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 andhuman 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 EC 5 0 of 1.2 nM. In contrast, dY-bombesin had a very poor potency for rat BRS-3 (EC 5 0 = 2μM). 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 (EC 5 0 = 1.8 ′ 0.5 nM). Within the E3 loop, mutation of Y 2 9 8 E 2 9 9 S 3 0 0 to S 2 9 8 Q 2 9 9 T 3 0 0 (RB3-SQT) or of D 3 0 6 V 3 0 7 P 3 0 8 to A 3 0 6 M 3 0 7 H 3 0 8 (RB3-AMH) only partially mimicked the effect of switching the entire E3 loop, and mutation of A 3 0 2 E 3 0 3 to V 3 0 2 D 3 0 3 or of V 3 1 0 V 3 1 1 to I 3 1 0 F 3 1 1 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.