Bombesin receptor in membranes from Swiss 3T3 cells: binding charcteristic, affinity labelling and modulation by guanine nucleotides

Abstract

Bombesin-like neuropeptides, including mammalian gastrin-releasing peptide (GRP), are potent mitogens for Swiss 3T3 cells. In this study, we have characterized the bombesin receptor in membrane preparations from these cells. Addition of Mg2+ during cell homogenization was essential to preserve 125I-GRP binding activity in the resulting membrane preparation. The effect of Mg2+ was concentration dependent, with a maximum at 5mM. Specific binding of 125I-GRP was saturable; Scatchard analysis indicated a single class of high-affinity sites of K(d) = (2.1±0.3) x 10-10 M at 15 °C and K(d) = (1.9±0.4) x 10-10 M at 37 °C, and a maximum binding capacity of 580±50 fmol/mg of protein (15 °C) or 604±40 fmol/mg of protein (37 °C). The kinetically derived dissociation constant was 1.5 x 10-10 M. 125I-GRP binding was inhibited in a concentration-dependent manner by various peptides containing the highly conserved C-terminal heptapeptide of the bombesin family, including bombesin, GRP, neuromedin B and the 8-14 fragment of bombesin. In contrast, a variety of structurally unrelated mitogens and neuropeptides had no effect. The cross-linking agent ethyleneglycolbis(succinimidylsuccinate) covalently linked 125I-GRP to a single M(r) 75000-85000 protein in membrane preparations of 3T3 cells. Affinity labelling of this molecule was specific and dependent on the presence of Mg2+ during membrane preparation. Finally, the non-hydrolysable GTP analogue guanosine-5'-[γ-thio]triphosphate (GTP[S]) caused a concentration-dependent inhibition of 125I-GRP binding and cross-linking to 3T3 cell membranes [concentration giving half-maximal inhibition (IC50)~0.2 μM]. The inhibitory effect was specific (GMP, ATP or ATP[S] had no effect at 10 μM) and was due to an increase in K(d) from (1.7±0.2) x 10-10 M to (4.3±0.6) x 10-10 M in the presence of 10 μM-GTP[S]. This modulation of ligand affinity and cross-linking implies that the bombesin receptors that mediate mitogenesis in Swiss 3T3 cells are coupled to a guanine-nucleotide-binding-protein signal-transduction pathway.