| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2002: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 2001: ¥2,700,000 (Direct Cost: ¥2,700,000)
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| Research Abstract |
Initially gastrin is well-characterized as a stimulant of acid secretion. The peptide hormone gastrin also exerts growth-promoting effects on normal and malignant gastrointestinal tissues. Progastrin is post-translationally processed to the glycine-extended forms of gastrin-34 and gastrin-17 (G34-Gly and G17-Gly, respectively) which are then processed further by the action of the peptide a-amidating enzyme to form carboxyl-terminally amidated peptides G34-NH2 and G17-NH2. Although G-Gly has been considered a biosynthetic processing intermediate devoid of biological activity, we recently reported that G-Gly has growth-promoting effects mediated by a selective receptor distinct from the gastrin/CCKB receptor. In view of the potential importance of this receptor as a mediator of cellular growth, we undertook these studies to characterize its pharmacological properties and isolate a cDMA encoding the receptor. For these studies we utilized a pancreatic acinar cell cancer cell line, AR4-2J,
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and a gastric cancer derived cell line, AGS, which exhibits a dose-dependent proliferative response to G-Gly. 125I-Leu15G(2-17)-Gly binding to AR4-2J cells was dose-dependently inhibited by G(2-17)-Gly, but not by G17-NH2, CCK8, nor by the gastrin/CCKB receptor antagonists L365,260 or PD-134308. Displacement of binding was also observed with G17-Gly ligands truncated at the amino (G(5-17)-Gly and G(12-17)-Gly) and carboxyl termini (G17(1-14)), however, these peptides were much less potent than G(2-17)-Gly. 125I-Leu15G(2-17)-Gly binding could not be inhibited by progastrin peptides with extensions beyond the carboxyl-terminus (G(12-17)-GlyArgArg and G(12-17)-GlyArgArgSerAla) nor G-Gly molecules extended at the amino-terminus of G17-Gly(G34-Gly). Further characterization of the G-Gly receptor was undertaken through affinity crosslinking of 125I-Leu15G(2-l7)-Gly to AR4-2J cell membranes using disuccinimidyl suberate as a linking reagent. Two crosslinked bands (159 kDa and 46 kDa) were obtained that exhibited dose-dependent inhibition of binding with G(2-17)-Gly but not with G17-NH2. Less
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