Molecular Basis of the Pharmacological Difference between Rat and Human Bombesin Receptor Subtype-3 (BRS-3)

We cloned the gene and cDNA for rat bombesin receptor subtype-3 (BRS-3) and characterized its mRNA expression pattern and pharmacological properties. Despite the high degree of sequence similarity (80% identical), rat and human BRS-3 differ markedly in their pharmacological properties. Although the natural ligand for BRS-3 is still unknown, a synthetic peptide, dY-Q-W-A-V-(β-A)-H-F-Nle-amide (dY-bombesin), activates human BRS-3 with an EC50 of 1.2 nM. In contrast, dY-bombesin had a very poor potency for rat BRS-3 (EC50 = 2 μM). To understand the molecular basis of this pharmacological difference, we constructed chimeric receptors in which individual extracellular loops of rat BRS-3 were replaced with the corresponding human sequences. Switching the N-terminal region or the second extracellular loop did not significantly change receptor properties. However, switching the third extracellular loop (E3) in the rat BRS-3 resulted in a chimeric receptor (RB3-E3) that behaved almost identically to human BRS-3. RB3-E3 bound dY-bombesin with high affinity (K i = 1.2 ± 0.7 nM), and was activated by dY-bombesin with high potency (EC50 = 1.8 ± 0.5 nM). Within the E3 loop, mutation of Y298E299S300 to S298Q299T300 (RB3-SQT) or of D306V307P308 to A306M307H308 (RB3-AMH) only partially mimicked the effect of switching the entire E3 loop, and mutation of A302E303 to V302D303 or of V310V311 to I310F311 had little effect on the dY-bombesin potency. These results indicate that the sequence variation in the E3 loop is responsible for the species difference between rat and human BRS-3, and multiple residues in the E3 loop are involved in interactions with the agonist dY-bombesin.