In the present study, we characterized specific binding of bombesin (BBS)/gastrin-releasing peptide (GRP) to mouse colon cancer (MC-26) cells. MC-26 cells were inoculated into male BALB/c mice subdermally, and tumors were harvested from mice 21-28 days postinoculation. Tumor membranes were analyzed for binding to GRP-related peptides, using either '"I-GRP or 12*l-tyrosine4-BBS. Under optimal binding assay con ditions, BBS displaced specific binding of both '"I-GRP and 'â€¢"!tyrosine4-BBS in a dose-dependent manner, and a curvilinear displace ment resulted. Specific binding data, analyzed by either a Scatchard or a Lineweaver-Burk plot, demonstrated presence of 2 classes of specific binding sites, arbitrarily named type I and type II sites. Type I sites had a high binding affinity | A,, 0.45 Â±0.05 IIM (SE)j and a relatively low capacity (226 Â±27 fmol/mg membrane protein), whereas type II sites had a 10-20-fold lower binding affinity and ~6-7-fold higher capacity. BBS/GRP binding sites were specific for GRP-related peptides and demonstrated no significant binding affinity for all other unrelated pep tides tested. Relative binding affinity of GRP analogues was in the order of GRP (14-27) > neuromedin C Â»BBS * GRP (1-27) > neuromedin B (for the later, P > 0.05 versus other peptides). Two BBS receptor antagonists, |D-Arg',n-trp7-',Leu"]-substance P (spantide) and jLeu1'1-^(CH2NH)Leu"JBBS also inhibited specific binding of '"I-GRP in a dosedependent manner. Molecular weight of GRP/BBS binding proteins on tumor membranes was determined by cross-linking methods. A major molecular form (>80-90%) (M, -75,000) and a minor M, -180,000 band were evident, both under reducing and nonreducing conditions. BBS (0.5-50 IIM)demonstrated a significant dose-dependent growth effect on MC-26 cells in vitro, in terms of | 'I I]ili\mulino and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide uptake; these studies indicate that the BBS/GRP binding sites on MC-26 cells may serve as functional receptors and mediate the growth effects of BBS on MC-26 cells.
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