molecular basis for agonism in the bb receptor –...

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MOLECULAR BASIS FOR AGONISM IN THE BB3 RECEPTOR

– AN EPITOPE LOCATED ON THE INTERFACE OF TM-III, -VI AND -VII.

F. Gbahou, B. Holst and T.W. Schwartz

Laboratory for Molecular Pharmacology, Institute of Neuroscience and

Pharmacology, University of Copenhagen, Blegdamsvej 3, DK-2200

Copenhagen, Denmark (F.G., B.H., T.W.S.);

Inserm U894 Centre de Psychiatrie et Neurosciences, Equipe de

Neurobiologie et Pharmacologie Moléculaire, 2 ter rue d’Alésia

75014 Paris, France (F.G.)

JPET Fast Forward. Published on January 11, 2010 as DOI:10.1124/jpet.109.162131

Copyright 2010 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on January 11, 2010 as DOI: 10.1124/jpet.109.162131

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Running title : Activating epitopes in the BB3 receptor

Corresponding author : Florence Gbahou

Inserm U894 Centre de Psychiatrie et Neurosciences,

Equipe de Neurobiologie et Pharmacologie Moléculaire,

2 ter rue d’Alésia

75014 Paris, France

Tel. +33 (0)1 40 78 92 78

Fax. +33 (0)1 45 80 72 93

Email: [email protected]

Number of text pages: 30

Number of tables : 2

Number of figures : 7

Number of references : 36

Number of words in abstract : 221

Number of words in Introduction : 424

Number of words in Discussion : 1465

Abbreviations : BB3 or BRS-3, Bombesin receptor subtype-3; Ac, acetyl; Apa, 3-

amino-propionic acid; Bzl, benzyl; Nle, norleucine; TM, transmembrane; PCR,

Polymerase chain reaction; IP3, inositol 1,4,5-triphosphate; LiCl, lithium chloride;

PBS, phosphate buffered saline; TMB, 3,3’,5’5 tetramethylbenzidine;

Section assignment : Cellular and Molecular


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ABSTRACT

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 centre of the pocket, i.e. the opposing faces of the

extracellular segments of 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, which 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 which was strongly influenced - both positively and negatively -

by a number of mutations in the binding pocket. The substitutions, which decreased

the constitutive signalling, included 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 which

corresponds to the site where, for example, activating metal-ion sites previously have

been constructed in 7TM receptors.


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INTRODUCTION

BB3, which used to be called BRS-3, belongs to the mammalian bombesin receptor

family of seven transmembrane (7TM) G protein-coupled receptors which also

comprises BB1, i.e. the receptor for neuromedin B, BB2 the receptor for gastrin-

releasing peptide (GRP), and neuromedin C (NMC) (Fathi et al., 1993; Jensen et al.,

2008; Ryan et al., 1998) (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 increase in

inositol phosphates. BB3 is located both peripherally and centrally including

expression in feeding centres of the hypothalamus as well as several other areas

within the CNS (Fathi et al., 1993; Ohki-Hamazaki et al., 1997a; Porcher et al., 2005;

Sano et al., 2004).

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 (Maekawa et al., 2004; Nakamichi et al.,

2004; Ohki-Hamazaki et al., 1997b). 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 (Maekawa et al., 2004; Ohki-Hamazaki

et al., 1997b). The BB3 deficient mice have impaired glucose tolerance and impaired

GLUT4 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 knock out 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).


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Based on the non-selective bombesin analog [D-Phe6,β-Ala11,Phe13,Nle14]-

Bombesin(6-14) of Jensen and coworkers a number of 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 (Boyle et al., 2005; Mantey et al., 2004; Mantey et al., 2001; 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 “non-

bombesin” 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 signalling activity of the BB3 receptor.


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METHODS

Materials

The synthetic Pep-1, D-Tyr-Gln-Trp-Ala-Val-(R)Apa-His-Phe-Nle-NH2 was

purchased from Phoenix Pharmaceuticals and Pep-2, Ac-Phe-Trp-Ala-His(tBzl)-Nip-

Gly-Arg-NH2 was synthesized by Alta Biosciences (University of Birmingham, UK).

Molecular Biology

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, La Jolla, CA). All PCR products were

performed using pfu polymerase (Stratagene, La Jolla, CA) according to the

instructions of the manufacturer. All mutations were verified by restriction

endonucleases 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 (DMEM)-

GlutaMAX (Invitrogen), supplement with 10% fetal bovine serum, 100 U/ml

penicilin, and 100 µg/ml streptomycin at 37ºC and 10% CO2. Cells were transfected

using the calcium phosphate precipitation method as described with some

modifications (Holst et al., 2007). Briefly, HEK-293 cells were seeded at a density of

8.106 cells/150 cm2 flask and grown overnight at 37ºC in growth medium. On the

following day, 40 µg of plasmid DNA were transfected using 2 M CaCl2, HBS buffer


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pH = 7.2 (VWR/Bie&Berntsen, Herlev, Denmark) and TE buffer (1 mM EDTA, 10

mM Tris pH = 7.5). After 5h incubation at 37ºC, transfection medium was changed by

fresh medium and cells incubated overnight at 37ºC, 10% CO2.

Phosphatidylinositol Turnover

One day after transfection, cells (seeding density, 2.105 cells/well) were incubated for

24 h with 5 µCi of myo-[3H]inositol (PT6-271, Amersham, 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 37ºC. After

stimulation with various concentrations of peptides for 45 min at 37ºC, cells were

extracted by addition of 1 ml of 10 mM formic acid to each well followed by

incubation on ice for 30 min 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 (ELISA)

Cells were transfected and seeded out in parallel with those used for IP accumulation

assay. The cells were washed twice with PBS, fixed for 10 min in 3.7%

formaldehyde. After three washes in PBS (3 x 10 min) cells were incubated in

blocking solution (3% dry milk, 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 Chemical Co., St.

Louis, MO) in 1:300 dilution in blocking solution. After three washes, cells were

incubated with goat anti-mouse horse radish peroxidase-conjugated secondary

antibody (Pierce) at 1:1250 dilution in the same buffer as the anti-FLAG antibody for


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1 h. After three washes in PBS (3 x 10 min), the immune reactivity was revealed by

addition of 100 µl TMB Plus substrate (Kem-En-Tec, Taastrup, Denmark) and the

reaction was stopped with 100 µl 0.2 M H2SO4. Absorbance was measured at 450 nm,

1 sec on a Wallac Victor 2 (Perkin Elmer Life Sciences).

Analysis of data

EC50 were determined by nonlinear regression using the Prism 4.0 software

(GraphPad Software, San Diego). Fmut indicates the fold shift in potency induced by

the mutated receptor as compared to the wild type receptor.


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RESULTS

The structures of the prototype BB3 selective peptide agonists D-Tyr-Gln-Trp-Ala-

Val-(R)Apa-His-Phe-Nle-NH2 (Pep-1) (Mantey et al., 2001) and Ac-Phe-Trp-Ala-

His(tBzl)-Nip-Gly-Arg-NH2 (Pep-2) (Boyle et al., 2005) are shown in Fig. 2. Peptide-

1 displayed a biphasic agonist profile in respect of stimulating IP3 turnover in

transiently transfected HEK-293 cells with an EC50 for the high potency component

being 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 non significantly different Emax (850 ± 82 dpm for Pep-1 vs 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).

A total of 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 signalling (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 which 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 WT) and the

GluIV:20 to Gln (28 ± 7%) mutations.

Mutational effects on BB3 constitutive signalling - The wild-type BB3 receptor

displayed a clear degree of constitutive signalling activity corresponding to 12 ± 1%


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(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 and Table 1). As

indicated by red symbols in Fig. 3, a number of the 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 4-7% of the maximal efficacy. However, in the case of the LeuIII:04 and

SerVI:24 to Ala substitutions, the ligand-independent signalling of the receptor was

totally eliminated (Table 1). Importantly, 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 signalling of the receptor.

Interestingly, four BB3 mutations instead increased the constitutive signalling 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 signalling to 26 ± 5 % and 36 ± 6 %

of the maximal efficacy respectively (Table 1).

Notably, none of the mutations affected the Emax for Pep-1 to a degree which could

explain the change in apparent constitutive activity, except for the case of the

LeuIII:04 as discussed above (Table 1).


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Mutational effects on agonist induced signalling – Most of the mutants had no or

only minor effects on the Pep-1 induced signalling (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, which were probed in the

present study – including the five positions, which 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 centre of

the more expanded epitope which 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 as

compared to Pep-1 were, however, minor hits, which 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 was also a

special case for Pep-2 as the Ala substitution mainly affected the efficacy, and in


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particularly for the high potency component as also observed for Pep-1 whereas the

Gln substitution totally eliminated the agonist curve for Pep-2 (Fig. 6).

In several of the mutants, the Emax for Pep-2 was reduced as compared to the wild-

type receptor and as compared to Pep-1 (Table 1). This is, for example, the TyrVI:20

to Ala mutant where the Emax for Pep-2 was only 26 % of that for observed in the

wild-type receptor and also lower than the Emax for Pep-1. The same case is observed

with the ArgVII:06 to Ala mutant for which the Emax for Pep-2 is also relatively low

(Table 1).


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DISCUSSION

In the present paper, 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., 2006; Elling et al., 1999). The BB3 receptor was found to

signal with 12% constitutive activity. Importantly, this ligand-independent activity

was strongly influenced both negatively and positively in a structurally systematic

fashion 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, which is a bombesin analog - [D-Tyr6,(R)-Apa11,

Phe13,Nle14]-Bombesin(6-14) - and Pep-2, which is a “non-bombesin” peptide - Ac-

Phe-Trp-Ala-His(TauBzl)-Nip-Gly-Arg-NH2, only share a Trp-Ala dipeptide

sequence. There are other structural similarities between the peptides in relation to

certain side chains being aromatic and imidazol-based however 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.


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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 centre of the more widespread

mutational map for Pep-2. The four major mutational hits for Pep-2, i.e. those

substitutions, which 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, in for example 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


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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 the case

of small organic acids such as lactate (GPR81), nicotinic acid/β-hydroxybutyrate

(GPR109A and B), oxoeicosanoid (TG1019) and kynerenic 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., 2006; Elling et al., 1999; Holst et al., 2000).

Notably, the endogenous agonist for the NK1 receptor, substance P was not affected

by these substitutions as it binds “above” this epitope (Holst et al., 2000). Moreover,

although, for example position III:08 is crucial both for monoamine ligand function

and for the activating metal-ion sites, and to some degree, for Pep-2 of 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., 2009; Holst et al., 2006; Holst et al., 2007). 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


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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 tilt inward in the main ligand-

binding pocket towards TM-III (Elling et al., 2006; Nygaard et al., 2009; Schwartz et

al., 2006).

Peptide agonist interaction also in the extracellular loops – In the case of 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 may also, 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

previously been demonstrated 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 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 in order to stabilize an active

conformation of the receptor (Nygaard et al., 2009; Schwartz et al., 2006).


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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 case of 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). Importantly, a naturally occurring mutation in this cluster,

PheVI:16 to Leu which selectively impairs the constitutive activity without affecting

the agonists induced signalling, is associated with a phenotype of short stature and

obesity in children (Pantel et al., 2006). This is strong evidence in favour 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 which identified the common epitope

being important for the agonist-induced activation of the BB3 receptor also are

important for the ligand-independent signalling, i.e. AsnVI:16, TyrVI:20, ThrVII:02,

ArgVII:06 and possibly LeuIII:04. However, also mutations of residues located more

superficially in the receptor, HisVI:23, SerVI:24 and Thr204 in ECL-2, impaired the

constitutive activity. Interestingly, 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 non-peptide agonists for the BB3 receptor were discovered

based on an omeprazole lead (Carlton et al., 2008). It will be interesting, whether such


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compounds are dependent upon the same epitopes in the BB3 receptor as the peptide-

based agonists characterized in the present study.


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LEGENDS FOR FIGURES

Fig. 1. Serpentine and helical wheel diagram of the human BB3 receptor. The

residues mutated in the present study are indicated in white on dark grey background.

The highly conserved, finger-print residues of each transmembrane segment are

indicated in white on blue.

Fig. 2. Structures and dose-response curves for the two prototype, selective BB3

agonist peptides (Pep-1 and Pep-2) in stimulating inositol phosphate

accumulation. Experiments were performed in HEK-293 cells transiently transfected

either with empty vector (mock) or wild-type (wt) BB3 receptor. The results were

normalized to the basal (mock cells) and maximum IP accumulation on wt receptor.

Each point represents the mean ± SEM of 15 to 17 independent experiments with

duplicate determinations. The position of the β-turn mimetic moiety is indicated by

dotted brackets. Ac, acetyl; Apa, 3-amino-propionic acid; Bzl, benzyl; Nle,

norleucine.

Fig. 3. Helical wheel diagram of the BB3 receptor displaying the effect of

mutations on the constitutive, ligand-independent signalling. The data concerning

the basal, constitutive signalling for the wild-type and each of the mutants are shown

in Table 1. In green are shown mutants which increased, in red mutants which

decreased, and in grey mutants which did not affect the constitutive signalling.

Fig. 4. Effect of two mutations in TM-V – SerV:05 to Ala (A) and CysV:08 to Ala

(B) – on Pep-1 and Pep-2 agonist-induced signalling through the BB3 receptor.


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Dose-response curves for Pep-1 (panels to the left) and Pep-2 (panels to the right)

were performed in transiently transfected HEK-293 cells. The results are expressed as

percent of the maximal effect observed in BB3 wild-type. Wild-type BB3 receptor data

shown in open squares and mutant data in filled triangles. Each point represents the

mean ± SEM of 3 to 17 independent experiments with duplicate determinations (see

Table 2 for details).

Fig. 5. Effect of selected mutations in TM-VI – AsnVI:16 to Ala (A) and

TyrVI:20 to Ala (B), and in TMVII – ArgVII:06 to Ala (C) – on Pep-1 and Pep-2

agonist-induced signalling through the BB3 receptor. Dose-response curves for

Pep-1 (panels to the left) and Pep-2 (panels to the right) were performed in transiently

transfected HEK-293 cells. The results are expressed as percent of the maximal effect

observed in BB3 wild-type. Wild-type BB3 receptor data shown in open squares and

mutant data in filled triangles. Each point represents the mean ± SEM of 4 to 17

independent experiments with duplicate determinations (see Table 2 for details).

Fig. 6. Effect of Ala and Gln substitutions in position IV:20 on Pep-1 and Pep-2

agonist-induced signalling through the BB3 receptor. GluIV:20 was substituted

both with Ala and Gln, and the mutated receptors were tested for their ability to

induce IP accumulation in the presence of increasing concentrations of Pep-1 and

Pep-2 in transiently transfected HEK-293 cells. Wild-type BB3 receptor data shown in

open squares and mutant data in filled triangles. Data are mean ± SEM of 3 to 17

independent experiments performed in duplicate.


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Fig. 7. Helical wheel diagram of the BB3 receptor displaying the effect of

mutations on the Pep-1 and Pep-2 agonist induced signalling. In grey are indicated

residues where mutagenesis had no or less than 10-fold effect on the potency of the

agonist peptide. In orange are indicated mutations which affected the potency of the

peptides between 10 and 100-fold, and in red those which affected the potency more

than 100-fold. The actual potencies are shown in Table 2. In the lower helical wheel

diagram are indicated the location of the positions, III:08, VI:16 and VII:06 which

upon substitution with metal-ion chelating residues - Asp, Cys or His – previously

have generated high affinity activating metal-ion sites in the β2-adrenoceptor and the

NK1 receptor (Elling et al., 2006; Elling et al., 1999; Holst et al., 2000).


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TABLE 1. Activity of BB3 mutations in transient HEK-293 cells. Cell surface expression of BB3 wild type (wt) and mutants were determined by ELISA. 100% equals the expression of BB3 wild-type receptor. Numbers are representative of at least two independent experiments with 2 determinations each. The constitutive activity is expressed both in dpm and as percentage of maximal signalling efficacy in response to Pep-1. Numbers are mean of 3 to 17 independent experiments with duplicate determinations. * p<0.05 compared to the BB3 wt receptor ** p<0.01 compared to the BB3 wt receptor *** p<0.001 compared to the BB3 wt receptor or BB3 wt compared to mock cells; n.s. non significant

Construct

Expression level

Constitutive activity

Emax (dpm ± SEM)

% ± SEM

dpm ± SEM (n)

% ± SEM

Pep-1

n

Pep-2

n

Mock cells

141 ± 3 (32)

BB3 wt

100

246 ± 12 (32)

12 ± 1***

850 ± 82

17

729 ± 73

15

Cys II : 17 Ala (C100)

84 ± 10

342 ± 9 (6)

24 ± 1***

603 ± 155

3

535 ± 82

3 His II : 24 Ala (H107) 97 ± 15 252 ± 9 (6) 13 ± 1n.s. 615 ± 50 3 590 ± 63 3

Leu III : 04 Ala (L123)

25 ± 2

142 ± 9 (6)

0***

8 ± 23

3

31 ± 6

3 Ser III : 05 Ala (S124) 116 ± 16 232 ± 47 (8) 11 ± 2n.s. 908 ± 77 4 826 ± 47 4 Arg III : 08 Ala (R127) 66 ± 10 288 ± 27 (8) 17 ± 3n.s. 472 ± 146 4 506 ± 111 4

Glu ECL2 : 19 Ala (E201)

133 ± 10

359 ± 41 (9)

26 ± 5***

969 ± 163

5

706 ± 185

4 Thr ECL2 : 22 Ala (T204) 53 ± 7 206 ± 11 (8) 8 ± 1*** 513 ± 97 4 359 ± 56 4

Glu IV : 20 Ala (E182)

49 ± 14

273 ± 22 (8)

16 ± 3n.s.

651 ± 104

4

515 ± 88

4 Glu IV : 20 Gln (E182) 28 ± 7 279 ± 29 (6) 16 ± 3n.s. 755 ± 232 3 260 ± 75 3

Ser V : 05 Ala (S218)

77 ± 14

261 ± 16 (6)

14 ± 2n.s.

721 ± 73

3

723 ± 60

3 Cys V : 08 Ala (C221) 78 ± 13 304 ± 32 (10) 19 ± 4* 640 ± 132 5 679 ± 159 5

Asn VI : 16 Ala (N287)

76 ± 6

196 ± 11 (10)

6 ± 1***

353 ± 94

5

127 ± 26

5 Tyr VI : 20 Ala (Y291) 107 ± 8 204 ± 17 (8) 7 ± 2** 751 ± 82 4 191 ± 57 4 His VI : 23 Ala (H294) 132 ± 10 180 ± 13 (11) 5 ± 1*** 733 ± 126 6 566 ± 116 5 Ser VI : 24 Ala (S295) 102 ± 10 128 ± 6 (9) 0*** 620 ± 106 5 513 ± 99 4

Thr VII : 02 Ala (T312)

130 ± 9

193 ± 14 (6)

6 ± 2*

580 ± 38

3

699 ± 129

3 Ile VII : 03 Ala (I313) 120 ± 17 217 ± 11 (8) 9 ± 1n.s. 971 ± 32 4 726 ± 86 4

Arg VII : 06 Ala (R316) 67 ± 12 174 ± 10 (8) 4 ± 1*** 361 ± 49 4 76 ± 21 4 Ala VII : 09 Val (A319)

70 ± 7 450 ± 53 (11) 36 ± 6*** 725 ± 139 6 653 ± 221 5

This article has not been copyedited and form

atted. The final version m

ay differ from this version.

JPET

Fast Forward. Published on January 11, 2010 as D

OI: 10.1124/jpet.109.162131

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JPET #162131

30

TABLE 2. Mutational mapping of the activation epitope for the BB3 selective agonist peptides 1 and 2 (Pep-1 and Pep-2) by use of a library of 20 mutants the BB3 receptor. The constructs were transiently transfected into HEK-293 cells and IP3 turnover was measured. The high potency component (EC50) of Pep-1 and Pep-2 is indicated (see text). Fmut indicates the fold shift in potency induced by mutation as compared with the wild type receptor. n = number of experiments.

Residue

Pep-1

Pep-2

EC50 nM ± SEM

Fmut

n

EC50 nM

Fmut

n

BB3 wt

0.3 ± 0.1

1

17

1.2 ± 0.6

1

15

Cys II : 17 Ala (C100)

0.4 ± 1.1

1.3

3

20 ± 12

17

3

His II : 24 Ala (H107) 0.4 ± 0.1 1.3 3 12 ± 6 10 3

Leu III : 04 Ala (L123)

–

>1000

3

–

>1000

3 Ser III : 05 Ala (S124) 0.15 ± 0.06 0.5 4 50 ± 8 42 4 Arg III : 08 Ala (R127) 0.1 ± 0.05 0.3 4 26 ± 9 22 4

Glu E2 : 19 Ala (E201)

0.3 ± 0.2

1

5

8.1 ± 2.6

7

3

Thr E2 : 22 Ala (T204) 0.3 ± 0.1 1 4 7.8 ± 3.9 6.5 4

Glu IV : 20 Ala (E182) 0.2 ± 0.1

0.7

4

0.6 ± 0.8

0.5

4

Glu IV : 20 Gln (E182) 0.17 ± 0.14 0.6 3 – >1000 3

Ser V : 05 Ala (S218)

0.2 ± 0.07

0.7

3

5.1 ± 1.1

4.3

3 Cys V : 08 Ala (C221) 0.2 ± 0.01 0.7 5 4.4 ± 2.5 3.7 5

Asn VI : 16 Ala (N287)

4.8 ± 3.8

16

5

–

>1000

5

Tyr VI : 20 Ala (Y291) 192 ± 78 640 4 – >1000 4 His VI : 23 Ala (H294) 2.1 ± 0.6 7 6 3.1 ± 1.6 2.6 5 Ser VI : 24 Ala (S295) 0.9 ± 0.4 3 5 25 ± 3 21 4

Thr VII : 02 Ala (T312)

9.6 ± 2.2

32

3

27 ± 8

23

3

Ile VII : 03 Ala (I313) 0.3 ± 0.1 1 4 23 ± 7 19 4 Arg VII : 06 Ala (R316) 132 ± 95 440 4 – >1000 4 Ala VII : 09 Val (A319)

1.1 ± 0.7 3.7 6 0.15 ± 0.2 0.1 5


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