Agonist-specific requirement for a glutamate in transmembrane helix 1 of the oxytocin receptor

Molecular and Cellular Endocrinology 333 (2011) 20–27

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Agonist-specific requirement for a glutamate in transmembrane helix 1 of the oxytocin receptor Denise L. Wootten a,1 , John Simms a,1 , Amelia J. Massoura a , Julie E. Trim b,2 , Mark Wheatley a,∗ a b

School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK Ferring Research Ltd., Southampton Science Park, 1 Venture Road, Southampton, UK

a r t i c l e

i n f o

Article history: Received 6 July 2010 Received in revised form 18 November 2010 Accepted 25 November 2010 Keywords: GPCR Oxytocin receptor Oxytocin Peptide hormone

a b s t r a c t Defining key differences between agonist and antagonist binding to hormone receptors is important and will aid rational drug design. Glu1.35 in transmembrane helix 1 (TM1) of the human oxytocin receptor (OTR) is absolutely conserved in all OTRs cloned to date. We establish that Glu1.35 is critical for high affinity binding of agonists (full and partial) but is not required for antagonist binding (peptide or non-peptide). Consequently, the mutant receptor [E1.35A]OTR exhibited markedly decreased OT affinity (>1200-fold) and disrupted second messenger generation. Substitutions of Glu1.35 by Asp, Gln or Arg were incapable of supporting wild-type OTR agonist binding or signaling. Molecular modeling revealed that Glu1.35 projects into the receptor’s central binding crevice and provides agonist-specific contacts not utilized by antagonists. This study explains why Glu is absolutely conserved at residue-1.35 in all receptors binding OT and related peptides, and provides molecular insight into key differences between agonist–receptor and antagonist–receptor binding modes. © 2010 Elsevier Ireland Ltd. All rights reserved.

1. Introduction The neurohypophysial peptide hormone oxytocin (OT) and oxytocin-like peptides, such as mesotocin and isotocin, facilitate reproduction in all vertebrates (Acher et al.,1995; Parry and Bathgate, 2000; Gimpl and Fahrenholz, 2001). Even in the relatively simple earthworm Eisenia foetida, the OT-related peptide annetocin induces egg-laying behaviour (Oumi et al., 1996). In humans, OT mediates a wide range of central and peripheral effects (Gimpl and Fahrenholz, 2001; Opar, 2008), including increasing the frequency and intensity of uterine contraction at parturition and contraction of the mammary gland myoepithelium during lactation (Gimpl and Fahrenholz, 2001; Soloff et al., 1979). The potent uterotonic role played by OT in birth has resulted in extensive use of this peptide clinically to induce and augment labor (Owen and Hauth, 1992). The physiological effects of OT are mediated

Abbreviations: AVP, [arginine8 ]vasopressin; ELISA, enzyme-linked immunosorbent assay; GPCR, G-protein-coupled receptor; InsP, inositol phosphate; InsP3 , inositol trisphosphate; OT, oxytocin; OTA, d(CH2 )5 Tyr(Me)2 Thr4 Orn8 Tyr(NH2 )9 vasotocin; OTR, oxytocin receptor; TM, transmembrane helix. ∗ Corresponding author. Tel.: +44 121 414 3981; fax: +44 121 414 5925. E-mail address: [email protected] (M. Wheatley). 1 Present address: Monash Institute of Pharmaceutical Sciences, 381 Royal Parade, Parkville, Melbourne, Victoria 3052, Australia. 2 Present address: Shire Pharmaceutical Development, Hampshire International Business Park, Chineham, Basingstoke RG24 8EP, UK. 0303-7207/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2010.11.029

by a specific oxytocin receptor (OTR) expressed by target tissues. As pregnancy approaches term there is an increase in the abundance of OTRs expressed by the myometrium which results in a specifically timed increased responsiveness of the uterus to OT (Fuchs et al., 1995; Kimura and Saji, 1995; Parry and Bathgate, 2000). Binding antagonists to the OTRs can effectively blockade the receptors, thereby reducing receptor availability to OT resulting in increased uterine quiescence. Both peptide antagonists such as Atosiban (d[DTyr(Et)2 ,Thr4 ,Orn8 ]OT) (Valenzuela et al., 2000) and non-peptide antagonists (Pettibone and Freidinger, 1997; Hawtin et al., 2005a) have been developed for this tocolytic purpose. The OTR is a Family A (rhodopsin-like) G-protein-coupled receptor (GPCR) and exhibits structural features typical of this family, including seven transmembrane (TM) helices (Kimura et al., 1992). Only one OTR subtype has been cloned from humans, implying that the wide range of physiological effects of OT is mediated by a single receptor which signals primarily by coupling to phospholipase C to generate inositol trisphosphate (InsP3 ) as second messenger (Gimpl and Fahrenholz, 2001). As agonists induce OTR signaling and antagonists do not, defining differences between the agonist–OTR interaction and the antagonist–OTR interaction at the molecular level, will provide insight into OT action and may aid rational drug design. In this study we establish that a Glu in TM1 of the human OTR is critical for agonist binding and signaling but is not required for antagonist binding (peptide antagonist or non-peptide antagonist). Furthermore, we demonstrate that there is a specific requirement for Glu

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at this locus which explains why this Glu is absolutely conserved in all receptors for OT and OT-related peptides cloned to date. 2. Materials and methods 2.1. Materials The radioligand [Tyr9 -125 I]OTA (specific activity of 2200 Ci/mmol) was from Perkin Elmer (Beaconsfield, UK). OT and AVP were purchased from Sigma (Poole, UK). The cyclic peptide antagonist [d(CH2 )5 Tyr(Me)2 Thr4 Orn8 TyrNH2 9 ]vasotocin (OTA) was from Bachem (St. Helens, UK) and L-368,899 was a kind gift from Dr. Douglas J. Pettibone (Merck Research Laboratories, West Point, PA). Cell culture media, buffers and supplements were purchased from Gibco (Uxbridge, UK). DpnI and Pfu polymerase were from New England Biolabs (Hitchin, UK). 2.2. Mutant receptor constructs Human OTR cDNA with an N-terminal haemagglutinin (HA) tag was subcloned into pcDNA3.1(+) (Invitrogen) prior to mutagenesis. Introduction of the epitope tag did not affect the pharmacology of the receptor. Mutants were engineered using the QuikChangeTM site-directed mutagenesis kit (Stratagene, Cambridge, UK) according to the manufacturer’s instructions using forward and reverse oligonucleotide primers synthesized by Invitrogen (UK). The forward primers for [E1.35A]OTR, [E1.35D]OTR, [E1.35Q]OTR and [E1.35R]OTR were 5 -C-CTG-GCG-CGC-GTG-GCA-GTG-GCG-GTG-CTG-3 , 5 -CTG-GCC-AAA-CTGGAC-ATC-GCC-GTG-CTG-3 , 5 -CTG-GCC-AAA-CTG-CAG-ATC-GCC-GTG-CTG-3 and 5 -CTG-GCC-AAA-CTG-CGC-ATC-GCC-GTG-CTG-3 , respectively, with appropriate base changes shown in bold. All receptor constructs were confirmed by automated fluorescent sequencing in their entirety in both sense and antisense directions (Functional Genomics Laboratory, University of Birmingham, Birmingham, UK) and subcloned using unique HindIII and KpnI restriction sites.

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2.6. Inositol phosphates production The assay for accumulation of inositol phosphates induced by OT or AVP was based on that previously described (Howl et al., 1995). Briefly, following pre-labeling of transfected cells with 1 ␮Ci/ml myo-[2-3 H]inositol (Perkin Elmer) in inositol free DMEM containing 1% (v/v) FCS, a mixed fraction containing mono-, bis-, and tris-, phosphates (InsP–InsP3 ) was collected following stimulation by agonist for 30 min, at the concentrations indicated, in the presence of 10 mM LiCl. EC50 values were determined by nonlinear regression after fitting of sigmoidal curves to experimental data. 2.7. Secondary structure prediction The sequence of the OTR was submitted to the hidden Markov model-based protein structure prediction, SAM-T02 (Karplus et al., 2003), which utilizes a hidden Markov model engine to search for homologous proteins from which a sequence alignment is produced and a structure prediction obtained. 2.8. Receptor modeling The OTR sequence was aligned against the crystal structure coordinates of bRho using CLUSTALW (Thompson et al., 1994). The alignment was then used to generate homology models using MODELLER version 6.2 (Sali and Blundell, 1993). A collection of 200 model structures was generated and ranked based on an objective function provided by MODELLER version 6.2. From this ensemble, a single structure was selected for further analysis. Further refinement of the homology model was achieved through molecular dynamics simulations of the receptor embedded in a hydrated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine bilayer. Molecular dynamics simulations were carried out using the GROMOS96 force-field parameters, with minor modifications, as implemented in GROMACS (Lindahl et al., 2001).

3. Results 2.3. Cell culture and transfection HEK 293T cells were routinely cultured in Dulbecco’s modified Eagles medium (DMEM) containing l-glutamine (2 mM), d-glucose (4500 mg/l) and sodium pyruvate (1 mM) supplemented with 10% (v/v) fetal calf serum (FCS) in humidified 5% (v/v) CO2 in air at 37 ◦ C. For radioligand binding assays, cells were seeded at a density of ∼5 × 105 cells/100 mm dish and transfected after 48 h. For measurements of cell-surface expression, cells were seeded at a density of 1.5 × 105 cells per poly dlysine-coated well (24-well plate) and transfected after 30 h. For measurement of agonist-induced inositol phosphates production, HEK 293T cells were seeded onto poly d-lysine-coated 12-well plates at a density of 2.5 × 105 cells per well and transfected after 30 h. Cells were transfected using a mixture (per 1 ␮g DNA) of 6 ␮l 10 mM polyethyleneimine and 45 ␮l 5% glucose solution, incubated for 30 min at room temperature and added to an appropriate final volume of full media. 12- and 24-well plates were treated with 0.5 ␮g and 1 ␮g DNA per well respectively and 100 mm dishes were treated with 5 ␮g DNA/dish. Characterization of expressed receptors was performed 48–72 h after transfection. 2.4. Radioligand binding assays A washed membrane fraction was prepared from transfected HEK 293T cells and the protein concentration determined using the BCA protein assay kit (Pierce Chemical Co., UK) with BSA as standard. Radioligand binding assays were performed as previously described (Hawtin et al., 2000) using the OTR-selective peptide antagonist [125 I]OTA (2200 Ci/mmol) as tracer ligand. Competition binding assays (final volume of 500 ␮l) containing radioligand (0.5–10 pM), cell membranes (100–500 ␮g) and competing ligand (at the concentration indicated) were incubated at 30 ◦ C for 90 min to establish equilibrium. Bound and free ligand were separated by centrifugation (13,000 × g, 10 min), membranes washed, dissolved in Soluene-350 (Packard) and the radioactivity quantified by liquid scintillation spectroscopy using HiSafe3 (Wallac, UK) as cocktail. Non-specific binding was determined in parallel incubations using OTA (1 ␮M). Binding data were analyzed by non-linear regression to fit theoretical Langmuir binding isotherms to the experimental data using GraphPad PRISM (Graphpad Software Inc., San Diego, CA). IC50 values for competing ligands were corrected for radioligand occupancy (Cheng and Prusoff, 1973) using the radioligand affinity (Kd ) experimentally determined for each individual construct. 2.5. Determination of cell-surface expression using enzyme-linked immunosorbent assay (ELISA) All receptor constructs incorporated an HA epitope tag in the N-terminus which enabled cell-surface expression to be determined 48 h after transfection, in fixed cells, using an ELISA as described previously (Hawtin et al., 2006). Results were normalized against a wild-type OTR control processed in parallel. Non-transfected cells were used to determine background. All experiments were performed in quadruplicate.

3.1. Glu1.35 provides critical agonist-specific binding contacts Elucidation of peptide hormone binding sites within the GPCR architecture is of fundamental importance for understanding the molecular basis of agonist-induced receptor activation. Furthermore, defining the specific ligand-receptor interactions which result in a ligand functioning as an agonist, versus an antagonist, will aid rational drug design. For the OTR, the ligand binding site is composed of residues located within both the TM helical bundle plus residues in extracellular domains (Gimpl and Fahrenholz, 2001). It has been shown previously that the N-terminus of the OTR is required for high affinity binding of OT but not for binding antagonists (Hawtin et al., 2001; Postina et al., 1996). Furthermore, this agonist-specific effect was attributed to Arg34 (residue 1.27 using standard GPCR residue nomenclature (Ballesteros and Weinstein, 1995)) located within the N-terminal domain proximal to the membrane and close to the top of TM1 (Wesley et al., 2002). Examination of all the known sequences of receptors for OT and OT-like peptides in the GPCR database (www.gpcr.org) revealed that a glutamic acid residue is absolutely conserved within the TM1 helix, at residue-1.35. Furthermore, molecular modeling indicates that the side-chain of this Glu1.35 orientates into the binding crevice within the TM helical bundle (Fig. 1) where it would be well placed to provide binding contacts for ligand interaction. To investigate the role of Glu1.35 in ligand recognition, a mutant human OTR was engineered in which Glu1.35 was replaced by Ala ([E1.35A]OTR). [E1.35A]OTR was expressed in HEK 293T cells, pharmacologically characterized and compared to wild-type OTR. Three different classes of ligand were employed to probe the contribution of Glu1.35 to the binding site: (i) the natural agonist OT; (ii) the cyclic peptide antagonist [d(CH2 )5 Tyr(Me)2 Thr4 Orn8 Tyr(NH2 )9 ]vasotocin (OTA), which is structurally related to OT (Elands et al., 1988); and (iii) a nonpeptide antagonist L-368,899 which is camphor-based and has no structural similarity to OT (Williams et al., 1994). The wildtype receptor and [E1.35A]OTR exhibited the same cell surface expression level of 1–2 pmol/mg protein (Table 1). Competition radioligand binding curves were determined for the different

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observed effects on potency, the Emax of both OT and AVP was unaffected by the E1.35A mutation (Fig. 2C). 3.3. Absolute requirement for glutamate at residue 1.35 It was important to define the properties of the Glu1.35 sidechain that underlie its importance in OTR function. The specific structural requirements at this locus were evaluated by systematic mutation. The following constructs were engineered and pharmacologically characterized; [E1.35D]OTR (preserves the negative charge but shortens the side-chain length by a single –CH2 ), [E1.35Q]OTR (removes the negative charge but maintains sidechain length and polarity) and [E1.35R]OTR (reverses the charge from negative to positive). All of the mutant receptors were expressed at the same level as wild-type OTR and consistent with the agonist-specific function of Glu1.35 , exhibited wild-type affinity for the peptide antagonist OTA and for the non-peptide antagonist L-368,899 (Table 1). In contrast, agonist affinity was greatly influenced by the identity of the residue at position-1.35. The affinity of OT for [E1.35D]OTR, [E1.35Q]OTR and [E1.35R]OTR was reduced by 800-fold, 1200-fold and 2000-fold respectively, compared with wild-type (Fig. 3A and Table 1). Likewise, binding of the partial agonist AVP to these mutant receptors was also markedly reduced (Fig. 3B and Table 1). The nature of the residue at position-1.35 also dictated second messenger generation in response to OT, with none of the Glu1.35 substitutions supporting wild-type intracellular signaling (Fig. 4A and B). Reversing the charge ([E1.35R]OTR) was particularly detrimental, raising the EC50 for InsP accumulation by >100-fold. Replacing the negative charge with a polar group in [E1.35Q]OTR resulted in a 56-fold increase in EC50 compared to wild-type. Interestingly, preserving the negative charge with the conservative substitution [E1.35D]OTR, greatly perturbed second messenger generation, resulting in a 47-fold increase in EC50 compared to wild-type OTR (Fig. 4A and B). All of these residue-1.35 substitutions were more detrimental to signaling than a simple alanine substitution ([E1.35A]OTR). The OT-induced maximal response for [E1.35A]OTR and [E1.35R]OTR was wild-type, despite the increased EC50 values exhibited. However, the Emax for [E1.35D]OTR and [E1.35Q]OTR was only 38% and 55% of wild-type respectively (Fig. 4A and B). All constructs were expressed at the cell-surface at similar levels to wild-type OTR (Table 1), indicating that the decreased Emax values were not due to a loss of receptor abundance at the cell-surface.

Fig. 1. Molecular model of the OTR indicating the orientation of Glu1.35 . The OTR is viewed from above. The side-chain of Glu1.35 is shown (in black) projecting towards the central binding crevice formed by the TM bundle (in grey) with individual TM helices numbered 1–7.

classes of ligand using [125 I]OTA as tracer (Fig. 2). The Ki values are presented in Table 1, corrected for radioligand occupancy. The binding affinity of both the peptide antagonist (OTA) and the nonpeptide antagonist (L-368,899) were unaffected by substitution of Glu1.35 by Ala (Fig. 2A). In contrast, this substitution resulted in a profound decrease in affinity for the natural agonist OT with the Ki increasing by 1200-fold (Fig. 2B). To investigate further the role of Glu1.35 in agonist binding, we utilized vasopressin (AVP). AVP is an analog of OT in which the residues Ile3 and Leu8 in OT have been replaced by Phe3 and Arg8 respectively. Moreover, AVP is a partial agonist at the OTR, not a full agonist like OT (Chini et al., 1996). Nevertheless, the affinity of AVP for [E1.35A]OTR was also markedly reduced (200-fold) compared to wild-type OTR (Fig. 2B, Table 1). Consequently Glu1.35 is essential for high affinity agonist binding (full or partial agonist), but the role of this residue is strictly agonist-specific and it does not have a role in binding antagonists (peptide or non-peptide). 3.2. A role for Glu1.35 in OTR signaling capability The intracellular signaling capability of the wild-type OTR and [E1.35A]OTR mutant was investigated. InsP accumulation dose–response curves were determined for OTR and [E1.35A]OTR using OT or AVP (Fig. 2C). As expected, AVP was a partial agonist with respect to InsP signaling, with the maximal response induced by AVP being only 58 ± 4% (n = 3) of the OT maximum response (Fig. 2C). The [E1.35A]OTR mutation perturbed intracellular signaling (Fig. 2C). The EC50 value for InsP–InsP3 accumulation (n = 3) increased from 2.9 ± 0.7 nM (OTR) to 74 ± 9 nM ([E1.35A]OTR) in response to OT, and from 11 ± 1 nM (OTR) to 109 ± 8 nM ([E1.35A]OTR) in response to AVP. In contrast to the

3.4. Glu1.35 and Arg1.27 are both required for high affinity agonist binding but do not participate in a mutual charge–charge interaction A marked similarity was noted between the functional roles of Glu1.35 and those reported previously for Arg1.27 (Wesley et al., 2002), in that both residues provide contacts critical for agonist

Table 1 Pharmacological profile of wild-type and mutant OTRs. Mutant OTRs were expressed in HEK 293T cells and characterized pharmacologically. IC50 values were derived from competition binding experiments. The affinity (Ki ) and cell surface expression were determined as described in Sections 2.4 and 2.5, respectively. Data shown are the mean ± SEM of three separate experiments performed in triplicate. OTA = peptide antagonist, L-368,899 = non-peptide antagonist. Receptor

Binding affinities Ki (nM)

Cell surface expression (% WT)

Agonists OT OTR E1.35A E1.35D E1.35Q E1.35R

1.1 1400 826 1350 2221

Antagonists AVP

± ± ± ± ±

0.1 490 89 137 362

10 1951 1531 1371 693

OTA ± ± ± ± ±

0.5 381 110 220 91

1.2 1.3 0.7 1.1 3.9

± ± ± ± ±

L-368,899 0.2 0.1 0.1 0.2 0.4

13 15 12 10 19

± ± ± ± ±

1.6 2.1 1.1 0.6 1.7

100 97 ± 5 105 ± 7 95 ± 6 99 ± 9

120 100

A

80 60 40 20 0 -12

120

-11

-10

-9

-8

-7

80 60 40 20 0 -12

-6

-11

100 80 60 40 20

-12

-8

-7

-6

-5

-7

-6

-5

120

-11

-10

-9

-8

-7

-6

-5

80 60 40 20 0 -12

-11

Log {[Ligand] (M)}

InsP-InsP 3 Accumulation (% OT-WT max )

-9

100

Specific Binding (%)

Specific Binding (%)

B

120

0

C

-10

Log {[OT] (M)}

Log {[Ligand] (M)}

B

23

100

Specific Binding (%)

A Specific Binding (%)

D.L. Wootten et al. / Molecular and Cellular Endocrinology 333 (2011) 20–27

-10

-9

-8

Log {[AVP] (M)}

120

Fig. 3. Ligand binding characteristics of mutant OTRs. Radioligand binding assays were performed using membranes of HEK 293T cells transfected with: wild-type OTR (), [E1.35A]OTR (䊉), [E1.35Q]OTR (),[E1.35D]OTR () or [E1.35R]OTR () and with either OT (A) or AVP (B) as competing ligand. Data are mean ± SEM of three separate experiments performed in triplicate.

100 80 60 40 20 0 -11

-10

-9

-8

-7

-6

-5

Log {[Ligand] M} Fig. 2. Comparison of the pharmacological characteristics of [E1.35A]OTR to wildtype OTR. HEK 293T cells were transfected with either wild-type OTR (open symbols) or [E1.35A]OTR (solid symbols). (A) Competition binding curves for the peptide antagonist OTA (, ) and the non-peptide antagonist L-368,899 (, ). (B) Competition binding curves for OT (, 䊉) and AVP (, ). (C) InsP–InsP3 dose-response curves for OT (, 䊉) and AVP (, ) presented as percent of maximum OT-induced signaling by wild-type OTR. All data shown are mean ± SEM of three separate experiments performed in triplicate.

binding and signaling but not for antagonist binding. As Glu1.35 and Arg1.27 are eight residues apart, extension of the ␣-helix of TM1 as far as residue-1.27 would position Glu1.35 and Arg1.27 two turns apart on the same face of the ␣-helix and perhaps facilitate a mutual charge–charge interaction (Fig. 5). To investigate this further, a double reciprocal mutant was engineered in which the positions of the glutamyl and arginyl were exchanged ([E1.35R/R1.27E]OTR). If Glu1.35 -Arg1.27 mutually interact in the wild-type OTR, then they would still be able to interact when their loci were reversed in Arg1.35 -Glu1.27 . The double mutant [E1.35R/R1.27E]OTR was pharmacologically characterized and compared to wild-type OTR

and also to the single mutants [E1.35R]OTR and [R1.27E]OTR. Agonist binding affinity was markedly decreased for all three mutant receptors with [E1.35R/R1.27E]OTR exhibiting the greatest decrease (>10,000-fold; Table 2). In contrast to agonist binding, antagonist binding affinity was only slightly affected (4-fold; Table 2). Intracellular signaling in response to either OT or AVP was also disrupted for these mutant receptors. In each case, the perturbation of signaling (reflected in both EC50 and Emax ) was greater for [E1.35R/R1.27E]OTR than for either [E1.35R]OTR or [R1.27E]OTR (Fig. 6 and Table 3). Differences in signaling capability did not reflect differences in expression as all the constructs were expressed at similar levels to wild-type OTR (Table 3).

Table 2 Pharmacological profile of wild-type and mutant OTRs. Mutant OTRs were expressed in HEK 293T cells and characterized pharmacologically. IC50 values were derived from competition binding experiments. The affinity (Ki ) was determined as described in Section 2.4. Data shown are the mean ± SEM of three separate experiments performed in triplicate. OTA = OT antagonist. Receptor

OTR E1.35R R1.27E R1.27E/E1.35R

Binding affinities Ki (nM) OT

AVP

OTA

1.1 ± 0.1 2221 ± 362 2900 ± 682 >10,000

10 ± 0.5 693 ± 91 1280 ± 103 >10,000

1.2 3.9 1.9 4.8

± ± ± ±

0.2 0.4 0.3 0.7

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D.L. Wootten et al. / Molecular and Cellular Endocrinology 333 (2011) 20–27

A

120 100

120 100

Specific Binding (%)

InsP-InsP3 Accumulation (% WT max)

A

80 60 40 20 0 -11

-10

-9

-8

-7

-6

80 60 40 20 0

-5

-12

-11

Log {[OT] (M)}

B

-10

-9

-8

-7

-6

-5

Log {[OT] M}

Stimulation of InsP−InsP3 EC50 values

Emax values

(nM)

(% wt)

OTR

2.9 ± 0.7

100

E1.35A

74 ± 8.6

98 ± 4

E1.35D

137 ± 11

38 ± 2

E1.35Q

173 ± 8

55 ± 3

E1.35R

336 ± 49

98 ± 6

B InsP-InsP3 Accumulation (% WT max)

Receptor

120 100 80 60 40 20 0 -11

Fig. 4. Intracellular signaling by mutant OTRs. (A) OT-induced accumulation of InsP–InsP3 in HEK 293T cells transfected with wild-type OTR (), [E1.35A]OTR (䊉), [E1.35Q]OTR (), [E1.35D]OTR () or [E1.35R]OTR (). Values are expressed as percent maximum OT-induced signaling by wild-type OTR. Data are mean ± SEM of three separate experiments performed in triplicate. (B) EC50 and Emax values of OTinduced accumulation of InsP–InsP3 in cells expressing wild-type or mutant OTRs. Emax values are expressed as percent maximum OT-induced signaling by wild-type OTR. Values are mean ± SEM of three separate experiments performed in triplicate.

-10

-9

-8

-7

-6

-5

Log {[OT] M} Fig. 6. Pharmacological characterization of mutant OTRs. HEK 293T cells were transfected with wild-type OTR (), [E1.35R]OTR (), [R1.27E]OTR () or [E1.35R/R1.27E]OTR (). (A) OT competition binding curves for each receptor. (B) OT-induced accumulation of InsP–InsP3 . Values are expressed as percent maximum OT-induced signaling by wild-type OTR. Data in both panels are mean ± SEM of three separate experiments performed in triplicate.

4. Discussion OT regulates a plethora of physiological processes including sexual response, social behaviour and emotions (Gimpl and Fahrenholz, 2001; Opar, 2008). In particular, OT has a welldocumented role in uterine contraction which has resulted in the hormone being used routinely in clinics for the induction of labor (Stubbs, 2000). However, preterm labor occurs in 10% of all births world-wide and is the single largest cause of neonatal morbidity and death (Goldenberg and Rouse, 1998). The abundance of OTRs in the myometrium increases towards term, which sensitizes the uterus to circulating levels of OT. Consequently, blockade of the OTR has emerged as a therapeutic strategy to prevent preterm labor by maintaining uterine quiescence. The peptide antagonist Atosiban has been used clinically and shown to be effective in the treatment

Fig. 5. Molecular model of the TM1 helix of the OTR revealing the alignment of Glu1.35 and Arg1.27 stacked on one face of the helix. The positions of the side-chains of Glu1.35 and Arg1.27 are indicated. (A) TM1 viewed from within the plane of the membrane and (B) TM1 viewed from above.

Table 3 Intracellular signaling by wild-type and mutant OTRs. EC50 and Emax values for accumulation of InsP–InsP3 , in response to OT or AVP, in cells expressing wild-type OTR or mutant OTRs. Emax values in response to OT, or AVP, are expressed as percent of maximum of wild-type OTR InsP–InsP3 induced by OT (1 ␮M), or AVP (1 ␮M), respectively. Data shown are the mean ± SEM of three separate experiments performed in triplicate. Receptor

OT-induced InsP–InsP3 EC50 values (nM)

OTR E1.35R R1.27E R1.27E/E1.35R

2.9 336 280 1067

± ± ± ±

0.7 49 56 239

AVP-induced InsP–InsP3 Emax values (% WT-OT) 100 98 ± 6 86 ± 7 22 ± 3

EC50 values (nM) 11 127 181 2087

± ± ± ±

0.9 9 26 296

Cell surface expression (% WT) Emax values (% WT-AVP) 100 68 ± 9 75 ± 6 28 ± 11

100 99 ± 9 89 ± 5 87 ± 6

D.L. Wootten et al. / Molecular and Cellular Endocrinology 333 (2011) 20–27

of imminent preterm birth (Valenzuela et al., 2000; Romero et al., 2000). In addition, non-peptide antagonists have been developed (Pettibone and Freidinger, 1997; Brown et al., 2007; Liddle et al., 2008) including L-368,899 (Pettibone et al., 1993; Williams et al., 1994). The initial event in receptor activation is binding the hormone. Therapeutic intervention often uses agonists, or antagonists, which have been specifically developed to either mimic, or block, the natural hormone. It is of fundamental importance to elucidate the receptor binding contacts, as defining key differences between the agonist–receptor complex and the antagonist–receptor complex will provide mechanistic insights into receptor activation and will aid rational drug design (Bellenie et al., 2009; Frantz et al., 2010). For the OTR, it has been shown previously that the N-terminus is required for agonist binding and signaling but not antagonist binding (Hawtin et al., 2001), with Arg34 being particularly important (Wesley et al., 2002). In general however, the binding site for Family A GPCRs with peptide ligands comprises residues from both extracellular domains and the TMs (Howl and Wheatley, 1996; Hawtin et al., 2006; Conner et al., 2007). In the current study we have investigated the role of a Glu1.35 in TM1 of the OTR, which is absolutely conserved in all receptors for OT and OT-like peptides, cloned to date from a wide range of species (www.gpcr.org/7tm old), excluding ‘hypothetical’ proteins. Alanine substitution established that Glu1.35 was crucial for high affinity OT binding and signaling. The importance of Glu1.35 was not restricted to full agonists however, as it was also required for binding and signaling of the partial agonist AVP (Fig. 2). Nevertheless, such a critical role was restricted to agonists as substitution of Glu1.35 had only minor effects on peptide antagonist (OTA) or non-peptide antagonist (L-368,899) binding (Fig. 2). This preservation of high affinity antagonist binding established that loss of agonist binding was not due to aberrant assembly of the mutant receptors, or to distortion of the mature protein and furthermore, enabled us to accurately quantifying changes in agonist affinity using binding assays with [125 I]antagonist as tracer. It has been reported for some GPCRs, such as the ␬-opioid receptor, that agonist affinity can be dependent on the nature of the radio-tracer employed in binding assays (Hjorth et al., 1996). However, it is unlikely that the use of [3 H]antagonist in our study is the explanation for the observed effects on agonist binding. Marked differences in agonist affinities were noted between wild-type OTR and mutant receptors even when the same [3 H]antagonist tracer and agonist combination was used in each case. Furthermore, in addition to the effects on agonist affinity, substitution of Glu1.35 also perturbed OT-induced signaling and these experiments were independent of antagonist. Employing a [3 H]agonist as tracer ligand was not a feasible option for our competition binding studies due to the loss of agonist affinity when Glu1.35 was substituted.

Residue

1

2

25

Detailed investigation revealed that the structural requirements at residue-1.35 are very specific. Reversing the charge at this position with arginine was particularly detrimental to OT binding (Table 1). The polar residue glutamine possesses a side-chain of similar length to glutamate but it is not charged and cannot maintain high affinity agonist binding. Aspartate possesses almost identical charge characteristics to glutamate but is one methylene shorter. Despite this chemical conservation, Asp cannot substitute for Glu1.35 . This establishes that negative charge alone at residue1.35 is not sufficient to support agonist binding. The charge has to be very precisely positioned and requires the length supplied by the extra –CH2 of glutamate to provide a high affinity agonist binding platform. The substitution of Glu1.35 by Arg, Gln or Asp also perturbed the binding of the partial agonist AVP, although the folddecrease in affinity was less than that observed for OT. In particular, E1.35R decreased AVP affinity by 70-fold but decreased OT affinity by >2000-fold (Table 1). It is noteworthy that the corresponding residue-1.35 provides important agonist binding contacts in other peptide-GPCRs including Arg57(1.35) in the cholecystokinin (CCK) receptor-2 (Silvente-Poirot et al., 1998; Anders et al., 1999; Marco et al., 2007), Glu54(1.35) in the V1a vasopressin receptor (Hawtin et al., 2005b) and Arg38(1.35) in the gonadotropin-releasing hormone (GnRH) receptor (Stewart et al., 2008). Substitution of Glu1.35 perturbed the OTR signaling capability, with the detailed effects being highly dependent on the nature of the substitution (Fig. 4). It might have been predicted that substitution of Glu1.35 by Asp (or Gln) would be less disruptive than Arg, and with respect to the potency of OT-induced InsPs signaling this was indeed correct. However, for the Emax this was not what was observed. Charge reversal (Glu → Arg) supported wildtype maximum response whereas the more subtle substitution with Asp did not (Emax only 40% of wild-type OTR). This implies that the shorter side-chain of Asp not only prevents the side-chain establishing important contacts for signaling but also positions the negative charge in a detrimental position within the TM bundle, which results in greater disruption to signaling than either removing the side-chain (Ala1.35 ) or replacing the negative charge with a positive charge (Arg1.35 ). The fact that only Glu can support wildtype OTR binding and signaling explains why Glu is absolutely conserved at residue-1.35 in all receptors cloned to date for OT and OT-related peptides, such as mesotocin, isotocin and annetocin. All of these OT-related peptides share a common structure of a 20membered ring formed by a disulfide bond between Cys1 and Cys6 and a tri-peptide tail (Fig. 7). A feasible mechanism for the importance of Glu1.35 is provided by molecular modeling (Fig. 1) which indicates that Glu1.35 is one of several residues delimiting the ligand binding crevice, therefore it is plausible that Glu1.35 provides direct agonist-specific binding contacts. Most of the published molecular models of OT-related

3

4

5

6

7

8

9

Oxytocin Mesotocin Isotocin Cephalotocin Annetocin Arg-Conopressin Arg-Vasopressin

Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-GlyNH2 Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Ile-GlyNH2 Cys-Tyr-Ile-Ser-Asn-Cys-Pro-Ile-GlyNH2 Cys-Tyr-Phe-Arg-Asn-Cys-Pro-Ile-GlyNH2 Cys-Phe-Val-Arg-Asn-Cys-Pro-Thr-GlyNH2 Cys-Ile-Ile-Arg-Asn-Cys-Pro-Arg-GlyNH2 Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-GlyNH2

Ancestral peptide

Cys-Xaa-Xaa-Xaa-Asn-Cys-Pro-Xaa-GlyNH2

Fig. 7. Sequence alignment of OT-like peptides. The sequences of several named OT-like peptides are presented with conserved residues indicated in bold. In each case, a disulfide bond exists between Cys1 -Cys6 which is indicated by the solid line connecting residue-1 and residue-6.

26

D.L. Wootten et al. / Molecular and Cellular Endocrinology 333 (2011) 20–27

agonists bound to the OTR are in approximate agreement that the hydrophobic part of the ring of OT/AVP is accommodated within the 7TM helical bundle and the tri-peptide tail of the ligand is orientated towards the extracellular ends of TM1–TM2, although details vary (Favre et al., 2005; Slusarz et al., 2006a; Frantz et al., 2010). However, it has also been proposed that OT binds in the reverse orientation with the tocin ring located between TM1–TM2–TM7 and the tri-peptide tail projecting towards TM4-TM5 (Slusarz et al., 2006b). In the case of AVP binding to the V1a R, modeling studies have suggested that E1.35, together with D2.65 in extracellular loop 1, form a negatively charged region that directly interacts with the positively charged Arg8 of AVP (Rodrigo et al., 2007). If AVP docks to the OTR with a similar binding mode as that proposed for AVP docking to the V1a R, then it is possible that E1.35 in the OTR anchors AVP via an analogous charge–charge interaction with Arg8 . However, the interaction of E1.35 with OT must be fundamentally different to this, as OT possesses a Leu, rather than an Arg, at residue-8 and therefore lacks the required positive charge at this position for such an interaction. This is also the case for many other OT-related peptides (Fig. 7). In these cases it is probable that E1.35 interacts with the peptide main-chain or C-terminal glycinamide of OT, and related peptides, to orientate the agonist in the binding crevice. Differences in the binding contacts established by OT and AVP when bound to the OTR might explain why the effect on affinity of substitution at residue 1.35 is different for these two agonists; such that with respect to OT affinity E > D > Q ≈ A > R whereas for AVP affinity E > R > Q ≈ D > A. When a ligand interacts with a receptor it displays two fundamental properties; affinity and efficacy. Affinity is the ability of the ligand to bind to the receptor whereas efficacy is the ability of the ligand to activate the receptor (Strange, 2008). Changes to the structure of either the ligand or the receptor can have different ramifications on affinity compared to efficacy. Consequently, it is theoretically possible to develop selective agonists for receptors based on either selective affinity, selective efficacy or a combination of both parameters (Baker, 2010). In the current study, substitution of residue-1.35 differentially affected the affinity of OT and the efficacy of OT. Consequently, it is not unexpected that for wild-type OTR, E1.35A, E1.35D, E1.35Q and E1.35R, the rank order of OT affinity (Ki ) did not correspond to the rank order of EC50 values, with the specific affect on affinity and efficacy being dictated by the nature of the residue at position 1.35. In this study we establish that Glu1.35 is required for high affinity agonist binding and signaling by the OTR but does not have a role in binding antagonists of any class. These are precisely the functional properties reported previously for Arg1.27 (Wesley et al., 2002). As Glu1.35 and Arg1.27 are eight residues apart they will be located two turns apart on the same face of an ␣-helix. Consequently, it was theoretically possible that a mutual charge–charge interaction between Glu1.35 and Arg1.27 stabilized an OTR conformation with high affinity for agonists and signaling capability. This possibility was investigated using a double reciprocal mutant ([E1.35R/R1.27E]OTR) which would preserve any mutual interaction between these two residues. This approach was employed previously to demonstrate interaction between Asn2.50 and Asp7.49 in the GnRH receptor (Zhou et al., 1994). However, there was no recovery of OTR function with the double reciprocal mutant (Fig. 6 and Table 2) indicating that Glu1.35 and Arg1.27 operate independently. An analogous situation has been reported for the V1a vasopressin receptor where Glu1.35 and Arg1.27 (located two turns apart) contribute independently to agonist binding (Hawtin et al., 2005b) and for the CCK receptor-2 where Arg1.35 and Tyr1.39 (located one turn apart) contribute independently to agonist binding (Anders et al., 1999; Marco et al., 2007). In conclusion, we have established that Glu1.35 of the OTR is critical for high affinity agonist binding and signaling but it is not

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