine the structural requirements regulating such coupling, we have synthesized several gastrin and choleeystokinin fragments (sulphated or unsulphated) elongated at the N-terminus of the common C-terminal tetrapeptide. We investigated their interaction with CCK s receptors in guinea pig brain membranes and Jurkat cells and their involvement in the G protein coupling. Their apparent binding affinities to CCK u receptors were measured by inhibition of [l~I]Bolton Hunter-CCK-8 (3-[125I]iodo-4-hydroxyphenyl)propionyl-CCK-8) binding in the presence or absence of GTPyS (guanosine 5'-O-(3-thio)triphosphate) or aluminium tetrafluoride (AIF~). Activation of the G proteins by GTPyS or AIF 4-led to a decrease in binding affinity for the gastrin related peptides, the common CCK-gastrin C-terminal forms, the cholecystokinin hexapeptide and the unsulphated cholecystokinin heptapeptide. Sulphated CCK-7, CCK-8, and cionin apparent binding affinities were not affected. These finding indicated that the sulphated tyrosine in position 7 in CCK (as counted from the C-terminus), provides the cholecystokinin selectivity for the CCK B receptor compared to gastrin. The results are discussed with the aim to better clarify the physiological relevance of gastrin and cholecystokinin toward CCK B receptors and their related intracellular events.
Anticancer research
Due to their high CCK-2/gastrin receptor selectivity, high affinity, and rapid background clearance, radiolabeled minigastrins (MG) are emerging as promising new tools in the diagnosis and therapy of CCK-2/gastrin receptor-positive tumors. In this study, the pharmacokinetic profile, particularly the excretion mode, of two 111In-labeled minigastrins was compared in rats. The first tracer, 111In-MG-0 is based on (D)Glu1-MG, while the second, 111In-MG-11, is its des-(Glu)5-derivative, expected to be less retained in renal tissue. The fate of 111In-MG-0 and 111In-MG-11 in the body of rats was investigated during biodistribution and bioelimination experiments, while the respective elimination parameters were determined in perfused rat liver and kidney models. During biodistribution both compounds were rapidly cleared from the blood and most non-target organs whereas activity levels in the bowel and stomach declined slowly. The overall contribution of hepatobiliary excretion of 111In-MG-0...
Gastroenterology, 2005
Background & Aims: The role of amidated gastrin 17 (G17) and the gastrin/CCKB/CCK2 receptor in colorectal carcinogenesis is still a controversial issue. Here, we investigated the effect of G17 on proliferation and apoptosis of CCK2 receptor-expressing human colon cancer cell lines in vitro and in vivo. Methods: Proliferation was determined by cell counting and cell cycle analysis. Apoptosis was analyzed by annexin V staining, TUNEL staining, caspase-3/7 assay, and JC1 (⌬⌿) assay. Signal-transduction pathways were analyzed by Western blotting and gelshift and luciferase assays. An in vivo tumor model with subcutaneously inoculated colon cancer cells in SCID mice was used, and systemic hypergastrinemia was induced by omeprazole. Results: In Colo320 cells stably transfected with the wild-type CCK2 receptor (Colo320wt) or in Lovo cells endogenously expressing CCK2 receptors, G17 treatment inhibited proliferation along with a G2/M cell cycle arrest. Furthermore, the administration of G17 significantly augmented apoptosis of CCK2 receptor-expressing cells. In contrast, G17 had no effect on proliferation and apoptosis in Colo320 cells stably transfected with a tumor-derived CCK2 receptor mutant (Colo320mut) or in cells lacking CCK2 receptor expression. Systemic hypergastrinemia in severe combined immunodeficiency (SCID) mice suppressed the growth of Colo320wt tumors accompanied by enhanced apoptosis as compared with untreated tumors. In contrast, omeprazole did not affect Colo320mut tumors reflecting a loss-offunction state of the CCK2(mut) receptor. This is supported by the observation that, in Colo320wt cells, but not in Colo320mut cells, G17 treatment induced the MAPK/ERK/AP-1 pathway and inhibited the activity of NF-B. Conclusions: G17 exerts an antiproliferative and proapoptotic effect on human colon cancer cells expressing the wild-type CCK2 receptor. This supports the view that amidated gastrin prevents rather than promotes colorectal carcinogenesis.
Cancer research, 2000
Serum gastrin is known to be elevated in patients with liver-metastasizing colon cancer; thus, cholecystokinin (CCK) B/gastrin receptors may also be up-regulated. A liver-invasive model of colon cancer was established with the human colonic cell line C170HM2, which expresses the CCKB/gastrin receptor at both the gene and protein level. An antiserum has been derived that is directed against the NH2-terminal 17 amino acids of the human CCKB/gastrin receptor coupled to diphtheria toxoid. The peptide was denoted gastrin receptor protein (GRP) 1. The therapeutic effect of GRP1 antiserum was evaluated on the liver invasion of C170HM2 tumors. Biodistribution studies revealed that GRP1 antiserum localized preferentially within the liver tumors when compared with normal liver tissue (1.5-fold increase after 24 h; P < 0.05). Antiserum against GRP1 inhibited both tumor take rate and final liver tumor weight when compared with treatment with control serum in mice with an increasing tumor bur...
Comparative biodistribution of 12 111In-labelled gastrin/CCK2 receptor-targeting peptides
European Journal of Nuclear Medicine and Molecular Imaging, 2011
Purpose Cholecystokinin 2 (CCK-2) receptor overexpression has been demonstrated in various tumours such as medullary thyroid carcinomas and small-cell lung cancers. Due to this high expression, CCK-2 receptors might be suitable targets for radionuclide imaging and/or radionuclide therapy. Several CCK-2 receptor-binding radiopeptides have been developed and some have been tested in patients. Here we aimed to compare the in vivo tumour targeting properties of 12 111 Inlabelled 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-conjugated gastrin/CCK2 receptor-binding peptides. Methods Two CCK8-based peptides and ten gastrin-based peptide analogues were tested. All peptides were conjugated with DOTA and labelled with 111 In. Biodistribution studies were performed in mice with subcutaneous CCK2/
Characterization of gastrin-cholecystokinin 2 receptor interaction in relation to c-fos induction
Endocrine Related Cancer, 2008
The interaction of gastrin with the cholecystokinin 2 (CCK2)/gastrin receptor has been studied extensively in relation to gastric acid secretion. However, not much is known about the contribution of individual amino acids of gastrin interacting with the CCK2 receptor, when gastrin is acting as a tumor growth factor. The purpose of the present study was to determine the significance of each individual amino acid residue of human gastrin-17 with respect to CCK2 receptor-mediated cell proliferation. Activation of this receptor was assessed using an in vitro bioassay based on gastrininduced expression of a c-fos-luciferase reporter, transfected in AR42JB13 and Colo 320 cells, a rat pancreatic and human colorectal cell line respectively. Gastrin-17 dose dependently increased c-fos induction in both cancer cell lines. L365,260, a known CCK2 receptor antagonist, completely blocked the gastrin signal, demonstrating the specificity of this assay. We demonstrated for the first time that four carboxy-terminal amino acids of gastrin-17 are essential for activation of the CCK2 receptor with respect to c-fos induction. Also other residues of gastrin-17, notably glycine-2 for the rat CCK2 receptor and glutamic acid 8-10 and tyrosine-12 for the human receptor, were found to be important, although to a lesser extent. Alanine-substitution variants of each of the four carboxy-terminal amino acids of gastrin-17 showed strongly reduced receptor activation but did not act as competitive inhibitors of gastrin-17. Identification of the essential role of the carboxyterminal tetrapeptide of gastrin-17 in CCK2 receptor-mediated c-fos induction indicates that gastrin inhibitory therapeutic strategies should mainly be targeted toward this region of gastrin.