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 EC(50) of 1.2 nM. In contrast, dY-bombesin had a very poor potency for rat BRS-3 (EC(50) = 2 microM). 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(50) = 1.8 +/- 0.5 nM). Within the E3 loop, mutation of Y(298)E(299)S(300) to S(298)Q(299)T(300) (RB3-SQT) or of D(306)V(307)P(308) 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 V(302)D(303) or of V(310)V(311) to I(310)F(311) 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.
Liu
Jie
J
Department of Obesity and Metabolic Research, Merck Research Laboratories, Rahway, NJ 07065, USA.
Lao
Zhege J
ZJ
Zhang
Jiaping
J
Schaeffer
M T
MT
Jiang
Michael M
MM
Guan
Xiao-Ming
XM
Van der Ploeg
Lex H T
LH
Fong
Tung M
TM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001839:Bombesin / Q000494:pharmacology; D003001:Cloning, Molecular; D017931:DNA Primers; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D051381:Rats; D018004:Receptors, Bombesin / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D015183:Restriction Mapping; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D018004:Receptors, Bombesin; C107120:bombesin receptor subtype 3; D001839:Bombesin