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-(beta-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 muM). 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 (YES300)-E-298-S-299 to S(298)Q(299)T(300) (RB3-SQT) or of (DVP308)-V-306-P-307 to A(306)M(307)H(308) (RB3-AMH) only partially mimicked the effect of switching the entire E3 loop, and mutation of A(302)E(303) to (VD303)-D-302 or of (VV311)-V-310 to (VF311)-F-310 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.