Abstract

The mammalian bombesin peptides [gastrin-releasing peptide (GRP) and neuromedin B (NMB)] are important in numerous biological and pathological processes. These effects are mediated by the heptahelical GRP receptor (GRPR) and NMB receptor (NMBR). GRP has high affinity for GRPR and lower affinity for NMBR. Almost nothing is known about the molecular basis for the selectivity of GRP. To address this question, we first studied four loss-of-affinity GRPR chimeric receptors formed by exchanging the four extracellular (EC) domains of GRPR with the corresponding NMBR EC domains. Receptors were transiently expressed, and affinities were determined by binding studies. Only substitution of the third EC domain (EC3) of GRPR markedly decreased GRP affinity. In the reverse study using gain-of-affinity NMBR chimeras, only replacement of EC3 of NMBR markedly increased GRP affinity. Replacing each of the 20 comparable EC3 amino acids that differed in the NMBR in GRPR showed that two separate NMBR substitutions in the GRPR, Ile for Phe¹⁸⁵ or Ile for Ala¹⁹⁸, markedly decreased GRP affinity. Additional point mutants demonstrated that an amino acid with an aromatic ring in position 185 of GRPR and the size of the backbone substitution in position 198 of GRPR were important for GRP selectivity. These results demonstrate that selectivity of GRP for GRPR over NMBR is primarily determined by two amino acid differences in the EC3 domains of the receptor. Our results suggest that an interaction between the aromatic ring of Phe¹⁸⁵ of the GRPR with GRP is the most important for GRP selectivity.