Biologically Active Recombinant Human Progastrin6–80Contains a Tightly Bound Calcium Ion

Abstract

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.

Intestinal expression of mutant and wild‐type progastrin significantly increases colon carcinogenesis in response to azoxymethane in transgenic mice

“…Our group and others recently described a high-affinity progastrin receptor (PG-R) that preferentially binds progastrinlike peptides and mediates the biologic effects of PG on target cells. 13,14,19 Thus, it appears likely that the cocarcinogenic effects of wildtype hPG and mutant hPG are mediated via the highaffinity PG-R binding sites that are present on colonic mucosal cells. 19 The results of the current study suggest that, in all probability, PG-R mediates the biologic effects of the wild-type and mutant forms of hPG equally well, however, the binding affinity of mtPG for the PG-R must be confirmed in an in vitro model.…”

“…8,9 In recent years, it has been discovered that nonamidated gastrins (e.g., G-Gly) exert potent growth factor effects in vitro on rat intestinal cells, 10 3T3 fibroblasts, 10 pancreatic carcinoma cells, 11 and colon carcinoma cells. 10,12 More recently, the growth factor effects of the full-length precursor peptide, PG, were demonstrated in vitro [13][14][15] and in vivo 16 -20 on small and large intestinal mucosal cells and on gastrointestinal carcinoma cell lines. Azoxymethane (AOM)-induced colonic tumors in rats, as well as a significant percentage of adenomas (Ads) and adenocarcinomas (AdCas) in humans, express the gastrin gene.…”

Annexin II binds progastrin and gastrin-like peptides, and mediates growth factor effects of autocrine and exogenous gastrins on colon cancer and intestinal epithelial cells

“…High-affinity binding sites (receptors?) were identified that were distinct from CCK 1 R, CCK 2 R and CCKC-R (Chicone et al, 1989;Narayan et al, 1992;Seva et al, 1994;Singh et al, 1995Singh et al, , 2003Baldwin et al, 2001). These novel binding sites remain unidentified to-date.…”

“…PG, GG and G17 exert proliferative effects on normal and cancerous gastrointestinal (GI) cells in vitro and in vivo (Wang et al, 1996;Hollande et al, 1997;Koh et al, 1999;Baldwin et al, 2001;Brown et al, 2003;Singh et al, 2003;Ottewell et al, 2005). PG and GG exert cocarcinogenic effects on colon carcinogenesis in response to azoxymethane (Singh et al, 2000a, b;Aly et al, 2001;Cobb et al, 2004).…”

Use of interfering RNA to investigate the role of endogenous gastrin in the survival of gastrointestinal cancer cells

“…However, in GI epithelial cells, processing is incomplete, resulting in secretion of the precursor peptide, progastrin, and a range of intermediate products, of which the most well-characterised is gly-gastrin (Rehfeld et al, 2004). Whereas amidated gastrin acts through the CCK-2 receptor (CCK-2R) (Shulkes and Baldwin, 1997), progastrin and gly-gastrin bind with low affinity to this receptor (Baldwin et al, 2001;Dockray et al, 2001). Furthermore, the biological effects of progastrin and gly-gastrin are not blocked by CCK-B receptor antagonists (Seva et al, 1994;Stepan et al, 1999;Baldwin et al, 2001), and amidated gastrin and gly-gastrin stimulate different downstream signalling events (Todisco et al, 2001).…”