The molecular basis for high affinity of a universal ligand for human bombesin receptor (BnR) family members

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• 作者：Hirotsugu Uehara ; Simon J. Hocart ; Nieves Gonz¨¢lez ; Samuel A. Mantey ; Tomoo Nakagawa ; Tatsuro Katsuno ; David H. Coy ; Robert T. Jensen
• 关键词：¦Â-Ala ; ¦Â-alanine replacement ; BB1 ; neuromedin B receptor ; BB2 ; gastrin-releasing peptide receptor ; BB4 ; bombesin receptor subtype 4 (amphibian) ; Bn ; bombesin ; BRS-3 ; bombesin receptor subtype 3 ; BnR ; member of bombesin receptor family ; BSA ; bovine serum
• 刊名：Biochemical Pharmacology
• 出版年：2012
• 出版时间：1 October, 2012
• 年：2012
• 卷：84
• 期：7
• 页码：936-948
• 全文大小：1294 K

文摘

There is increased interest in the Bn-receptor family because they are frequently over/ectopically expressed by tumors and thus useful as targets for imaging or receptor-targeted-cytotoxicity. The synthetic Bn-analog, [d-Tyr⁶, ¦Â-Ala¹¹, Phe¹³, Nle¹⁴]Bn(6-14) [Univ.Lig] has the unique property of having high affinity for all three human BNRs (GRPR, NMBR, BRS-3), and thus could be especially useful for this approach. However, the molecular basis of this property is unclear and is the subject of this study. To accomplish this, site-directed mutagenesis was used after identifying potentially important amino acids using sequence homology analysis of all BnRs with high affinity for Univ.Lig compared to the Cholecystokinin-receptor (CCK_AR), which has low affinity. Using various criteria 74 amino acids were identified and 101 mutations made in GRPR by changing each to those of CCK_AR or to alanine. 22 GRPR mutations showed a significant decrease in affinity for Univ.Lig (>2-fold) with 2 in EC2[D97N, G112V], 1 in UTM6[Y284A], 2 in EC4[R287N, H300S] showing >10-fold decrease in Univ.Lig affinity. Additional mutations were made to explore the molecular basis for these changes. Our results show that high affinity for Univ.Lig by human Bn-receptors requires positively charged amino acids in extracellular (EC)-domain 4 and to a lesser extent EC2 and EC3 suggesting charge-charge interactions may be particularly important for determining the general high affinity of this ligand. Furthermore, transmembrane amino acids particularly in UTM6 are important contributing both charge-charge interactions as well as interaction with a tyrosine residue in close proximity suggesting possible receptor-peptide cation-¦Ð or H-bonding interactions are also important for determining its high affinity.