Molecular basis for high affinity and selectivity of peptide antagonist, Bantag-1, for the orphan BB3 receptor
| Autor: | Paola Moreno, Robert T. Jensen, Samuel A. Mantey, Tatiana Iordanskaia, Taichi Nakamura, Irene Ramos-Álvarez |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Recombinant Fusion Proteins Peptide CHO Cells Biochemistry Article 03 medical and health sciences Mice Cricetulus Functional selectivity Animals Humans 5-HT5A receptor Receptor Protease-activated receptor 2 G protein-coupled receptor Pharmacology chemistry.chemical_classification Neuromedin B Amino acid Receptors Bombesin 030104 developmental biology chemistry Mutagenesis Peptides Protein Binding |
| Zdroj: | Biochemical pharmacology. 115 |
| ISSN: | 1873-2968 |
| Popis: | Bombesin-receptor-subtype-3 (BB3 receptor) is a G-protein-coupled-orphan-receptor classified in the mammalian Bombesin-family because of high homology to gastrin-releasing peptide (BB2 receptor)/neuromedin-B receptors (BB1 receptor). There is increased interest in BB3 receptor because studies primarily from knockout-mice suggest it plays roles in energy/glucose metabolism, insulin-secretion, as well as motility and tumor-growth. Investigations into its roles in physiological/pathophysiological processes are limited because of lack of selective ligands. Recently, a selective, peptide-antagonist, Bantag-1, was described. However, because BB3 receptor has low-affinity for all natural, Bn-related peptides, little is known of the molecular basis of its high-affinity/selectivity. This was systematically investigated in this study for Bantag-1 using a chimeric-approach making both Bantag-1 loss-/gain-of-affinity-chimeras, by exchanging extracellular (EC) domains of BB3/BB2 receptor, and using site-directed-mutagenesis. Receptors were transiently expressed and affinities determined by binding studies. Bantag-1 had >5000-fold selectivity for BB3 receptor over BB2/BB1 receptors and substitution of the first EC-domain (EC1) in loss-/gain-of affinity-chimeras greatly affected affinity. Mutagenesis of each amino acid difference in EC1 between BB3 receptor/BB2 receptor showed replacement of His(107) in BB3 receptor by Lys(107) (H107K-BB3 receptor-mutant) from BB2 receptor, decreased affinity 60-fold, and three replacements [H107K, E11D, G112R] decreased affinity 500-fold. Mutagenesis in EC1's surrounding transmembrane-regions (TMs) demonstrated TM2 differences were not important, but R127Q in TM3 alone decreased affinity 400-fold. Additional mutants in EC1/TM3 explored the molecular basis for these changes demonstrated in EC1, particularly important is the presence of aromatic-interactions by His(107), rather than hydrogen-bonding or charge-charge interactions, for determining Bantag-1 high affinity/selectivity. In regard to Arg(127) in TM3, both hydrogen-bonding and charge-charge interactions contribute to the high-affinity/selectivity for Bantag-1. |
| Databáze: | OpenAIRE |
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