Molecular Basis for Agonist Selectivity and Activation of the Orphan Bombesin Receptor Subtype 3 Receptor

Abstract

Bombesin receptor subtype (BRS)-3, a G-protein-coupled orphan receptor, shares 51% identity with the mammalian bombesin (Bn) receptor for gastrin-releasing peptide. There is increasing interest in BRS-3 because it is important in energy metabolism, glucose control, motility, and tumor growth. BRS-3 has low affinity for all Bn-related peptides; however, recently synthetic high-affinity agonists, [D-Tyr 6 /D-Phe 6 ,␤Ala 11 ,Phe 13 ,Nle 14 ]Bn-(6 -14), were described, but they are nonselective for BRS-3 over other Bn receptors. Based on these peptides, three BRS-3-selective ligands were developed: peptide 2, [D-Tyr ]Bn(6 -14); and peptide 4, acetyl-Phe-Trp-Ala-His-(tBzl)-piperidine-3 carboxylic acid-Gly-Arg-NH 2 . Their molecular determinants of selectivity/high affinity for BRS-3 are unknown. To address this, we used a chimeric/site mutagenesis approach. Substitution of extracellular domain 2 (EC2) of BRS-3 by the comparable gastrin-releasing peptide receptor (GRPR) domain decreased 26-, 4-, and 0-fold affinity for peptides 4, 3, and 2. Substitution of EC3 decreased affinity 4-, 11-, and 0-fold affinity for peptides 2 to 4. Ten-point mutations in the EC2 and adjacent transmembrane regions (TM2) 2 and 3 of BRS-3 were made. His107 (EC2-BRS-3) for lysine (H107K) (EC2-GRPR) decreased affinity (25-and 0-fold) for peptides 4 and 1; however, it could not be activated by either peptide. Its combination with Val101 (TM2), Gly112 (EC2), and Arg127 (TM3) resulted in complete loss-ofaffinity of peptide 4. Receptor-modeling showed that each of these residues face inward and are within 4 Å of the binding pocket. These results demonstrate that Val101, His107, Gly112, and Arg127 in the EC2/adjacent upper TMs of BRS-3 are critical for the high BRS3 selectivity of peptide 4. His107 in EC2 is essential for BRS-3 activation, suggesting amino-aromatic ligand/ receptor interactions with peptide 4 are critical for both binding and activation. Furthermore, these result demonstrate that even though these three BRS-3-selective agonists were developed from the same template peptide,

Molecular Basis for the Selectivity of the Mammalian Bombesin Peptide, Neuromedin B, for Its Receptor

“…Studies were performed in transiently transfected Balb-3T3 cells as described in Table 1 binding site conformation. Our results showing a synergetic effect on agonist affinity of multiple substitutions are similar to findings reported for the selectivity of peptide histidine isoleucinamide for the human vasoactive intestinal peptide receptor 1 (Couvineau et al, 1996), GRP for GRPR over human BRS-3 (Nakagawa et al, 2005), the BRS-3-selective agonist Ac-Phe-Trp-Ala-His(tBzl)-Nip-Gly-Arg-NH 2 for BRS-3 over GRPR (Gonzalez et al, 2008), and the peptide antagonists JMV594/JMV641 for GRPR over NMBR (Tokita et al, 2001b) and the nonpeptide antagonist CP96345 for human over the rat neurokinin-1 receptor (Fong et al, 1992). Our finding of the importance of an isoleucine in NMBR instead of serine in GRPR in TM5 (Ile 216 NMBR) or Ala in EC3 (Ile 199 ) has both similarities and differences from findings with a number of other G-protein-coupled receptors (Greenfeder et al, 1999).…”

“…4 (Ile 216 ), instead of serine in GRPR (Ser 215 ), was confirmed to be important for NMB selectivity by making a GRPR gainof-affinity mutant (i.e., [S215I]GRPR), as proposed in NMBR loss-of-affinity studies . Compared with GRPR/BRS-3, our results are similar in that both extracellular and upper TM regions are important for selectivity of Bn, GRP, and some BRS-3-selective peptide agonists for GPRR or BRS-3 Tokita et al, 2002;Nakagawa et al, 2005;Gonzalez et al, 2008;Jensen et al, 2008). Compared with other G-protein-coupled receptors, our results are similar to the interaction of the peptide agonists, substance P with NK-1 receptors (Li et al, 1996) and CCK-8 with CCK-B receptors (Silvente-Poirot et al, 1998), where both the extracellular and TM domains play a marked role in selectivity.…”

“…In contrast, differences in EC2 are primarily important for the high affinity of some BRS-3-selective peptide agonists (Gonzalez et al, 2008), and differences in EC4 are important for GRPR selectivity of the highly selective GRPR peptide antagonists, JMV641 and JMV594 (Tokita et al, 2001b). Likewise, the high affinity of GRP or Bn for GRPR is primarily determined by differences from other Bn receptors in the EC3 region Tokita et al, 2002).…”

“…The structural basis for the selectivity of GRPR agonists/ antagonists and high affinity for the GRPR has been extensively studied Tokita et al, 2002;Nakagawa et al, 2005;Gonzalez et al, 2008). However, little is known about the molecular basis of selectivity and high affinity of NMB for the NMBR.…”

“…We made an NH 2 -terminal truncated NMBR mutant and used a chimeric NMBR-GRPR receptor approach. The latter approach was used because it has been proven to be useful in elucidating the structural basis of other G-protein-coupled receptor interaction with their ligands Tokita et al, 2002;Gonzalez et al, 2008). The receptor chimeric approach was accomplished by exchanging extracellular NMBR and GRPR domains (NMB loss-and gainof-affinity chimeras).…”