High-affinity binding sites for bombesin on mouse colonic mucosal membranes

Bombesin (BBS) has specific biological effects on colonic mucosal cells, but the presence of BBS receptors on colonic mucosa have not been described to-date. In the present study we examined the mouse colonic mucosal membranes for the presence of specific binding sites for BBS/gastrin releasing peptides (GRP), and characterized the binding kinetics and molecular weight of the specific binding proteins. The radiolabeled ligand (¹²⁵I-Tyr⁴-BBS), in the absence or presence of a 1000-fold excess of BBS, was used to establish the optimal binding assay conditions of time, pH and temperature for measuring the maximum number of specific binding sites for BBS related peptides. Under the optimal binding assay conditions, BBS displaced the binding of ¹²⁵I-Tyr⁴-BBS in a dose-related manner. A single class of high-affinity binding sites (Kd = 0.23 ±0.02 nM) for BBS were measured, with a binding capacity of 27.3 + 4.6 fmoles/mg membrane protein. The binding sites were specific for binding BBS/GRP related peptides, since all structurally related peptides inhibited the binding of ¹²⁵I-Tyro-BBS in a dose-dependent manner, while structurally unrelated peptides did not compete for the ¹²⁵I-Tyro-BBS binding sites. The relative binding affinity (RBA) of BBS/GRP related peptides was determined to be in the order of GRP (14-27) = GRP (18-27) > GRP (1-27) > neuromedin B > BBS. The BBS-receptor antagonists, [Leu¹³-ψ-(CH₂NH) Lcu¹⁴]-BBS (LL-BBS) and D-Phe⁶, BN(6-13) propylamide (D-Phe⁶,BN(6-13)-PA), inhibited the specific binding of ¹²⁵I-Tyr⁴-BBS to colonic mucosal membranes in a dose-dependent manner. The inhibitory potency of D-Phe⁶,BN[6-13]PA was significantly greater than that of LL-1313S. Molecular mass of the specific binding proteins for BBS/GRP was determined to be 70-80 KDa, by chemical cross-linking methods. The 70-80 KDa binding proteins were specific for binding GRP-related peptides and were displaced in dose-dependent manner by increasing doses of BBS. These results thus suggest that the colonic mucosa may be yet another target for GRP related peptides.