González Nieves, Nakagawa Tomoo, Mantey Samuel A, Sancho Veronica, Uehara Hirotsugu, Katsuno Tatsuro, Jensen Robert T
Digestive Diseases Branch, NIDDK, NIH, Bethesda, MD 20892-1804, USA.
J Pharmacol Exp Ther. 2009 Oct;331(1):265-76. doi: 10.1124/jpet.109.154245. Epub 2009 Jul 23.
The mammalian bombesin (Bn) peptides, neuromedin B (NMB) and gastrin-releasing peptide (GRP), have widespread actions in many tissues, and their effects are mediated by two closely related G-protein-coupled receptors, the NMBR and GRPR. Little is known about the structural determinants of NMBR selectivity for NMB, in contrast to GRP selectivity for the GRPR, which has been extensively studied. To provide insight, chimeric NMBR-GRPR loss-of-affinity and gain-of-affinity mutants were made, as well as NH(2)-terminally truncated NMBR and point mutants using site-directed mutagenesis. Receptors were expressed in Balb-3T3-cells or CHOP cells, and affinities were determined. NMB had 115-fold greater affinity for NMBR than GRPR. Receptor-chimeric studies showed that NMBR selectivity for NMB was primarily determined by differences in the third extracellular (EC3) regions of GRPR-NMBR and adjacent upper-transmembrane-5 (TM5) region. In this region, 24 NMB gain-of-affinity GRPR mutants or NMBR loss-of-affinity point/combination mutants were made. Three gain-of-affinity mutant GRPRs [[A198I] (EC3), [H202Q] (EC3), [S215I] (upper TM5)] had increased NMB affinity (2.4-21-fold), and these results were confirmed with NMBR loss-of-affinity mutants [I199A,Q203H,I215S-NMBR]. The combination mutant [A198I,S215]GRPR had the greatest effect causing a complete NMB gain-of-affinity. The importance of differences at position 199NMBR or 203NMBR was studied by substituting amino acids with various properties. Our results show that NMBR selectivity for NMB is due to differences in the EC3 of NMBR-GRPR and the adjacent upper-TM5 region. Within these regions, isoleucines in NMBR [position 199 (EC3)] (instead of A198GRPR) and in 215NMBR (TM5) (instead of S214GRPR), as well as Q203NMBR (instead of H202GRPR) are responsible for high NMB-affinity/selectivity of NMBR. The effect at position 199 is primarily due to differences in hydrophobicity of the substitution, whereas steric factors and charge of the substitution at position 203 were important determinants of NMB selectivity.
哺乳动物铃蟾肽（Bn）家族的神经介素B（NMB）和胃泌素释放肽（GRP）在许多组织中具有广泛作用，其效应由两个密切相关的G蛋白偶联受体——NMBR和GRPR介导。与对GRPR的GRP选择性已得到广泛研究相反，关于NMBR对NMB的选择性的结构决定因素知之甚少。为了深入了解，构建了嵌合的NMBR - GRPR亲和力丧失和亲和力增加突变体，以及使用定点诱变技术构建的N端截短的NMBR和点突变体。受体在Balb - 3T3细胞或CHOP细胞中表达，并测定亲和力。NMB对NMBR的亲和力比对GRPR高115倍。受体嵌合研究表明，NMBR对NMB的选择性主要由GRPR - NMBR的第三个细胞外（EC3）区域和相邻的跨膜5区（TM5）上部的差异决定。在该区域，构建了24个NMB亲和力增加的GRPR突变体或NMBR亲和力丧失的点突变/组合突变体。三个亲和力增加的突变体GRPR [[A198I]（EC3），[H202Q]（EC3），[S215I]（TM5上部）]的NMB亲和力增加（2.4 - 21倍），这些结果通过NMBR亲和力丧失突变体[I199A，Q203H，I215S - NMBR]得到证实。组合突变体[A198I，S215]GRPR产生的影响最大，导致NMB完全获得亲和力。通过用具有各种特性的氨基酸进行替换，研究了199NMBR或203NMBR位置差异的重要性。我们的结果表明，NMBR对NMB的选择性归因于NMBR - GRPR的EC3和相邻的上部TM5区域的差异。在这些区域内，NMBR中199位（EC3）的异亮氨酸（而非GRPR的A198）、215位（TM5）的异亮氨酸（而非GRPR的S214）以及203位的谷氨酰胺（而非GRPR的组氨酸）是NMBR具有高NMB亲和力/选择性的原因。199位的影响主要归因于替换氨基酸的疏水性差异，而203位替换氨基酸的空间因素和电荷是NMB选择性的重要决定因素。
