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

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• 作者：Jie Liu ; Zhege J. Lao ; Jiaping Zhang ; M. T. Schaeffer ; Michael M. Jiang ; Xiao-Ming Guan ; Lex H. T. Van der Ploeg ; and Tung M. Fong
• 刊名：Biochemistry
• 出版年：2002
• 出版时间：July 16, 2002
• 年：2002
• 卷：41
• 期：28
• 页码：8954 - 8960
• 全文大小：179K
• 年卷期：v.41,no.28(July 16, 2002)
• ISSN：1520-4995

文摘

We cloned the gene and cDNA for rat bombesin receptor subtype-3 (BRS-3) and characterizedits 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 thenatural ligand for BRS-3 is still unknown, a synthetic peptide, dY-Q-W-A-V-(mages/gifchars/beta2.gif" BORDER=0 ALIGN="middle">-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 poorpotency for rat BRS-3 (EC₅₀ = 2 mages/entities/mgr.gif">M). To understand the molecular basis of this pharmacological difference,we constructed chimeric receptors in which individual extracellular loops of rat BRS-3 were replacedwith the corresponding human sequences. Switching the N-terminal region or the second extracellularloop 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 humanBRS-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 toS²⁹⁸Q²⁹⁹T³⁰⁰ (RB3-SQT) or of D³⁰⁶V³⁰⁷P³⁰⁸ to A³⁰⁶M³⁰⁷H³⁰⁸ (RB3-AMH) only partially mimicked theeffect of switching the entire E3 loop, and mutation of A³⁰²E³⁰³ to V³⁰²D³⁰³ or of V³¹⁰V³¹¹ to I³¹⁰F³¹¹ hadlittle effect on the dY-bombesin potency. These results indicate that the sequence variation in the E3 loopis responsible for the species difference between rat and human BRS-3, and multiple residues in the E3loop are involved in interactions with the agonist dY-bombesin.