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, there is little known of the molecular basis of its high-affinity/selectivity. This was systematic 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 His107 in BB3 receptor by Lys107(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 His107, rather than hydrogen-bonding or charge-charge interactions, for determining Bantag-1 high affinity/selectivity. In regard to Arg127 in TM3, both hydrogen- bonding and charge-charge interactions contribute to the high-affinity/selectivity for Bantag-1.

Understanding Peptide Binding in Class A G Protein-Coupled Receptors

“…Mutagenesis studies of the bombesin receptors (BB 2 and BB 3 ) indicate the importance of asparagine or arginine 3.32 for bombesin binding (Akeson et al, 1997;Nakamura et al, 2016). Mutation of tyrosine at position 6.55 in BB 2 is critical for the activity of the peptide (Lin et al, 2000).…”

Peptide receptors as cancer drug targets

“…For example, 68 Ga‐sarabesin 3 binds with high affinity to prostate cancer cells and imaged prostate tumors in mice . Also, Bantag‐1, a new BRS‐3 antagonist, has been shown to inhibit lung cancer proliferation . Traditionally, SCLC is treated with chemotherapy as well as radiation therapy, and NSCLC is treated with chemotherapy; however, the 5‐year survival rate is 16%.…”

Development and Characterization of a Novel, High-Affinity, Specific, Radiolabeled Ligand for BRS-3 Receptors

“…BALB 3T3 (mouse fibroblast) cells; NCI-N417 and NCI-H69 (human small cell lung cancer cells); and NCI-H520, NCI-H460, NCI-H720, and NCI-H727 (human non-small cell lung cancer cells) were purchased from the American Type Culture Collection (Gaithersburg, MD); Dulbecco's minimum essential medium (DMEM), RPMI 1640, PBS, FBS, Dulbecco's phosphate-buffered saline, trypsin-EDTA 1Â, penicillin streptomycin, and GENETICIN-selective antibiotic (G418 sulfate) were from Invitrogen (Carlsbad, CA); GRP and NMB were from Bachem (Torrance, CA); the Bn analogs [D-Tyr 6 , b-Ala 11 , Phe 13 , Nle 14 ]Bn-(6-14) (Peptide-1) (Mantey et al, 1997;Pradhan et al, 1998;Moreno et al, 2013) and other BnR ligands were obtained from Dr. David H. Coy, Tulane University (New Orleans, LA); Bantag-1, a BRS-3 antagonist (Guan et al, 2010;Feng et al, 2011;Nakamura et al, 2016) was from Sigma-Aldrich (Allentown, PA);…”

“…The best binding fractions were stored with 0.5% BSA at 220°C. 125 I-Peptide-1 ( 125 I-[D-Tyr 6 , b-Ala 11 , Phe 13 , Nle 14 ] Bn-(6-14)), with a specific activity of 2200 Ci/mmol, was prepared as described previously (Mantey et al, 1997;Moreno et al, 2013;Nakamura et al, 2016).…”