Molecular Basis of the Selectivity of Gastrin-Releasing Peptide Receptor for Gastrin-Releasing Peptide

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 Phe 185 or Ile for Ala 198 , 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 Phe 185 of the GRPR with GRP is the most important for GRP selectivity.In contrast to adrenergic and muscarinic cholinergic receptors, with many receptors for gastrointestinal (GI) hormones/ neurotransmitters, little is known about the molecular basis of their agonist selectivity. Some of the most important GI hormone/neurotransmitter receptors are those mediating the actions of the mammalian bombesin (Bn)-related peptides, gastrin-releasing peptide (GRP), and neuromedin B (NMB). These peptides mediate a wide spectrum of biological activities, including stimulating the growth of both normal and neoplastic tissues (Willey et al

Bombesin Inhibits Alveolarization and Promotes Pulmonary Fibrosis in Newborn Mice

“…It is also known that bombesin does not appreciably bind to the BRS3 receptor (26,34). In some experimentally mutated receptors, however, bombesin has markedly higher affinity than does GRP (35), suggesting that a novel bombesin receptor could, in fact, mediate effects of bombesin that might persist in GRPR KO mice, as observed with bombesin-induced interstitial fibrosis and decreased alveolarization in the present study.…”

“…In fact, amphibian bombesin binds to and functions better at the mammalian bombesin/GRP receptor than does GRP itself, probably due to conformational differences related to its smaller molecular weight that permit greater access to the cell-surface GRPR in tissues (23)(24)(25). Three of the four amino acids required for high-affinity bombesin binding to GRPR are also required for high-affinity GRP binding (34,35). The peptide-specific binding site for GRP has been mapped to the third extracellular domain (35), whereas NMB selectivity has been mapped to the fifth transmembrane region (36).…”

“…Three of the four amino acids required for high-affinity bombesin binding to GRPR are also required for high-affinity GRP binding (34,35). The peptide-specific binding site for GRP has been mapped to the third extracellular domain (35), whereas NMB selectivity has been mapped to the fifth transmembrane region (36). Four times as much GRP or 300 times as much NMB is required to elicit the same responses as bombesin (on a molar basis) at the bombesin/GRP-preferring receptor (23).…”

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

“…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.…”

Molecular cloning and characterization of avian bombesin‐like peptide receptors: new tools for investigating molecular basis for ligand selectivity

“…These results coincide with the high affinity of chGRP-R and moderate affinity of chBRS-3.5 for BN. Moreover, two residues, Phe 185 and Ala 198 , have been shown to be indispensable for the selectivity of mouse GRP-R for GRP (Tokita et al, 2002). These residues are conserved in both chGRP-R and chBRS-3.5 that showed selectivity for GRP rather than NMB.…”