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Bombesin-receptor-subtype-3 (BB 3 receptor) is a G-protein-coupled-orphan-receptor classified in the mammalian Bombesin-family because of high homology to gastrin-releasing peptide (BB 2 receptor)/neuromedin-B receptors (BB 1 receptor). There is increased interest in BB 3 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 BB 3 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 BB 3 /BB 2 receptor, and using site-directed-mutagenesis. Receptors were transiently expressed and affinities determined by binding studies. Bantag-1 had >5000-fold selectivity for BB 3 receptor over BB 2 /BB 1 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 BB 3 receptor/BB 2 receptor showed replacement of His 107 in BB 3 receptor by Lys 107 (H107K-BB 3 receptor-mutant) from BB 2 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. [ABSTRACT FROM AUTHOR]

Abstract: There is increased interest in the Bn-receptor family because they are frequently over/ectopically expressed by tumors and thus useful as targets for imaging or receptor-targeted-cytotoxicity. The synthetic Bn-analog, [d-Tyr6, β-Ala11, Phe13, Nle14]Bn(6–14) [Univ.Lig] has the unique property of having high affinity for all three human BNRs (GRPR, NMBR, BRS-3), and thus could be especially useful for this approach. However, the molecular basis of this property is unclear and is the subject of this study. To accomplish this, site-directed mutagenesis was used after identifying potentially important amino acids using sequence homology analysis of all BnRs with high affinity for Univ.Lig compared to the Cholecystokinin-receptor (CCKAR), which has low affinity. Using various criteria 74 amino acids were identified and 101 mutations made in GRPR by changing each to those of CCKAR or to alanine. 22 GRPR mutations showed a significant decrease in affinity for Univ.Lig (>2-fold) with 2 in EC2[D97N, G112V], 1 in UTM6[Y284A], 2 in EC4[R287N, H300S] showing >10-fold decrease in Univ.Lig affinity. Additional mutations were made to explore the molecular basis for these changes. Our results show that high affinity for Univ.Lig by human Bn-receptors requires positively charged amino acids in extracellular (EC)-domain 4 and to a lesser extent EC2 and EC3 suggesting charge-charge interactions may be particularly important for determining the general high affinity of this ligand. Furthermore, transmembrane amino acids particularly in UTM6 are important contributing both charge-charge interactions as well as interaction with a tyrosine residue in close proximity suggesting possible receptor–peptide cation–π or H-bonding interactions are also important for determining its high affinity. [Copyright &y& Elsevier]

Abstract: The bombesin (Bn) receptor family includes the gastrin-releasing peptide (GRPR) and neuromedin B (NMBR) receptors, Bn receptor subtype 3 (BRS-3) and Bn receptor subtype 4 (BB4). They share 50% homology, yet their affinities for gastrin-releasing peptide (GRP) differ. The determinants of GRP high affinity for GRPR and BB4, and low affinity for BRS-3 are largely unknown. To address this question we made an analysis of structural homologies in Bn receptor members correlated with their affinities for GRP to develop criteria to identify amino acids important for GRP selectivity. Fourteen differences were identified and each was mutated singly in GRPR to that found in hBRS-3. Eleven mutants had a loss of GRP affinity. Furthermore, three of four amino acids in the GRPR selected used a similar approach and previously reported to be important for high affinity Bn binding, were important for GRP affinity. Some GRPR mutants containing combinations of these mutations had greater decreases in GRP affinity than any single mutation. Particularly important for GRP selectivity were K101, Q121, A198, P199, S293, R288, T297 in GRPR. These results were confirmed by making the reverse mutations in BRS-3 to make GRP gain of affinity mutants. Modeling studies demonstrated a number of the important amino acids had side-chains oriented inward and within 6Å of the binding pocket. These results demonstrated this approach could identify amino acids needed for GRP affinity and complemented results from chimera/mutagenesis studies by identifying which differences in the extracellular domains of Bn receptors were important for GRP affinity. [Copyright &y& Elsevier]