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

• Published in: The Journal of pharmacology and experimental therapeutics 2007-11, Vol.324 (2), p.463-474
• Main Authors: Gonzalez, Nieves, Hocart, Simon J., Portal-Nuñez, Sergio, Mantey, Samuel A., Nakagawa, Tomoo, Zudaire, Enrique, Coy, David H., Jensen, Robert T.
• Format: Article
• Language: English
• ISSN: 0022-3565; 1521-0103
• DOI: 10.1124/jpet.107.132332

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-Tyr 6 /D-Phe 6 ,βAla 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 6 (R)-Apa 11 ,Phe 13 ,Nle 14 ]Bn(6–14); peptide#3,[D-Tyr 6 ,(R)-Apa 11 ,4Cl-Phe 13 ,Nle 14 ]Bn(6–14); peptide #4,Ac-Phe-Trp-Ala-His(tBzl)-Nip-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 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. His 107 (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 Val 101 (TM2),Gly 112 (EC2),Arg 127 (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 [Val 101 ,His 107 ,Gly 112 ,Arg 127 ] in the EC2/adjacent upper TMs of BRS-3 are critical for the high BRS3-selectivity of peptide#4. His 107 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-Phe 6 ,βAla 11 ,Phe 13 ,Nle 14 ]Bn-(6–14), their molecular determinants of selectivity/high affinity varied considerably.