title: Molecular basis for high affinity and selectivity of peptide antagonist, Bantag-1, for the orphan BB3 receptor.
abstract: 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.
authors.affiliation: Digestive Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-1804, USA.
authors.forename: Taichi
authors.initials: T
authors.lastname: Nakamura
authors.affiliation: Digestive Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-1804, USA.
authors.forename: Irene
authors.initials: I
authors.lastname: Ramos-Álvarez
authors.affiliation: Digestive Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-1804, USA.
authors.forename: Tatiana
authors.initials: T
authors.lastname: Iordanskaia
authors.affiliation: Digestive Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-1804, USA.
authors.forename: Paola
authors.initials: P
authors.lastname: Moreno
authors.affiliation: Digestive Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-1804, USA.
authors.forename: Samuel A
authors.initials: SA
authors.lastname: Mantey
authors.affiliation: Digestive Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-1804, USA. Electronic address: robertj@bdg10.niddk.nih.gov.
authors.forename: R T
authors.initials: RT
authors.lastname: Jensen
mesh_terms: D000818:Animals; D016466:CHO Cells; D003412:Cricetulus; D006801:Humans; D051379:Mice; D016296:Mutagenesis; D010455:Peptides / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011485:Protein Binding; D018004:Receptors, Bombesin / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism
chemical_list: D010455:Peptides; D018004:Receptors, Bombesin; D011993:Recombinant Fusion Proteins; C000617994:bantag-1; C107120:bombesin receptor subtype 3
keywords: Bombesin; Gastrin-releasing peptide; Neuromedin B; Obesity; Satiety