Identification of a 70-kDa gastrin-binding protein on DLD-1 human colorectal carcinoma cells.

Gastrin17gly acts as a growth factor for the colonic mucosa. Studies of the receptor involved have generally been restricted to its binding properties, and no investigation of the structure of gastrin17gly receptors on human colorectal carcinoma cell lines has yet been reported. The aim of this study was to optimise the conditions for binding of gastrin17gly to the human colorectal carcinoma cell line DLD-1, and to investigate the structure of the receptor responsible. Binding of 125I[Met15]gastrin17gly to DLD-1 cells was measured in competition experiments with increasing concentrations of either gastrin17gly or gastrin17, or with single concentrations of gastrin receptor antagonists. The molecular weights of the gastrin17gly binding proteins were determined by gel electrophoresis and autoradiography after covalent cross-linking of 125I[Nle15]gastrin2,17gly to cells or membranes with disuccinimidyl suberate. The IC50 value for binding of gastrin17gly to DLD-1 cells was 2.1+/-0.4 microM. Binding was inhibited by the non-selective gastrin/cholecystokinin receptor antagonists proglumide and benzotript, but not by the cholecystokinin-A receptor antagonist L364,718, or the gastrin/cholecystokinin-B receptor antagonist L365,260. The molecular weight of the major gastrin binding protein on DLD-1 cells or membranes was 70,000. We conclude that the major gastrin17gly binding site on the human colorectal carcinoma cell line DLD-1 is clearly distinct from the cholecystokinin-A and gastrin/cholecystokinin-B receptors, but is similar in some respects to the gastrin/cholecystokinin-C receptor.

Binding sites for progastrin-derived peptides in colonic crypts.

BACKGROUND AND AIMS: Gastrin17gly acts as a growth factor for the colonic mucosa. Studies on the binding properties of the receptor involved in transducing the proliferative effects have generally been confined to colorectal carcinoma cell lines, and no investigation of gastrin17gly receptors on normal colonocytes has yet been reported. The aim of this study was to investigate the binding of 125I-[Met15]-gastrin17gly to normal colonic crypts. METHODS: Crypts were released from normal rat and rabbit colonic mucosa by treatment with EDTA and isolated by centrifugation. The binding of 125I-[Met15]-gastrin17gly was measured in displacement experiments with increasing concentrations of either gastrin17gly, gastrin17 or gastrin receptor antagonists. The concentrations required for 50% inhibition were determined by the use of curve fitting. RESULTS: 125I-[Met15]-Gastrin17gly bound to both rat and rabbit crypts, and displacement experiments with unlabeled gastrin17gly revealed that the IC50 values were 1.0 +/- 0.6 and 0.6 +/- 0.2 micromol/L, respectively. Binding was also competed by gastrin17, with IC50 values of 2.4 +/- 1.7 and 2.4 +/- 0.7 micromol/L, respectively. Binding was inhibited by the non-selective gastrin/CCK receptor antagonists proglumide and benzotript, but not by the cholecystokinin (CCK)-A receptor antagonist L364 718, or the gastrin/CCK-B receptor antagonist L365 260. CONCLUSION: We conclude that the gastrin17gly binding site on normal colonic crypts has properties consistent with the gastrin/CCK-C receptor.

Biologically active recombinant human progastrin(6-80) contains a tightly bound calcium ion.

Evidence is accumulating that gastrin precursors may act as growth factors for the colonic mucosa in vivo. The aims of this study were to prepare recombinant human progastrin(6-80) and to investigate its structure and biological activities in vitro. Human progastrin(6-80) was expressed in Escherichia coli as a glutathione S-transferase fusion protein. After thrombin cleavage progastrin(6-80) was purified by reverse phase high pressure liquid chromatography and characterized by radioimmunoassay, amino acid sequencing, and mass spectrometry. Assays for metal ions by atomic emission spectroscopy revealed the presence of a single tightly bound calcium ion. Progastrin(6-80) at concentrations in the pm to nm range stimulated proliferation of the conditionally transformed mouse colon cell line YAMC. The observations that progastrin(6-80) did not bind to either the cholecystokinin (CCK)-A or the gastrin/CCK-B receptor expressed in COS cells and that antagonists selective for either receptor did not reverse the proliferative effects of progastrin(6-80) suggested that progastrin(6-80) stimulated proliferation independently of either the CCK-A or the gastrin/CCK-B receptor. We conclude that recombinant human progastrin(6-80) is biologically active and contains a single calcium ion. With the exception of the well known zinc-dependent polymerization of insulin and proinsulin, this is the first report of selective, high affinity binding of metal ions to a prohormone.

Overexpression of sense or antisense human gastrin mRNA does not affect proliferation of normal rat kidney fibroblasts.

Progastrin-derived peptides have been reported to stimulate mitogenesis in Swiss 3T3 fibroblasts [P. Singh, A. Owlia, R. Espeijo, B. Dai, Novel gastrin receptors mediate mitogenic effects of gastrin and processing intermediates of gastrin on Swiss 3T3 fibroblasts: Absence of detectable cholecystokinin (CCK)-A and CCK-B receptors. J. Biol. Chem. 270 (1995) 8429-8438]. The aim of the present study was to determine the generality of these findings, by investigating the effect of endogenous and exogenous progastrin-derived peptides on the proliferation of the normal rat kidney fibroblast cell line NRK. Levels of endogenous progastrin-derived peptides were modified by stable transfection of NRK cells with tetracycline-repressible plasmids containing sequences encoding human gastrin in either the sense or antisense orientation. Expression of sense and antisense gastrin mRNA was demonstrated by reverse transcriptase PCR and by radioimmunoassay, and cell proliferation rates were determined by the colorimetric MTT assay. Sense clones produced full length human progastrin, but significant quantities of glycine-extended or amidated gastrin17 were not detected. Concentrations of endogenous rat progastrin in antisense clones were significantly lower than concentrations in clones transfected with vector only. However no difference in proliferation rate was observed between sense, antisense and vector-transfected clones. No stimulation of proliferation was observed in synchronised untransfected NRK cells after supplementation of media with gastrin17 or gastrin17gly in the concentration range 0.3 to 100 nM. Our results do not provide evidence in support of the hypothesis that endogenous or exogenous progastrin-derived peptides act as growth factors in NRK fibroblasts.