Characterization of a bombesin/gastrin-releasing peptide receptor on a human gastric-cancer cell line

This study examined the expression of receptors of the bombesin (BBS) family in human gastric-cancer cell lines. Of 5 cell lines screened, only one, St42, demonstrated specific binding sites for ¹²⁵I-Tyr⁴-BBS, which have been further characterized. This binding was saturable, and temperature- and time-dependent. Scatchard analysis of displacement data performed at 37°C revealed 2 binding sites: a high-affinity, low-capacity site (K_D = 0.13 nM, B_max = 1500 sites/cell) and a lower-affinity, higher-capacity site (K_D = 11 nM, B_max = 35,000 sites/cell); the latter was lost when internalization of peptide was prevented, suggesting that it may be an artefact. Displacement assays with gastrin-releasing peptide (GRP) and neuromedin B (NMB) revealed that the receptor was of the GRP-preferring sub-type (GRP IC₅₀ = 0.35 nM; NMB IC₅₀ = 112 nM). Co-valent cross-linking of ¹²⁵I-Tyr⁴-BBS to the receptor demonstrated the presence of a single band corresponding to a molecular weight of 37 to 44 kDa on SDS-PAGE, similar to that of the cloned GRP receptor protein core. G-protein linkage of this receptor was demonstrated by selective inhibition of ¹²⁵I-Tyr⁴-BBS binding by guanosine nucleotides. The binding of BBS to the receptor resulted in a rise in intracellular calcium. Three of four structurally distinct BBS antagonists bound to the receptor with high affinity, but [DPhe¹², Leu¹⁴]-bombesin did not cause any displacement of ¹²⁵I-Tyr⁴-BBS even at 10 mM. The functional significance of GRP receptors on human gastric-cancer cells is as yet unknown, but further studies may determine whether such receptors have importance in the therapy of gastric cancer.