Identification of a unique ligand which has high affinity for all four bombesin receptor subtypes
Introduction
Recently three different subclasses of bombesin receptors have been described including two identified in mammals (gastrin-releasing peptide receptor (Spindel et al., 1990; Battey et al., 1991; Anonymous, 1996) and neuromedin B receptor (Von Schrenck et al., 1989; Wada et al., 1991)) and one identified in frog brain (bombesin receptor subtype 4 (BB4 receptor) (Nagalla et al., 1995)), but not yet identified in mammals. A possible fourth subtype has been proposed which is a 399 amino acid orphan receptor which, because of its high degree of homology to mammalian bombesin receptors (51–52% for the gastrin-releasing peptide receptor and 47% for the neuromedin B receptor (Gorbulev et al., 1992; Fathi et al., 1993)), was named BRS-3 for bombesin receptor subtype-3 in one study (Fathi et al., 1993) and BB3 in one classification (Anonymous, 1996). After expression in Xenopus oocytes, BRS-3 receptors could be activated by bombesin, albeit at concentrations several orders of magnitude higher than those required to activate the gastrin-releasing peptide receptor or neuromedin B receptor in the same assay (Fathi et al., 1993). All four receptors are members of the heptahelical superfamily of G protein-coupled receptors (Spindel et al., 1990; Battey et al., 1991; Gorbulev et al., 1992; Nagalla et al., 1995).
Whereas the pharmacology and cell biology of the gastrin-releasing peptide receptor has been extensively studied (Kroog et al., 1995), much less is known about the neuromedin B receptor and only a few studies have dealt with these aspects of the BRS-3 receptor (Gorbulev et al., 1992; Wu et al., 1996) or the BB4 receptor (Nagalla et al., 1995). In the case of the BRS-3 receptor this lack of information is because of a lack of a high affinity ligand, with the BB4 receptor it is because this receptor was only recently described and with the neuromedin B receptor it is in part due to the fact that its ligand (-[d-Tyr0]neuromedin B or -Bolton–Hunter-neuromedin B (Von Schrenck et al., 1989; Wang et al., 1993; Lin et al., 1996) is not generally available. In the process of screening synthetic bombesin analogues that could interact with the hBRS-3 receptor, we recently discovered that [d-Phe6,βAla11,Phe13,Nle14]bombesin-(6–14) [d-Phe6,βAla11,Phe13,Nle14]bombesin-(6–14) had high affinity for the hBRS-3 receptor (Mantey et al., 1997). In the non-small cell lung cancer cell H1299 we found that this peptide also interacted with human gastrin-releasing peptide receptor cells natively expressed in low numbers in these cells (Corjay et al., 1991). In the present study we have investigated the ability of this peptide and an analogue which can be radiolabeled -[d-Tyr6,βAla11,Phe13,Nle14]bombesin-(6–14) to interact with all four subtypes of bombesin receptors. We find this ligand is unique in that it has selective high affinity for all four bombesin receptor subtypes, but does not interact with receptors mediating the action of unrelated peptides.
Section snippets
Materials
Male Sprague–Dawley rats weighing 80–100 g were purchased from Taconic Farms of New York. N-2-hydroxyethyl piperazine-N′-2-ethanesulfonic acid (HEPES) and albumin bovine fraction V were from ICN Biomedical (Aurora, OH); soybean trypsin inhibitor type 1-S, bacitracin, atropine sulfate and carbachol were from Sigma (St. Louis, MO); basal medium Eagle vitamin and basal medium Eagle amino acids solutions (100-times concentrated), Dulbecco's phosphate buffered saline (DPBS), Dulbecco's modified
Results
The ability of peptides to interact with the gastrin-releasing peptide receptor has been characterized using either the radiolabeled agonists [-Tyr4]bombesin or -gastrin-releasing peptide, or the radiolabeled antagonist -[d-Phe6]bombesin-(6–13)methyl ester (Ladenheim et al., 1991; Vigna and Mantyh, 1991; Mantey et al., 1993; Shapira et al., 1993). To investigate the ability of -[d-Tyr6,βAla11,Phe13,Nle14]bombesin-(6–14) to interact with this receptor, we compared the abilities
Discussion
In this study we report for the first time the identification of a unique, specific ligand that interacts with high affinity with each of the four reported subtypes of bombesin receptors. The evidence that the ligand, -[d-Tyr6,βAla11,Phe13,Nle14]bombesin-(6–14), is interacting with the bombesin receptors is supported by a number of findings. First, for each of the three subclasses of bombesin receptors (gastrin-releasing peptide receptor, neuromedin B receptor and BB4 receptor) for which
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