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.

Keywords: Bombesin; Gastrin-releasing peptide; Neuromedin B; Obesity; Satiety.

Figure 1

Comparison of the ability of the antagonist Bantag-1 and the nonselective Bn analog agonist (peptide #1) to inhibit binding to cells containing wild type BB₃, BB₃*, wild type BB₂ or wild type BB₁ receptors. The peptides were incubated with 50 pM ¹²⁵I- [D-Tyr⁶, β-Ala¹¹, Phe¹³, Nle¹⁴Bn-(6–14) for 60 minutes at 21°C in 300 μl of binding buffer with BB₃ receptor cells (3 x 10⁶ cells/ml), BB₃* receptor cells (1.1 x 10⁶ cells/ml), BB₂ receptor cells (7 x 10⁶ cells/ml) or BB₁ receptor cells (0.1 x 10⁶ cells/ml) and the saturable binding was determined as described in Materials and Methods. The results are expressed as the percentage of saturable binding without unlabeled peptide added (percentage control). The results are the mean and S.E.M. from at least three separate experiments and in each experiment the data points were determined in duplicated. Abbreviations: Bantag-1, Boc-Phe-His-4-amino-5-cyclohexyl-2,4,5-trideoxypentonyl-Leu- (3-dimethylamino) benzylamide N-methylammonium trifluoroacetate; BB₃ or BB₃ receptor, Bombesin receptor subtype 3; BB₃*or BB₃* receptor, His²⁹⁴ in BB₃ receptor substituted for by Arg²⁸⁸ in comparable position of BB₂ receptor which increases expression of the receptor but does not change affinity for Bantag-1 alone; CHOP, polyoma large T antigen-expressing Chinese hamster ovary; BB₂ or BB₂ receptor, gastrin-related peptide receptor; BB₃, BB₃*, BB₂ or BB₁ receptors stably transfected into CHOP cells; BB₁ receptor, neuromedin B receptor; peptide #1, [D-Tyr⁶, β-Ala¹¹, Phe¹³, Nle¹⁴]Bn-(6–14).

Figure 2

Affinities of the antagonist, Bantag-1 for loss-of-affinity BB₃ chimeric receptors and BB₂ expressed in CHOP cells. The diagrams of the chimeric receptors formed are shown at the top. The chimeric BB₃ receptors were formed by replacing each of the extracellular domains of BB₃* receptor one at a time by the comparable BB₂ receptor extracellular domain as described in Material and Methods. The peptides were incubated with 50 pM ¹²⁵I- [D-Tyr⁶, β-Ala¹¹, Phe¹³, Nle¹⁴]Bn-(6–14) for 60 minutes at 21°C in 300 μl of binding buffer with BB₃* receptor cells (1.1 x 10⁶ cells/ml), (e1-BB₂) BB₃* cells (4.2 x 10⁶ cells/ml), (e2-BB₂) BB₃* cells (4.8 x 10⁶ cells/ml), (e3-BB₂) BB₃* cells (2.1 x 10⁶ cells/ml) or BB₂ receptor cells (7 x 10⁶ cells/ml), and the saturable binding was determined as described under Materials and Methods. The results are expressed as the percentage of saturable binding without unlabeled peptide added (percentage control). The results are the mean and S.E.M. from at least three separate experiments and in each experiment the data points were determined in duplicated. The arrows indicate large changes in affinity from the BB₃ receptor. Abbreviations: e or EC, extracellular; for other, Fig. 1 legend.

Figure 3

Affinities of the antagonist Bantag-1 for BB₂ receptor gain-of-affinity BB2 chimeric receptors and BB₃ receptor expressed in CHOP cells. The diagrams of the chimeric receptors formed are shown at the top. The chimeras BB₂ receptors were formed by replacing each of the extracellular domains of BB₂ receptor one at a time by the comparable BB₃* receptor extracellular domain as described in Material and Methods. The different concentrations of Bantag-1 were incubated with 50 pM ¹²⁵I- [D-Tyr⁶, β-Ala¹¹, Phe¹³, Nle¹⁴]Bn- (6–14) for 60 minutes at 21°C in 300 μl of binding buffer with (e1-BB₃) BB₂ cells (0.6 x 10⁶ cells/ml), (e2-BB₃) BB₂ cells (2 x 10⁶ cells/ml), (eC3-BB₃) BB₂ cells (1.4 x 10⁶ cells/ml) or BB₂ receptor cells (7.0 x 10⁶ cells/ml), and the saturable binding was determined as described under Materials and Methods. The results are expressed as the percentage of saturable binding without unlabeled peptide added (percentage control). The results are the mean and S.E.M. from at least three separate experiments and in each experiment the data points were determined in duplicated. The arrow indicates large change in affinity from the wild type BB₂ receptor. Abbreviations: see Fig. 1 legend.

Figure 4

Effect of single point mutations in the first extracellular domain of BB₃ receptor on affinity for Bantag-1 (loss-of-affinity BB₃ receptor point mutants). Top, alignment of amino acid sequences in the first extracellular domain of BB₃ and BB₂ receptor. The boxes indicate divergent amino acids between these two receptors in these regions. Arrows indicate the position of the point mutations made in BB₃ receptor by substituting into BB₃* receptor the divergent amino acid from the comparable position in BB₂ receptor. (A) Results with the four BB₃ receptor mutants made to explore the importance each of the four amino acid differences in EC1 of BB₂ and BB₃ receptor for determining the selectivity of Bantag-1. (B) Importance of the presence of a charged amino acid or with an aromatic group in position 107 of BB₃ receptor for determining selectivity of Bantag-1. The experimental conditions were the same as described in Fig. 1 legend. The results are expressed as the percentage of saturable binding without unlabeled peptide added (percentage control). The results are the mean and S.E.M. from at least three separate experiments and in each experiment the data points were determined in duplicated. Abbreviations: E111D refers to the replacement of glutamic acid in EC1 position 111 in BB₃ receptor by aspartic acid; TM, transmembrane; for other, see in Fig. 1 legend.

Figure 5

Effect of various point mutations in combination in the first extracellular domain and the third transmembrane domain of BB₃ receptor on affinity for Bantag-1 (loss-of-affinity combination mutants). (A) Effect of multiple mutations in BB₃ receptor in the EC1 on the affinity of Bantag-1. (B) Effect of combination mutations in TM3 of BB₃ receptor on determining selectivity of Bantag-1. The experimental conditions were the same as described in Fig. 1 legend. In each case either one or multiple mutations were made in the wild type BB₃ or BB₃* receptor. The results are expressed as the percentage of saturable binding without unlabeled peptide added (percentage control). The results are the mean and S.E.M. from at least three separate experiments and in each experiment the data points were determined in duplicated. Abbreviations: See in Fig. 1, 2 and 4 legends.

Figure 6

Effect of single point mutations in the third transmembrane domain of BB₃ receptor on affinity for Bantag-1 (loss-of-affinity BB₃ receptor mutations). Top, alignment of amino acid sequences in the second and third transmembrane domain of BB₃ and BB₂ receptor. The boxes indicate divergent amino acids between these two receptors in these regions. Arrows indicate the position of the point mutations made in BB₃ receptor by substituting into BB₃* receptor the divergent amino acid from the comparable position in BB₂ receptor. (A) Effect of four point mutants in BB₃ receptor made to explore the importance of four amino acid differences in TM3 between BB₃ and BB₂ receptors for determining selectivity of Bantag-1. (B) Importance of the presence of a of charged amino acid in position 127 for determining selectivity of Bantag-1. The experimental conditions were the same as described in Fig. 1 legend. The results are expressed as the percentage of saturable binding without unlabeled peptide added (percentage control). The results are the mean and S.E.M. from at least three separate experiments and in each experiment the data points were determined in duplicated. Abbreviations: See in Fig. 1, 2 and 4 legends.