Identification of Four Amino Acids in the Gastrin-releasing Peptide Receptor That Are Required for High Affinity Agonist Binding

Abstract

The bombesin family of G-protein-coupled receptors includes the gastrin-releasing peptide receptor (GRP-R), the neuromedin B receptor (NMB-R), bombesin receptor subtype 3 (BRS-3), and bombesin receptor subtype 4 (bb4). All species homologues of GRP-R, NMB-R, and bb4 bind bombesin with dissociation constants in the nanomolar range; by comparison, human BRS-3 binds bombesin at much lower affinity (K d > 1 M). We used this difference to help identify candidate residues that were potentially critical for forming the bombesin binding pocket. We reasoned that amino acids essential for bombesin binding would be conserved among all homologues of bb4, NMB-R, and GRP-R; conversely, at least one of these amino acids would not be conserved among homologues of BRS-3. Amino acid sequence alignment revealed nine residues that fit this model. We replaced each of these amino acids in mouse GRP-R with the homologous amino acid in human BRS-3. Four substitutions resulted in a significant decrease in bombesin affinity (R288H, Q121R, P199S, and A308S). The analogous mutations in BRS-3 (R127Q, H294R, S205P, and S315A) together resulted in a receptor with a 100-fold increase in bombesin and GRP affinities relative to wildtype BRS-3. From this, we propose a preliminary map of some of the amino acids comprising the agonist binding pocket.Bombesin is a tetradecapeptide that was originally isolated from frog skin (1). Numerous polypeptides with structurally related carboxyl termini were subsequently isolated from amphibians and classified into three subfamilies (bombesin, ranatensin, and phyllolitorin) based upon the sequence of the last three residues in their amidated carboxy-terminal domains (2). To date, two bombesin-like peptides have been isolated from mammalian tissue: NMB 1 in the ranatensin subfamily (3) and GRP in the bombesin subfamily (4). Bombesin, NMB, and GRP bind to a family of G-proteincoupled receptors known to include at least four receptor subtypes: the GRP-preferring receptor (GRP-R (or bb2)) (5, 6), the NMB-preferring receptor (NMB-R (or bb1)) (5, 7), bombesin receptor subtype 3 (BRS-3, (or bb3)) (8, 9), and bombesin receptor subtype 4 (bb4) (10). Although the four subtypes share about 50% identity when their primary amino acid sequences are aligned, the pharmacology of BRS-3 is significantly different from the pharmacology of the others.2 Notably, GRP-R, NMB-R, and bb4 all bind bombesin with affinities in the nanomolar range, whereas BRS-3 binds bombesin with much lower affinity (Ͼ10 M).We hypothesized that amino acids essential for high affinity bombesin binding would be conserved among all known GRP-R, NMB-R, and bb4 sequences and would diverge in the BRS-3 sequences. Nine amino acids in extracellular loops or in the outer half of transmembrane helices fulfilled these conditions. The importance of each of these residues for agonist binding to GRP-R was determined by using site-directed mutagenesis to exchange the residue found in GRP-R with its divergent counterpart in BRS-3. The affinities of BRS-3 or GRP-R mutan...

Mitogenic effects of gastrin-releasing peptide in head and neck squamous cancer cells are mediated by activation of the epidermal growth factor receptor

“…Moreover, Liebow et al showed that bombesin and EGF seemed to promote phosphorylation of similar substrates (Liebow et al, 1994). Bombesin, which binds with high affinities to multiple BLP receptors, including NMBR and bombesin receptor subtype 4 (Akeson et al, 1997), has been shown to transactivate EGFR in the PC-1 prostate cancer cell line (Prenzel et al, 1999). However, the biologic consequences of bombesin-induced EGFR trans;-activation in prostate cancer were not investigated.…”

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

“…The human hBRS-3 cDNA was inserted into pcDNA3.1 ϩ at the EcoRI site, and BRS-3 mutant receptors were constructed by using the QuikChange Site-Directed Mutagenesis Kit, following the manufacturer's instructions, with minor modifications. The mouse GRPR used in this study was identical to that described previously (Fathi et al, 1993a;Akeson et al, 1997). Nucleotide sequence analysis of the entire coding region was performed.…”

International Union of Pharmacology. LXVIII. Mammalian Bombesin Receptors: Nomenclature, Distribution, Pharmacology, Signaling, and Functions in Normal and Disease States

“…The human BB 1 receptor (Moody et al, 1986(Moody et al, , 1992(Moody et al, , 1995aCorjay et al, 1991;Benya et al, 1995b), as well as the rat BB 1 receptor Jones et al, 1992;Wang et al, 1992;Dobrzanski et al, 1993;Lach et al, 1995;Akeson et al, 1997;Tsuda et al, 1997b;Vigne et al, 1997;Hou et al, 1998) is coupled to phospholipase C, resulting in breakdown of phosphoinositides, mobilization of cellular calcium, and activation of protein kinase C. BB 1 receptor activation also results in the stimulation phospholipase A 2 (Moody et al, 1995a) and phospholipase D by a PKC-dependent and -independent mechanism (Tsuda et al, 1997b) but does not activate adenylate cyclase (Benya et al, 1992). BB 1 receptor stimulation also results in activation of tyrosine kinases (Lach et al, 1995;Tsuda et al, 1997b) stimulating tyrosine phosphorylation of p125…”