- Email: [email protected]
Intracellular third loop–C-terminal tail interaction in prostaglandin EP3β receptor
Biochemical and Biophysical Research Communications 371 (2008) 846–849
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Intracellular third loop–C-terminal tail interaction in prostaglandin EP3b receptor Yoshiaki Yano, Takuya Shimbo, Yukihiko Sugimoto, Katsumi Matsuzaki * Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida-Shimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan
a r t i c l e
i n f o
Article history: Received 25 April 2008 Available online 12 May 2008
Keywords: G-protein-coupled receptor Intracellular loop Peptide–peptide interaction Prostaglandin EP3b receptor Self-association
a b s t r a c t The secondary structures of and the interactions between the intracellular domains (the three loops and the C-terminal tail) of the mouse-derived prostaglandin E2 receptor EP3b subtype were investigated using peptides mimicking the domains. The N-termini of the peptides were palmitoylated to anchor on unilamellar vesicles composed of phosphatidylserine, enriched in the cytoplasmic leaflet of mammalian plasma membranes. Circular dichroism spectroscopy revealed that the peptides corresponding to the intracellular third loop (i3) and the C-terminus (C-term) assumed b-sheet and associated a-helical structures, respectively. A structural change was observed when i3 was mixed with C-term, indicating an interaction between them. Fluorescence experiments showed that i3 suppressed the self-association of C-term, confirming the interaction. These results demonstrate for the first time specific interaction between the intracellular third loop and the C-terminus. A model is proposed for the activation of the receptor. Ó 2008 Elsevier Inc. All rights reserved.
G-protein-coupled receptors (GPCRs), the most important drug target, have a characteristic membrane topology with seven transmembrane helices, an extracellular N-terminus, an intracellular Cterminus, three extracellular loops, and three intracellular loops [1,2]. The terminal and loop domains are tethered to the water– membrane interface by the neighboring transmembrane domains. Although X-ray crystal structures have been recently reported for two GPCRs (rhodopsin [3] and b2-adrenergic receptor [4]), considerable structural information for a variety of GPCRs has been obtained by non-crystallographic approaches using fragment peptides [5]. Particularly, structural study using fragment peptides mimicking intracellular loops and C-terminal domains, which are crucial for coupling to the downstream G-proteins, is a promising approach. For example, peptides mimicking C-terminal domains of GPCRs often assume helical structures [6,7], corresponding to the helix 8 of rhodopsin in the X-ray structure [3]. Another rationale of this approach is that the fragment peptides mimicking intracellular domains actually have biological activities [8–12], supporting the hypothesis that the domains are independent functional units.
Abbreviations: CD, circular dichroism; DMF, N-dimethylformamide; EP3bR, mouse-derived prostaglandin E2 receptor EP3b subtype; Fmoc, 9-fluorenylmethyloxycarbonyl; LUVs, large unilamellar vesicles; Mtt, methyltrityl; POPS, 1palmitoyl-2-oleoyl-phosphatidyl-L-serine; SUVs, small unilamellar vesicles; TMR, 5(6)-carboxytetramethylrhodamine; TFE, 2,2,2-trifluoroethanol. * Corresponding author. Fax: +81 75 753 4578. E-mail address: [email protected] (K. Matsuzaki). 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.04.180
Here we report the secondary structures of the intracellular domains of the Gi-coupled, mouse-derived prostaglandin E2 receptor EP3b subtype (EP3bR) [13,14] on lipid bilayers mimicking the inner leaflet of plasma membranes. We also found that the domains could self-associate and/or interact with each other. Peptides mimicking the intracellular loops (designated as i1, i2, and i3 for the first, second, and third loops, respectively) and the C-terminal domain (C-term) of EP3bR were synthesized and the N-termini of the peptides were palmitoylated to anchor on membranes [12] (Table 1). Circular dichroism (CD) and fluorescence experiments indicate that C-term tends to self-associate and i3 could suppress the self-association by interacting with C-term. Implications of these interactions in the activation mechanism of the receptor will be discussed. Materials and methods Peptide synthesis. Peptides mimicking the intracellular domains for EP3bR were synthesized by a standard 9-fluorenylmethyloxycarbonyl (Fmoc)-based solid phase method using NovaSyn TGR resin (Novabiochem) on a 0.2-mmol scale. For coupling, Fmoc amino acid (1 mmol), 1-hydroxybenzotriazole (1 mmol), and N-N0 -diisopropylcarbodiimide (1 mmol) dissolved in N, N-dimethylformamide (DMF) were reacted for 2 h. The reaction was monitored using the ninhydrin test. The Fmoc group was removed by treatment with 20% piperidine in DMF for 20 min. The N-termini of the peptides were palmitoylated by treatment with palmitic acid
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Y. Yano et al. / Biochemical and Biophysical Research Communications 371 (2008) 846–849 Table 1 Sequences and net charges of peptides mimicking intracellular domains of the mouse-derived EP3b receptor Peptide
Sequencea
Net charge (pH7.4)
i1 i2 i3 C-term TMR-C-termb
pal-RRESKRKKSFLLC-NH2 pal-ERALAIRAPHWYASHMKTRAT-NH2 pal-KALVSRCRAKAAVSQSSAQWGRITTETAIQ-NH2 pal-RKILLRKFCQMMNNLKWTFIAVPVSLGLRISSPREG-NH2 pal-K(TMR)RKILLRKFCQMMNNLKWTFIAVPVSLGLRISSPREG-NH2
+5 +3 +4 +6 +6
a b
Pal and NH2 indicate palmitoylation and amidation, respectively. TMR was attached to the side chain of the Lys at the N-terminus.
(5 equivalent) and 1-hydroxybenzotriazole (10 equivalent) overnight. The fluorophore 5(6)-carboxytetramethylrhodamine (TMR) was specifically attached to the side chain of the lysine at the Nterminus of C-term (TMR-C-term). After introduction of N-a-(9-fluorenylmethoxycarbonyl)-N-e-p-methyltrityl-L-lysine (Lys (Mtt)) (Watanabe chemical) and the palmitoylation of the N-terminus, the Mtt group was deprotected by treatment with dichloromethane/1,1,1,3,3,3-hexafluoro-2-propenol/2,2,2-trifluoroethanol (TFE)/
Fig. 1. CD spectra of peptides mimicking intracellular domains of EP3bR (i1, i2, i3, and C-term) in the presence of POPS SUVs at 37 °C. The concentrations of the lipids and the peptides were 1 mM and 10 lM, respectively.
triethylsilane = 6.5/2/1/0.5 (v/v) for 2 h. The deprotected amino group was labeled with TMR by treatment with the succinimidyl ester derivative of TMR (Invitrogen, 2 equivalent) in DMF containing 5% N,N-diisopropylethylamine for 48 h. The peptide was removed from the resin by treatment with trifluoroacetic acid/ ethaneditiol/m-cresol/thioanisole/H2O = 12.5/1/1/1/1 (v/v) for 3 h, precipitated by diethylether, and purified by reversed phase HPLC. The purity of the synthesized peptides (higher than 95%) was determined by analytical HPLC and ion spray mass spectroscopy. Preparation of vesicles. Small unilamellar vesicles (SUVs) composed of 1-palmitoyl-2-oleoyl-phosphatidyl-L-serine (POPS) were produced by sonication of freeze–thawed multilamellar vesicles in ice-water under a nitrogen atmosphere for 15 min (5 min, three times) using a probe-type UD-201 sonicator (Tomy Seiko, Tokyo). A buffer containing 10 mM Tris, 150 mM NaF, and 1 mM EDTA (pH 7.4) was used. Large unilamellar vesicles (LUVs) composed of POPS were prepared by an extrusion method as described elsewhere [15] using a buffer containing 10 mM Tris, 150 mM NaCl, and 1 mM EDTA (pH 7.4). The lipid concentration was determined in triplicate by phosphorus analysis [16]. CD measurements. CD spectra were measured at 37 °C on a Jasco J-820 apparatus, using a 1-mm-pathlength quartz cell. The lipid and peptide concentrations were 1 mM and 10 lM, respectively. The measurements were performed after mixing peptides dis-
Fig. 2. CD spectra of mixed peptides in the presence of POPS SUVs at 37 °C. (A–C) Mixture of a loop peptide (i1, i2, or i3) and C-term. (D) Mixture of the three loop peptides. The spectra for the mixed peptides (obsd) were compared with the arithmetic sum of the spectra for the component peptides (cald). The concentration of each peptide was 10 mM. The lipid concentration was 1 mM.
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solved in TFE with SUVs (final TFE concentration: 1%) for 3 h. Sixtyeight scans were averaged for each sample. The averaged blank spectra (SUVs containing 1% TFE) were subtracted. TMR fluorescence. The fluorescence intensity at 579 nm was measured at an excitation wavelength of 540 nm on a Shimadzu RF-5300 spectrofluorometer at 37 °C. The blank intensity due to scattering from LUVs was subtracted. The concentrations of lipid, TMR-C-term, and unlabeled peptides in buffer (containing 1% TFE) were 5 lM, 50 nM, and 250 nM, respectively. The measurements were performed after incubation for 3 h. Results and discussion Secondary structure of intracellular domains The membrane environment is often critical for domain-mimicking peptides to assume biologically relevant structures. For example, electrostatic interactions of positively charged peptides mimicking intracellular domains of b-adrenergic receptors with negatively charged phosphatidylserine enriched in the cytoplasmic leaflet of plasma membranes of mammalian cells are important for membrane affinity and folding of the peptides [17]. Therefore we measured CD spectra of the intracellular domains of EP3bR in the presence of SUVs composed of POPS (Fig. 1). The i1 peptide exhibited a CD spectrum characteristic of random structures. The conformation of i2 was also mainly unordered. In contrast, i3 assumed a b-sheet structure as characterized by a minimum near 220 nm. Although a peptide corresponding to the N-terminal half of i3 of the human prostaglandin EP3a receptor could activate Gi directly [10], the observed b-sheet structure implying intermolecular interaction between the loops might not be biologically relevant. On the other hand, the CD spectrum of C-term with double minima at 208 nm and 222 nm indicates that the peptide partially assumed a helical structure. The helicity (fH) was estimated from molar ellipticity at 222 nm ([h]222) to be 16%, using the equation, fH = ([h]222 2340)/30,300 100 [18]. The N-terminal region of C-term (KILLRK) could assume an amphiphilic helical structure, similar to C-terminal tails of other GPCRs [7]. The ratio of [h]222/ [h]208 was greater than 1, indicating association between the helices as observed in two-stranded a-helical coiled-coil structures [19]. The helical association was not observed in the presence of SUVs composed of a typical zwitterionic lipid, 1-palmitoyl-2oleoyl-L-a-phosphatidylcholine (data not shown). A peptide corresponding to the C-terminus of the angiotensin II AT1A receptor was reported to form helical aggregates at higher concentrations in acidic water, although the ratio of [h]222/[h]208 did not support the formation of coiled-coil structures [6].
unlabeled peptides (i1, i2, i3, and C-term), fluorescence enhancement of TMR was measured (Fig. 3). Mixing unlabeled C-term with TMR-C-term resulted in a 1.9-fold increase in TMR fluorescence, indicating that TMR-C-term had been self-quenched because of self-association. Addition of unlabeled i3 to TMR-C-term recovered TMR fluorescence by 1.7-fold whereas addition of i1and i2 did not, indicating that only i3 interacts with C-term, consistent with the results of CD measurements. These results for the first time demonstrate specific interaction between the third intracellular loop and the C-terminal tail of EP3bR. We propose an activation mechanism of the receptor (Fig. 4). In the absence of the agonist, the C-terminal tail covers the activating domain within the third intracellular loop, preventing the access of Gi- protein. Upon stimulation by the agonist, a conformational change of the receptor results in the dissociation of the Cterminal tail from the activating domain, allowing the access of Gi-protein. Furthermore, the self-associating property of the C-terminal tail may drive receptor–receptor interaction (oligomeriza-
Fig. 3. Fluorescence recovery of TMR-C-term by addition of intracellular domain peptides in the presence of POPS LUVs at 37 °C. Fluorescence intensities of TMR in the presence of unlabeled peptides (i1, i2, i3, and C-term) relative to those in the absence are shown. Error bars indicate SEM. The concentrations of the lipid, TMR-Cterm, and the non-labeled peptides were 5 lM, 50 nM, and 250 nM, respectively.
Interactions between intracellular domains The C-terminal tail of the EP3 receptor appears to play a role in constraining basal activity by preventing the access of Gi-protein to the activating domain of the receptor, because a mutant EP3 receptor in which the C-term tail is truncated exhibits agonist-independent constitutive activity [20]. Therefore, we examined interactions between loop domains and C-term, as detected by structural changes. Peptides mimicking the loop domains (i1, i2, and i3) were mixed with C-term and CD spectra were recorded. The observed spectra (obsd) were compared with the arithmetic sums of the spectra for the component peptides (cald) (Fig. 2A– C). A clear structural change was observed only when i3 and Cterm were mixed. No structural change was observed by mixing the three loop domains (Fig. 2D). These data indicate specific interaction between i3 and C-term. Interactions between the intracellular domains were further examined by fluorescence dequenching of self-associating TMR-C-term. After mixing TMR-C-term with
Fig. 4. Possible role of third intracellular loop–C-terminal tail interaction in the activation mechanism of EP3bR. In the absence of the agonist, the third intracellular loop–C-terminal tail interaction prevents the access of Gi-protein to the activating domain of the receptor. Upon stimulation by the agonist, a conformational change of the receptor (EP3bR*) results in the dissociation of the tail from the activating domain, allowing the access of Gi-protein.
Y. Yano et al. / Biochemical and Biophysical Research Communications 371 (2008) 846–849
tion), which appears to be promoted after the activation of the receptor. Acknowledgment This work was supported by MEXT (Targeted Proteins Research Program), Japan. References [1] K.L. Pierce, R.T. Premont, R.J. Lefkowitz, Seven-transmembrane receptors, Nat. Rev. Mol. Cell Biol. 3 (2002) 639–650. [2] E. Jacoby, R. Bouhelal, M. Gerspacher, K. Seuwen, The 7 TM G-protein-coupled receptor target family, ChemMedChem 1 (2006) 761–782. [3] K. Palczewski, T. Kumasaka, T. Hori, C.A. Behnke, H. Motoshima, B.A. Fox, I. Le Trong, D.C. Teller, T. Okada, R.E. Stenkamp, M. Yamamoto, M. Miyano, Crystal structure of rhodopsin: a G protein-coupled receptor, Science 289 (2000) 739–745. [4] V. Cherezov, D.M. Rosenbaum, M.A. Hanson, S.G. Rasmussen, F.S. Thian, T.S. Kobilka, H.J. Choi, P. Kuhn, W.I. Weis, B.K. Kobilka, R.C. Stevens, High-resolution crystal structure of an engineered human b2-adrenergic G protein-coupled receptor, Science 318 (2007) 1258–1265. [5] P.L. Yeagle, A.D. Albert, G-protein coupled receptor structure, Biochim. Biophys. Acta 1768 (2007) 808–824. [6] L. Franzoni, G. Nicastro, T.A. Pertinhez, M. Tato, C.R. Nakaie, A.C. Paiva, S. Schreier, A. Spisni, Structure of the C-terminal fragment 300–320 of the rat angiotensin II AT1A receptor and its relevance with respect to G-protein coupling, J. Biol. Chem. 272 (1997) 9734–9741. [7] M. Katragadda, M.W. Maciejewski, P.L. Yeagle, Structural studies of the putative helix 8 in the human b2 adrenergic receptor: an NMR study, Biochim. Biophys. Acta 1663 (2004) 74–81. [8] D. Palm, G. Munch, C. Dees, M. Hekman, Mapping of b-adrenoceptor coupling domains to Gs-protein by site-specific synthetic peptides, FEBS Lett. 254 (1989) 89–93.
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[9] M. Merkouris, I. Dragatsis, G. Megaritis, G. Konidakis, C. Zioudrou, G. Milligan, Z. Georgoussi, Identification of the critical domains of the d-opioid receptor involved in G protein coupling using site-specific synthetic peptides, Mol. Pharmacol. 50 (1996) 985–993. [10] T. Kikkou, O. Matsumoto, T. Ohkubo, Y. Kobayashi, G. Tsujimoto, NMR structure of an intracellular loop peptide derived from prostaglandin EP3a receptor, Biochem. Biophys. Res. Commun. 345 (2006) 933–937. [11] C.A. Johnston, D.P. Siderovski, Structural basis for nucleotide exchange on Gai subunits and receptor coupling specificity, Proc. Natl. Acad. Sci. USA 104 (2007) 2001–2006. [12] L. Covic, A.L. Gresser, J. Talavera, S. Swift, A. Kuliopulos, Activation and inhibition of G protein-coupled receptors by cell-penetrating membranetethered peptides, Proc. Natl. Acad. Sci USA 99 (2002) 643–648. [13] Y. Sugimoto, T. Namba, A. Honda, Y. Hayashi, M. Negishi, A. Ichikawa, S. Narumiya, Cloning and expression of a cDNA for mouse prostaglandin E receptor EP3 subtype, J. Biol. Chem. 267 (1992) 6463–6466. [14] Y. Sugimoto, S. Narumiya, Prostaglandin E receptors, J. Biol. Chem. 282 (2007) 11613–11617. [15] K. Matsuzaki, O. Murase, N. Fujii, K. Miyajima, An antimicrobial peptide, magainin 2, induced rapid flip-flop of phospholipids coupled with pore formation and peptide translocation, Biochemistry 35 (1996) 11361–11368. [16] G.R. Bartlett, Phosphorus assay in column chromatography, J. Biol. Chem. 234 (1959) 466–468. [17] K. Shinagawa, M. Ohya, T. Higashijima, K. Wakamatsu, Circular dichroism studies of the interaction between synthetic peptides corresponding to intracellular loops of b-adrenergic receptors and phospholipid vesicles, J. Biochem. 115 (1994) 463–468. [18] Y.H. Chen, J.T. Yang, H.M. Martinez, Determination of the secondary structures of proteins by circular dichroism and optical rotatory dispersion, Biochemistry 11 (1972) 4120–4131. [19] N.E. Zhou, B.Y. Zhu, C.M. Kay, R.S. Hodges, The two-stranded a-helical coiledcoil is an ideal model for studying protein stability and subunit interactions, Biopolymers 32 (1992) 419–426. [20] H. Hasegawa, M. Negishi, A. Ichikawa, Two isoforms of the prostaglandin E receptor EP3 subtype different in agonist-independent constitutive activity, J. Biol. Chem. 271 (1996) 1857–1860.
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Intracellular third loop–C-terminal tail interaction in prostaglandin EP3b receptor Yoshiaki Yano, Takuya Shimbo, Yukihiko Sugimoto, Katsumi Matsuzaki * Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida-Shimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan
a r t i c l e
i n f o
Article history: Received 25 April 2008 Available online 12 May 2008
Keywords: G-protein-coupled receptor Intracellular loop Peptide–peptide interaction Prostaglandin EP3b receptor Self-association
a b s t r a c t The secondary structures of and the interactions between the intracellular domains (the three loops and the C-terminal tail) of the mouse-derived prostaglandin E2 receptor EP3b subtype were investigated using peptides mimicking the domains. The N-termini of the peptides were palmitoylated to anchor on unilamellar vesicles composed of phosphatidylserine, enriched in the cytoplasmic leaflet of mammalian plasma membranes. Circular dichroism spectroscopy revealed that the peptides corresponding to the intracellular third loop (i3) and the C-terminus (C-term) assumed b-sheet and associated a-helical structures, respectively. A structural change was observed when i3 was mixed with C-term, indicating an interaction between them. Fluorescence experiments showed that i3 suppressed the self-association of C-term, confirming the interaction. These results demonstrate for the first time specific interaction between the intracellular third loop and the C-terminus. A model is proposed for the activation of the receptor. Ó 2008 Elsevier Inc. All rights reserved.
G-protein-coupled receptors (GPCRs), the most important drug target, have a characteristic membrane topology with seven transmembrane helices, an extracellular N-terminus, an intracellular Cterminus, three extracellular loops, and three intracellular loops [1,2]. The terminal and loop domains are tethered to the water– membrane interface by the neighboring transmembrane domains. Although X-ray crystal structures have been recently reported for two GPCRs (rhodopsin [3] and b2-adrenergic receptor [4]), considerable structural information for a variety of GPCRs has been obtained by non-crystallographic approaches using fragment peptides [5]. Particularly, structural study using fragment peptides mimicking intracellular loops and C-terminal domains, which are crucial for coupling to the downstream G-proteins, is a promising approach. For example, peptides mimicking C-terminal domains of GPCRs often assume helical structures [6,7], corresponding to the helix 8 of rhodopsin in the X-ray structure [3]. Another rationale of this approach is that the fragment peptides mimicking intracellular domains actually have biological activities [8–12], supporting the hypothesis that the domains are independent functional units.
Abbreviations: CD, circular dichroism; DMF, N-dimethylformamide; EP3bR, mouse-derived prostaglandin E2 receptor EP3b subtype; Fmoc, 9-fluorenylmethyloxycarbonyl; LUVs, large unilamellar vesicles; Mtt, methyltrityl; POPS, 1palmitoyl-2-oleoyl-phosphatidyl-L-serine; SUVs, small unilamellar vesicles; TMR, 5(6)-carboxytetramethylrhodamine; TFE, 2,2,2-trifluoroethanol. * Corresponding author. Fax: +81 75 753 4578. E-mail address: [email protected] (K. Matsuzaki). 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.04.180
Here we report the secondary structures of the intracellular domains of the Gi-coupled, mouse-derived prostaglandin E2 receptor EP3b subtype (EP3bR) [13,14] on lipid bilayers mimicking the inner leaflet of plasma membranes. We also found that the domains could self-associate and/or interact with each other. Peptides mimicking the intracellular loops (designated as i1, i2, and i3 for the first, second, and third loops, respectively) and the C-terminal domain (C-term) of EP3bR were synthesized and the N-termini of the peptides were palmitoylated to anchor on membranes [12] (Table 1). Circular dichroism (CD) and fluorescence experiments indicate that C-term tends to self-associate and i3 could suppress the self-association by interacting with C-term. Implications of these interactions in the activation mechanism of the receptor will be discussed. Materials and methods Peptide synthesis. Peptides mimicking the intracellular domains for EP3bR were synthesized by a standard 9-fluorenylmethyloxycarbonyl (Fmoc)-based solid phase method using NovaSyn TGR resin (Novabiochem) on a 0.2-mmol scale. For coupling, Fmoc amino acid (1 mmol), 1-hydroxybenzotriazole (1 mmol), and N-N0 -diisopropylcarbodiimide (1 mmol) dissolved in N, N-dimethylformamide (DMF) were reacted for 2 h. The reaction was monitored using the ninhydrin test. The Fmoc group was removed by treatment with 20% piperidine in DMF for 20 min. The N-termini of the peptides were palmitoylated by treatment with palmitic acid
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Y. Yano et al. / Biochemical and Biophysical Research Communications 371 (2008) 846–849 Table 1 Sequences and net charges of peptides mimicking intracellular domains of the mouse-derived EP3b receptor Peptide
Sequencea
Net charge (pH7.4)
i1 i2 i3 C-term TMR-C-termb
pal-RRESKRKKSFLLC-NH2 pal-ERALAIRAPHWYASHMKTRAT-NH2 pal-KALVSRCRAKAAVSQSSAQWGRITTETAIQ-NH2 pal-RKILLRKFCQMMNNLKWTFIAVPVSLGLRISSPREG-NH2 pal-K(TMR)RKILLRKFCQMMNNLKWTFIAVPVSLGLRISSPREG-NH2
+5 +3 +4 +6 +6
a b
Pal and NH2 indicate palmitoylation and amidation, respectively. TMR was attached to the side chain of the Lys at the N-terminus.
(5 equivalent) and 1-hydroxybenzotriazole (10 equivalent) overnight. The fluorophore 5(6)-carboxytetramethylrhodamine (TMR) was specifically attached to the side chain of the lysine at the Nterminus of C-term (TMR-C-term). After introduction of N-a-(9-fluorenylmethoxycarbonyl)-N-e-p-methyltrityl-L-lysine (Lys (Mtt)) (Watanabe chemical) and the palmitoylation of the N-terminus, the Mtt group was deprotected by treatment with dichloromethane/1,1,1,3,3,3-hexafluoro-2-propenol/2,2,2-trifluoroethanol (TFE)/
Fig. 1. CD spectra of peptides mimicking intracellular domains of EP3bR (i1, i2, i3, and C-term) in the presence of POPS SUVs at 37 °C. The concentrations of the lipids and the peptides were 1 mM and 10 lM, respectively.
triethylsilane = 6.5/2/1/0.5 (v/v) for 2 h. The deprotected amino group was labeled with TMR by treatment with the succinimidyl ester derivative of TMR (Invitrogen, 2 equivalent) in DMF containing 5% N,N-diisopropylethylamine for 48 h. The peptide was removed from the resin by treatment with trifluoroacetic acid/ ethaneditiol/m-cresol/thioanisole/H2O = 12.5/1/1/1/1 (v/v) for 3 h, precipitated by diethylether, and purified by reversed phase HPLC. The purity of the synthesized peptides (higher than 95%) was determined by analytical HPLC and ion spray mass spectroscopy. Preparation of vesicles. Small unilamellar vesicles (SUVs) composed of 1-palmitoyl-2-oleoyl-phosphatidyl-L-serine (POPS) were produced by sonication of freeze–thawed multilamellar vesicles in ice-water under a nitrogen atmosphere for 15 min (5 min, three times) using a probe-type UD-201 sonicator (Tomy Seiko, Tokyo). A buffer containing 10 mM Tris, 150 mM NaF, and 1 mM EDTA (pH 7.4) was used. Large unilamellar vesicles (LUVs) composed of POPS were prepared by an extrusion method as described elsewhere [15] using a buffer containing 10 mM Tris, 150 mM NaCl, and 1 mM EDTA (pH 7.4). The lipid concentration was determined in triplicate by phosphorus analysis [16]. CD measurements. CD spectra were measured at 37 °C on a Jasco J-820 apparatus, using a 1-mm-pathlength quartz cell. The lipid and peptide concentrations were 1 mM and 10 lM, respectively. The measurements were performed after mixing peptides dis-
Fig. 2. CD spectra of mixed peptides in the presence of POPS SUVs at 37 °C. (A–C) Mixture of a loop peptide (i1, i2, or i3) and C-term. (D) Mixture of the three loop peptides. The spectra for the mixed peptides (obsd) were compared with the arithmetic sum of the spectra for the component peptides (cald). The concentration of each peptide was 10 mM. The lipid concentration was 1 mM.
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solved in TFE with SUVs (final TFE concentration: 1%) for 3 h. Sixtyeight scans were averaged for each sample. The averaged blank spectra (SUVs containing 1% TFE) were subtracted. TMR fluorescence. The fluorescence intensity at 579 nm was measured at an excitation wavelength of 540 nm on a Shimadzu RF-5300 spectrofluorometer at 37 °C. The blank intensity due to scattering from LUVs was subtracted. The concentrations of lipid, TMR-C-term, and unlabeled peptides in buffer (containing 1% TFE) were 5 lM, 50 nM, and 250 nM, respectively. The measurements were performed after incubation for 3 h. Results and discussion Secondary structure of intracellular domains The membrane environment is often critical for domain-mimicking peptides to assume biologically relevant structures. For example, electrostatic interactions of positively charged peptides mimicking intracellular domains of b-adrenergic receptors with negatively charged phosphatidylserine enriched in the cytoplasmic leaflet of plasma membranes of mammalian cells are important for membrane affinity and folding of the peptides [17]. Therefore we measured CD spectra of the intracellular domains of EP3bR in the presence of SUVs composed of POPS (Fig. 1). The i1 peptide exhibited a CD spectrum characteristic of random structures. The conformation of i2 was also mainly unordered. In contrast, i3 assumed a b-sheet structure as characterized by a minimum near 220 nm. Although a peptide corresponding to the N-terminal half of i3 of the human prostaglandin EP3a receptor could activate Gi directly [10], the observed b-sheet structure implying intermolecular interaction between the loops might not be biologically relevant. On the other hand, the CD spectrum of C-term with double minima at 208 nm and 222 nm indicates that the peptide partially assumed a helical structure. The helicity (fH) was estimated from molar ellipticity at 222 nm ([h]222) to be 16%, using the equation, fH = ([h]222 2340)/30,300 100 [18]. The N-terminal region of C-term (KILLRK) could assume an amphiphilic helical structure, similar to C-terminal tails of other GPCRs [7]. The ratio of [h]222/ [h]208 was greater than 1, indicating association between the helices as observed in two-stranded a-helical coiled-coil structures [19]. The helical association was not observed in the presence of SUVs composed of a typical zwitterionic lipid, 1-palmitoyl-2oleoyl-L-a-phosphatidylcholine (data not shown). A peptide corresponding to the C-terminus of the angiotensin II AT1A receptor was reported to form helical aggregates at higher concentrations in acidic water, although the ratio of [h]222/[h]208 did not support the formation of coiled-coil structures [6].
unlabeled peptides (i1, i2, i3, and C-term), fluorescence enhancement of TMR was measured (Fig. 3). Mixing unlabeled C-term with TMR-C-term resulted in a 1.9-fold increase in TMR fluorescence, indicating that TMR-C-term had been self-quenched because of self-association. Addition of unlabeled i3 to TMR-C-term recovered TMR fluorescence by 1.7-fold whereas addition of i1and i2 did not, indicating that only i3 interacts with C-term, consistent with the results of CD measurements. These results for the first time demonstrate specific interaction between the third intracellular loop and the C-terminal tail of EP3bR. We propose an activation mechanism of the receptor (Fig. 4). In the absence of the agonist, the C-terminal tail covers the activating domain within the third intracellular loop, preventing the access of Gi- protein. Upon stimulation by the agonist, a conformational change of the receptor results in the dissociation of the Cterminal tail from the activating domain, allowing the access of Gi-protein. Furthermore, the self-associating property of the C-terminal tail may drive receptor–receptor interaction (oligomeriza-
Fig. 3. Fluorescence recovery of TMR-C-term by addition of intracellular domain peptides in the presence of POPS LUVs at 37 °C. Fluorescence intensities of TMR in the presence of unlabeled peptides (i1, i2, i3, and C-term) relative to those in the absence are shown. Error bars indicate SEM. The concentrations of the lipid, TMR-Cterm, and the non-labeled peptides were 5 lM, 50 nM, and 250 nM, respectively.
Interactions between intracellular domains The C-terminal tail of the EP3 receptor appears to play a role in constraining basal activity by preventing the access of Gi-protein to the activating domain of the receptor, because a mutant EP3 receptor in which the C-term tail is truncated exhibits agonist-independent constitutive activity [20]. Therefore, we examined interactions between loop domains and C-term, as detected by structural changes. Peptides mimicking the loop domains (i1, i2, and i3) were mixed with C-term and CD spectra were recorded. The observed spectra (obsd) were compared with the arithmetic sums of the spectra for the component peptides (cald) (Fig. 2A– C). A clear structural change was observed only when i3 and Cterm were mixed. No structural change was observed by mixing the three loop domains (Fig. 2D). These data indicate specific interaction between i3 and C-term. Interactions between the intracellular domains were further examined by fluorescence dequenching of self-associating TMR-C-term. After mixing TMR-C-term with
Fig. 4. Possible role of third intracellular loop–C-terminal tail interaction in the activation mechanism of EP3bR. In the absence of the agonist, the third intracellular loop–C-terminal tail interaction prevents the access of Gi-protein to the activating domain of the receptor. Upon stimulation by the agonist, a conformational change of the receptor (EP3bR*) results in the dissociation of the tail from the activating domain, allowing the access of Gi-protein.
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