Structure-activity requirements of bombesin for gastrin-releasing peptide- and neuromedin B-preferring bombesin receptors in rat brain

Abstract The pharmacological profile of [ 125 I][Tyr 4 ]bombesin binding to gastrin-releasing peptide- and neuromedin B-preferring sites has been investigated in rat cerebral cortex and olfactory bulb membranes, respectively. [ 125 I][Tyr 4 ]bombesin specific binding to cerebral cortex membranes was displaced biphasically by gastrin releasing peptide and [D-Phe 6 ]bombesin-(6–13)-ethyl amide. In the presence of 10 nM neuromedin B, displacement curves for bombesin-related peptides were monophasic with gastrin releasing peptide displaying approximately 100-fold higher affinity than neuromedin B. In olfactory bulb membranes, [ 125 I][Tyr 4 ]bombesin binding was also displaced biphasically by gastrin releasing peptide, [D-Phe 6 ]bombesin-(6–13)-ethyl amide and neuromedin B. In the presence of 10 μM [D-Phe 6 ]bombesin-(6–13)-ethyl ester, displacement curves were monophasic with neuromedin B possessing approximately 10-fold higher affinity than gastrin-releasing peptide. Under these conditions, successive deletion of N-terminal amino acids from bombesin-(1–14) was well tolerated at both sites, with little loss in affinity up to bombesin-(5–14). A 5- to 10-fold drop in affinity was observed at both sites with bombesin-(6–14), whilst the octapeptide acetyl-bombesin-(7–14) displayed similar affinities to bombesin-(1–14). Bombesin-(8–14), -(9–14) and -(10–14) were essentially inactive (IC 50 > 10 μM). C-terminal deletion of Met 14 (bombesin-(1–13)) resulted in 100-fold loss of affinity at the gastrin-releasing peptide site and complete loss of affinity at the neuromedin B site. Fragments smaller than bombesin-(1–13) were virtually inactive at either site. Replacement of consecutive amino acids in the minimal active fragment, acetyl-bombesin-(7–14), with L-alanine revealed the critical importance of Trp 8 and Leu 13 for binding to both sites.