Epitopes determining the agonist property of two structurally distinct selective ligands for the human bombesin receptor subtype 3 (BB3), [d-Tyr6,(R)-Apa11,Phe13, Nle14]-bombesin(6-14) (Pep-1) and Ac-Phe-Trp-Ala-His(TauBzl)-Nip-Gly-Arg-NH2 (Pep-2), were mapped through systematic mutagenesis of the main ligand-binding pocket of the receptor. The mutational map for the smaller Pep-2 spanned the entire binding pocket of the BB3 receptor. In contrast, the much fewer mutational hits for the larger Pep-1 were confined to the center of the pocket, i.e., the opposing faces of the extracellular segments of transmembrane (TM)-III, TM-VI, and TM-VII. All the residues, which upon mutation affected Pep-1, were also hits for Pep-2 and included those that were most essential for the function of Pep-2: LeuIII:04 (Leu123), TyrVI:16 (Tyr291), and ArgVII:06 (Arg316). The BB3 receptor was found to signal with 12% ligand-independent activity that was strongly influenced both positively and negatively by several mutations in the binding pocket. The substitutions, which decreased the constitutive signaling, included not only the major mutational hits for the peptide agonists but also mutations more superficially located in the receptor. It is concluded that activation of the BB3 receptor is dependent upon an epitope in the main ligand-binding pocket at the interface between TM-III, TM-VI, and TM-VII that corresponds to the site where, for example, activating metal ion sites have been constructed previously in 7TM receptors.
BB3, which used to be called BRS-3, belongs to the mammalian bombesin receptor family of seven-transmembrane (7TM) G protein-coupled receptors that also comprises BB1, i.e., the receptor for neuromedin B; BB2, the receptor for gastrin-releasing peptide and neuromedin C (Fathi et al., 1993; Ryan et al., 1998; Jensen et al., 2008) (Fig. 1). BB3 has a very low affinity for the amphibian bombesin peptide as it has for any known mammalian peptide, and it is consequently still considered an orphan receptor (Fathi et al., 1993; Jensen et al., 2007). It couples through Gq and phospholipase C, leading to calcium mobilization and an increase in inositol phosphates. BB3 is located both peripherally and centrally, including expression in feeding centers of the hypothalamus as well as several other areas within the central nervous system (Fathi et al., 1993; Ohki-Hamazaki et al., 1997a; Sano et al., 2004; Porcher et al., 2005).
Despite its status as an orphan receptor, BB3 has attracted much attention mainly based on the metabolic phenotype of BB3-deficient mice resembling type-2 diabetes as characterized by Wada and coworkers (Ohki-Hamazaki et al., 1997b; Maekawa et al., 2004; Nakamichi et al., 2004). Thus, BB3 knockout mice develop late onset, moderate obesity associated with insulin and leptin resistance as well as increased feeding efficiency and reduced metabolic rate (Ohki-Hamazaki et al., 1997b; Maekawa et al., 2004). The BB3-deficient mice have impaired glucose tolerance and impaired glucose transporter 4 translocation in adipocytes, conceivably associated with their insulin resistance (Nakamichi et al., 2004). Centrally, BB3 may balance appetite via inhibition of the MCH system as BB3 knockout mice have an enhanced hyperphagic responses to MCH and display increased expression of both MCH and the MCH type-1 receptor (Maekawa et al., 2004).
Based on the nonselective bombesin analog [d-Phe6,β-Ala11,Phe13,Nle14]-bombesin(6-14) of Jensen and coworkers, several high-affinity selective BB3 peptide ligands were designed. Some of these peptides are clearly bombesin analogs, whereas the resemblance to bombesin was eliminated in the primary structure of other peptides (Mantey et al., 2001, 2004; Boyle et al., 2005; Mantey et al., 2006). In the present study, we characterize by receptor mutagenesis epitopes in the main ligand-binding pocket of the BB3 receptor through which two such high-potency selective agonist peptides act: [d-Tyr6,(R)-Apa11,Phe13,Nle14]-bombesin(6-14) (Pep-1; compound 14 of Mantey et al., 2001) and the “nonbombesin” Ac-Phe-Trp-Ala-His(TauBzl)-Nip-Gly-Arg-NH2 (Pep-2; compound 34 of Boyle et al., 2005; Fig. 2). Surprisingly, this work also provide novel, interesting information about both loss-of-function and gain-of-function mutations in respect of ligand-independent, constitutive signaling activity of the BB3 receptor.
Materials and Methods
The synthetic Pep-1, d-Tyr-Gln-Trp-Ala-Val-(R)Apa-His-Phe-Nle-NH2, was purchased from Phoenix Pharmaceuticals (Belmont, CA), and Pep-2, Ac-Phe-Trp-Ala-His(tBzl)-Nip-Gly-Arg-NH2, was synthesized by Alta Biosciences (University of Birmingham, Birmingham, UK).
The cDNA for the human BB3 receptor was kindly provided by Kate Hansen (7TM Pharma A/S, Hørsholm, Denmark). The cDNA was cloned into the eukaryotic expression vector pCMV-Tag(2B) made by Stratagene (La Jolla, CA) for epitope tagging of proteins with a FLAG epitope. Mutations were constructed by PCR using the overlap extension method (Horton et al., 1989). The PCR products were digested with appropriate restriction endonucleases BamHI/EcoRI, purified, and cloned into the vector pCMV-Tag(2B) (Stratagene). All PCR products were performed using Pfu polymerase (Stratagene) according to the instructions of the manufacturer. All mutations were verified by restriction endonuclease mapping and subsequent DNA sequence analysis using an ABI Prism 310 automated sequencer (Applied Biosystems, Foster City, CA).
Transfection and Tissue Culture.
HEK-293 cells were grown in Dulbecco's modified Eagle's medium-GlutaMAX (Invitrogen, Carlsbad, CA), supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C and 10% CO2. Cells were transfected using the calcium phosphate precipitation method as described with some modifications (Holst et al., 2007). In brief, HEK-293 cells were seeded at a density of 8 × 106 cells/150-cm2 flask and grown overnight at 37°C in growth medium. On the following day, 40 μg of plasmid DNA was transfected using 2 M CaCl2, HBS buffer, pH 7.2 (VWR/Bie & Berntsen, Herlev, Denmark) and TE buffer (1 mM EDTA and 10 mM Tris, pH 7.5). After 5-h incubation at 37°C, transfection medium was changed by fresh medium, and cells were incubated overnight at 37°C and 10% CO2.
One day after transfection, cells (seeding density, 2 × 105 cells/well) were incubated for 24 h with 5 μCi of myo-[3H]inositol (GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK) in 1 ml of growth medium. Cells were washed twice in buffer [20 mM HEPES, pH 7.4, supplemented with 140 mM NaCl, 5 nM KCl, 1 mM MgSO4, 1 mM glucose, and 0.05% (w/v) bovine serum albumin] and were incubated in 0.5 ml of buffer supplemented with 10 mM LiCl for 30 min at 37°C. After stimulation with various concentrations of peptides for 45 min at 37°C, cells were extracted by addition of 1 ml of 10 mM formic acid to each well followed by incubation on ice for 30 to 60 min. The generated [3H]inositol phosphates were purified on AG 1-X8 anion exchange resin (Bio-Rad Laboratories, Hercules, CA). Determinations were made in duplicate.
Cell Surface Expression Measurement (Enzyme-Linked Immunosorbent Assay).
Cells were transfected and seeded out in parallel with those used for IP accumulation assay. The cells were washed twice with PBS and then fixed for 10 min in 3.7% formaldehyde. After three washes in PBS (three 10-min washes), cells were incubated in blocking solution (3% dry milk and 50 mM Tris-HCl, pH 7.5, in PBS) for 1 h at room temperature. Cells were kept at room temperature for all subsequent steps. The cells were incubated for 2 h with anti-FLAG (M2) antibody (Sigma-Aldrich, St. Louis, MO) at 1:300 dilution in blocking solution. After three washes, cells were incubated with goat anti-mouse horseradish peroxidase-conjugated secondary antibody (Pierce Chemical, Rockford, IL) at 1:1250 dilution in the same buffer as the anti-FLAG antibody for 1 h. After three washes in PBS (three 10-min washes), the immune reactivity was revealed by addition of 100 μl of TMB Plus substrate (Kem-En-Tec, Taastrup, Denmark), and the reaction was stopped with 100 μl of 0.2 M H2SO4. Absorbance was measured at 450 nm for 1 s on a Wallac VICTOR2 plate reader (PerkinElmer Life and Analytical Sciences, Boston, MA).
EC50 values were determined by nonlinear regression using the Prism 4.0 software (GraphPad Software Inc., San Diego, CA). Fmut indicates the fold shift in potency induced by the mutated receptor compared with the wild-type receptor.
The structures of the prototype BB3-selective peptide agonists Pep-1 (Mantey et al., 2001) and Pep-2 (Boyle et al., 2005) are shown in Fig. 2. Pep-1 displayed a biphasic agonist profile in respect of stimulating IP3 turnover in transiently transfected HEK-293 cells, with an EC50 value for the high-potency component of 0.3 nM (Fig. 2). Pep-2 had a similar biphasic agonist profile, albeit with a slightly lower potency for the high-potency component (EC50 = 1.2 nM), and a slightly lower but nonsignificantly different Emax value (850 ± 82 dpm for Pep-1 versus 729 ± 73 dpm for Pep-2; Table 1). Neither of the peptides stimulated IP3 turnover in mock-transfected cells, indicating that both the high- and the low-potency agonist components are mediated through the BB3 receptor (Fig. 2).
In total, 18 positions facing the main ligand-binding pocket of the BB3 receptor were probed mainly by Ala substitutions to identify residues being involved in the agonist-induced signaling (Fig. 1; Tables 1 and 2). GluIV:20 was substituted both with Ala and Gln, and AlaVII:09 was substituted with Val as a steric hindrance approach (Holst et al., 1998). Two of the mutants were located in extracellular loop 2 close to the conserved Cys residue that forms a disulfide bridge with CysIII:01 (Fig. 1). As shown in Table 1, all mutants were expressed relatively well as determined by cell surface ELISA, except for the LeuIII:04 to Ala [25 ± 2% of wild type (wt)] and the GluIV:20 to Gln (28 ± 7%) mutations.
Mutational Effects on BB3 Constitutive Signaling.
The wild-type BB3 receptor displayed a clear degree of constitutive signaling activity corresponding to 12 ± 1% (n = 32) of the maximally achievable efficacy in response to Pep-1 as determined by IP turnover in the transiently transfected HEK-293 cells (Fig. 1; Table 1). As indicated by red symbols in Fig. 3, several mutations clustering on the opposing faces of TM-III, TM-VI, and TM-VII decreased the constitutive activity of the BB3 receptor (Table 1). In most cases, the constitutive activity was reduced from 12 to 4 to 7% of the maximal efficacy. However, in LeuIII:04 and SerVI:24 to Ala substitutions, the ligand-independent signaling of the receptor was eliminated (Table 1). It is important to note that, as opposed to the LeuIII:04 mutation, the expression level of the SerVI:24 mutation was not affected at all as determined by cell surface ELISA (Table 1), indicating that at least in this case the mutation is truly impairing the constitutive signaling of the receptor.
It is interesting that four BB3 mutations instead increased the constitutive signaling of the BB3 receptor (Table 1). These gain-of-function mutations were located at very different and distinct positions in the receptor, as shown in green symbols in Fig. 3. That is, two gain-of-function mutations were found at each end of the main ligand-binding pocket, i.e., on the inner face of TM-II and TM-V, respectively, at positions II:17 and V:08 (Fig. 3). A third gain-of-function was located in extracellular loop 2, i.e., Glu201 to Ala; and the last was a steric hindrance mutation deep in the pocket at position VII:09, which increased the constitutive signaling to 26 ± 5 and 36 ± 6% of the maximal efficacy, respectively (Table 1). Notably, none of the mutations affected the Emax value for Pep-1 to a degree that could explain the change in apparent constitutive activity, except for the case of the LeuIII:04 as discussed above (Table 1).
Mutational Effects on Agonist-Induced Signaling.
Most of the mutants had no or only minor effects on the Pep-1-induced signaling (Tables 1 and 2), as exemplified by the SerV:05 and CysV:08 mutants shown in Fig. 4. In fact, only five mutations impaired the potency of Pep-1: LeuIII:04 (>1000-fold), AsnVI:16 (16-fold), TyrVI:20 (640-fold), ThrVII:02 (32-fold), and ArgVII:06 (440-fold) (Fig. 5; Table 2). The GluIV:20 mutations constituted a special case as both Ala and Gln substitution at this position impaired the efficacy of especially the high-potency component of Pep-1 without affecting the potency of the peptide (Fig. 6).
In contrast to Pep-1, the potency of Pep-2 was impaired 10-fold or more by mutations at 12 of the 18 positions in the main ligand-binding pocket that were probed in the present study, including the five positions that also were hits for Pep-1 (Table 2). As shown by helical wheel diagrams in Fig. 7, the epitope in the main ligand-binding pocket, which is essential for the function of Pep-1, i.e., the interface between the extracellular ends of TM-III, TM-VI, and TM-VII, constitutes the core or the center of the more expanded epitope that is essential for the function of Pep-2. In addition, Pep-2 is dependent also upon residues in the minor pocket at the interface between TM-II and TM-VII and residues on the inner face of TM-III, i.e., SerIII:05 and ArgIII:08 as well as SerVI:24 at the border between the extracellular end of TM-VI and extracellular loop 3 (Table 2; Fig. 7). All of these “extra” hits for Pep-2 compared with Pep-1 were, however, minor hits that only shifted the dose-response curve for Pep-2 between 16- and 40-fold to the right (Table 2). Among the common hits for the two agonist peptides, AsnVI:16 was only a minor hit for Pep-1 (16-fold), whereas it was a major hit for Pep-2 (>1000-fold). Mutations of GluIV:20 were also a special case for Pep-2 as the Ala substitution mainly affected the efficacy; and, in particular, for the high-potency component as also observed for Pep-1, whereas the Gln substitution eliminated the agonist curve for Pep-2 (Fig. 6).
In several of the mutants, the Emax value for Pep-2 was reduced compared with the wild-type receptor and Pep-1 (Table 1); e.g., the TyrVI:20-to-Ala mutant for which the Emax value for Pep-2 was only 26% of that for observed in the wild-type receptor and also was lower than the Emax value for Pep-1. The same case is observed with the ArgVII:06-to-Ala mutant for which the Emax value for Pep-2 is also relatively low (Table 1).
In the present study, we find that two structurally distinct selective agonist peptides for BB3 are dependent upon overlapping epitopes in the main ligand-binding pocket of the receptor. This common epitope located at the opposing faces of the extracellular segments of TM-III, TM-VI, and TM-VII, corresponds spatially to the epitope where, for example, activating metal ion sites previously have been built into 7TM receptors (Elling et al., 1999, 2006). The BB3 receptor was found to signal with 12% constitutive activity. It is important that this ligand-independent activity was strongly influenced both negatively and positively in a structurally systematic manner by the amino acid substitutions in the main ligand-binding pocket of the BB3 receptor.
Mutational Map of Activation Epitope for the BB3 Agonist Peptides.
As shown in Fig. 2, Pep-1, a bombesin analog, and Pep-2, a “nonbombesin” peptide, only share a Trp-Ala dipeptide sequence. There are other structural similarities between the peptides in relation to certain side chains being aromatic and imidazole-based but placed at different positions. The two peptides also both contain a β-turn mimetic moiety, but again, it is structurally different in the two peptides (Fig. 2). Another major difference is the C-terminal Arg residue of Pep-2 for which there is no similar moiety in Pep-1. Nevertheless, despite their apparent structural differences, these synthetic peptides are both potent and full agonists on the BB3 receptor.
The mutational map of residues being important for the ability of the smaller Pep-2 to act as an agonist on BB3 basically covers all parts of the main ligand-binding pocket contrary to the larger Pep-1 for which the mutational map is surprisingly much more restricted as it is confined to the opposing faces of TM-III, TM-VI, and TM-VII (Fig. 7). The mutational map for Pep-1 constitutes the center of the more widespread mutational map for Pep-2. The four major mutational hits for Pep-2, i.e., those substitutions that had more than 100-fold effect on its potency, were all located at this interface between TM-III, TM-VI, and TM-VII. The mutations at three of these positions were also the major hits for Pep-1, i.e., LeuIII:04, TyrVI:20, and ArgVII:06 (Fig. 7).
In a similar study involving mutational analysis in the ghrelin receptor, the map for its endogenous 28-amino acid residue acylated peptide agonist was found to be confined to only six positions located spatially at the same interface between TM-III, TM-VI, and TM-VII, even though this is a much larger peptide (Holst et al., 2009). Nevertheless, although the BB3 agonist peptides and ghrelin have similar activation epitopes on their respective receptors, they are to a large degree dependent upon different residues within this common epitope. For example, the only overlap between the map for ghrelin on its receptor and the mutational hits for the two synthetic agonist peptides on the BB3 receptor of the present study are positions VI:16 and VI:20.
The classical binding site for small monoamine agonists, for example in the β2-adrenoceptor, is also found at the interface between TM-III, TM-VI, and TM-VII. In this case, the major anchor point for the monoamine function is AspIII:08 in combination with AsnVII:06 and with AsnVI:20 being the presumed hydrogen bond partner for the important β-hydroxyl group. However, in this case, TM-V is also an important part with serine residues believed to be interaction partners for the hydroxyl groups of the catechol ring at the other end of the ligand (Del Carmine et al., 2004; Nygaard et al., 2009). As recently described by Liu and coworkers (2009), in small organic acids such as lactate (GPR81), nicotinic acid/β-hydroxybutyrate (GPR109A and B), oxoeicosanoid (TG1019), and kynurenic acid (GPR35), their almost identical, presumed binding site is also located at the interface between TM-III, TM-VI, and TM-VII. In these cases, a conserved ArgIII:12 is presumed to function as the main anchor point for the acid moiety.
In relation to mutational mapping of the activation epitopes in 7TM receptors, it is important that also gain-of-function in respect of ligand efficacy has been obtained. Thus, receptor activation by a small and geometrically well defined metal ion was obtained through engineering of a metal ion site into the corresponding epitope of the β2-adrenoceptor and the tachykinin NK1 receptor, i.e., at positions III:08, VI:16, and VII:06, as shown in Fig. 7 (Elling et al., 1999, 2006; Holst et al., 2000). Notably, substance P, the endogenous agonist for the NK1 receptor, was not affected by these substitutions as it binds “above” this epitope (Holst et al., 2000). Moreover, for example, although position III:08 is crucial both for monoamine ligand function and for the activating metal ion sites, and to some degree for Pep-2, in the present study (Fig. 7), this position is not at all important for Pep-1 (Table 2) or for ghrelin on its receptor (Holst et al., 2006, 2007, 2009). This is in agreement with the notion that there is no “common lock” for all the agonists “keys” in 7TM receptor (Schwartz et al., 2006), although agonists often are dependent upon residues located on the interface between TM-III, TM-VI, and TM-VII as demonstrated in the present study for BB3. The way this may function was summarized in the global toggle switch model for 7TM receptor activation (Schwartz et al., 2006). According to this model, agonists simply act by stabilizing the active conformation of the receptor in which, in particular, the extracellular segment of TM-VI but also to some extent TM-VII and TM-V, tilts inward in the main ligand-binding pocket toward TM-III (Elling et al., 2006; Schwartz et al., 2006; Nygaard et al., 2009).
Peptide Agonist Interaction Also in the Extracellular Loops.
In larger ligands such as peptides, it is believed that they may interact also with the extracellular loops of the receptor and the N-terminal extension. Through this interaction, they may not only function as “glue” between the helices but also may, or instead, depending on the peptide, function as “Velcro” at the surface of the receptor (Schwartz et al., 2006). Specifically concerning the BB3 peptide agonists, it has been demonstrated previously that they also are dependent upon residues located in the extracellular loop regions (Gonzalez et al., 2008). This fits very well with similar observations in for example the NK1 and the angiotensin AT1 receptor systems (Fong et al., 1992; Hjorth et al., 1994). It should be noted that 7TM receptors even can be activated by antibodies developed against peptides corresponding to the extracellular loops (Schwartz et al., 2006) or by a small zinc ion binding in the extracellular domain of the receptor as shown for GPR39 (Storjohann et al., 2008). This underlines the notion that an agonist in a 7TM receptor does not have to interact directly with a particular residue or epitope deep in the receptor pocket to stabilize an active conformation of the receptor (Schwartz et al., 2006; Nygaard et al., 2009).
Mutational Map of the Epitopes Influencing the Constitutive Activity of the BB3 Receptor.
The present study adds the BB3 receptor to the list of 7TM receptors displaying a clear—in this case 12%—degree of constitutive activity. Other receptors involved in the control of food intake and energy expenditure, such as the cannabinoid CB1 and ghrelin receptor, signal with close to 50% of their maximal efficacy in the absence of the endogenous ligand (Bouaboula et al., 1997; Holst and Schwartz, 2003). In the ghrelin receptor, a cluster of aromatic residues at the interface of TM-VI and -VII was identified to be particularly important for its constitutive activity (Holst et al., 2004). It is important to note that a naturally occurring mutation in this cluster, PheVI:16 to Leu, which selectively impairs the constitutive activity without affecting the agonists-induced signaling, is associated with a phenotype of short stature and obesity in children (Pantel et al., 2006). This is strong evidence in favor of the notion that the constitutive activity, at least of the ghrelin receptor, is of physiological importance in intact organism (Holst and Schwartz, 2006).
In the present study, we find that the mutations that identified the common epitope being important for the agonist-induced activation of the BB3 receptor also are important for the ligand-independent signaling, i.e., AsnVI:16, TyrVI:20, ThrVII:02, ArgVII:06, and possibly LeuIII:04. However, mutations of residues located more superficially in the receptor, e.g., HisVI:23, SerVI:24, and Thr204 in ECL-2, also impaired the constitutive activity. It is interesting to note that several mutations increased the constitutive activity of the BB3 receptor (Fig. 3), which is only rarely seen in, for example the ghrelin receptor.
Recently, small-molecule nonpeptide agonists for the BB3 receptor were discovered based on an omeprazole lead (Carlton et al., 2008). It will be interesting to determine whether such compounds are dependent upon the same epitopes in the BB3 receptor as the peptide-based agonists characterized in the present study.
- Received September 29, 2009.
- Accepted January 6, 2010.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
- BB3 or BRS-3
- bombesin receptor subtype-3
- melanin-concentrating hormone
- [d-Tyr6,(R)-Apa11,Phe13, Nle14]-bombesin(6-14)
- polymerase chain reaction
- human embryonic kidney
- inositol phosphate
- phosphate-buffered saline
- inositol 1,4,5-triphosphate
- fold shift in potency induced by mutation compared with the wild-type receptor
- enzyme-linked immunosorbent assay
- wild type
- 3-amino-propionic acid
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics