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 degrees C revealed 2 binding sites: a high-affinity, low-capacity site (KD = 0.13 nM, Bmax = 1500 sites/cell) and a lower-affinity, higher-capacity site (KD = 11 nM, Bmax = 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.
Preston
S R
SR
Academic Unit of Surgery, St. James's University Hospital, Leeds, UK.
Woodhouse
L F
LF
Gokhale
J
J
Miller
G V
GV
Primrose
J N
JN
D000230:Adenocarcinoma / Q000737:chemistry* / Q000378:metabolism; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D004705:Endocytosis; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D008970:Molecular Weight; D018004:Receptors, Bombesin / Q000737:chemistry* / Q000378:metabolism; D015290:Second Messenger Systems; D015398:Signal Transduction; D013274:Stomach Neoplasms / Q000737:chemistry* / Q000378:metabolism; D014407:Tumor Cells, Cultured
D018004:Receptors, Bombesin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D002118:Calcium