Received for publication October 1, 2007.
Revised November 14, 2007.
Accepted for publication November 14, 2007.

Molecular basis for agonist selectivity and activation of the orphan BRS-3-receptor

Abstract

Bombesin receptor subtype-3(BRS-3), a G protein-coupled orphan receptor, shares 51% identity with the mammalian bombesin(Bn) receptor for gastrin-releasing peptide(GRPR). 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-Tyr⁶/D-Phe⁶,Ala¹¹,Phe¹³,Nle¹⁴]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-Tyr⁶(R)-Apa¹¹,Phe¹³,Nle¹⁴]Bn(6-14); peptide#3,[D-Tyr⁶,(R)-Apa¹¹,4Cl-Phe¹³,Nle¹⁴]Bn(6-14); peptide #4,Ac-Phe-Trp-Ala-His(tBzl)-Nip-Gly-Arg-NH₂. 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 domain2(EC2) of BRS-3 by the comparable GRPR domain decreased 26-,4,0-fold affinity for peptides#4,3,2. Substitution of EC3 decreased affinity 4-,11-,0-fold affinity for peptides#2,3,4. 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-,0-fold) for peptide#4,1; however it could not be activated by either peptide. Its combination with Val¹⁰¹(TM2),Gly¹¹²(EC2),Arg¹²⁷(TM3) resulted in complete loss-of-affinity of peptide#4. Receptor-modeling showed that each of these residues face inward and are within 4Å of the binding-pocket. These results demonstrate [Val¹⁰¹,His¹⁰⁷,Gly¹¹²,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/ activation. Furthermore, these result demonstrate that even though these three BRS-3 selective agonists were developed from the same template peptide,[D-Phe⁶,Ala¹¹,Phe¹³,Nle¹⁴]Bn-(6-14), their molecular determinants of selectivity/high affinity varied considerably.

Key words: BRS-3, bombesin, gastrin-releasing peptide, ligand affinity, neuromedin b, receptor structure-function