Homodimer formation by the ATP/UTP receptor P2Y2 via disulfide bridges

Homodimer formation by the ATP/UTP receptor P2Y2 via disulfide bridges Abstract Many class C G-protein coupled receptors (GPCRs) function as homo- or heterodimers and several class A GPCRs have also been shown to form a homodimer. We expressed human P2Y2 receptor (P2Y2R) in cultured cells and compared SDS-PAGE patterns under reducing and non-reducing conditions. Under non-reducing conditions, approximately half of the P2Y2Rs were electrophoresed as a dimer. We then produced Cys to Ser mutants at four sites (Cys25, Cys106, Cys183 and Cys278) in the extracellular domains of P2Y2R and examined the effect on dimer formation and receptor activity. All single mutants formed dimers similarly to the wild-type protein, but C25S, C106S and C183S P2Y2R lost activity, while C278S P2Y2R maintained weak activity. Coexpression with wild-type P2Y2R recovered the activity of the C25S mutant. These results show that Cys106 and Cys183 are required for monomer or homodimer activity; Cys25 is required for monomer activity, but it is not needed in one protomer for homodimer activity; and Cys278 can be replaced in the monomer and homodimer. Approximately, half of C25S/C278S double mutants were electrophoresed as a dimer, similarly to the wild-type and single mutants, and dimers with the wild-type protein were active. These results suggest involvement of Cys106 and Cys183 in disulfide bonding between protomers in homodimer formation. α-complementation assay, arrestin recruitment, disulfide bond, GPCR homodimer, GPCR signal cross-talk Some G-protein coupled receptors (GPCRs) form homodimer or heteromers. Among the many class A GPCRs, rhodopsin (1), bradykinin receptor B2 (B2R) (2), purinergic adenosine A1 receptor (A1R) and P2Y2 receptors (P2Y2R) (3) form homodimers. Disulfide bridges are present in B2R (4), but the bridging details are unknown and the quaternary structure has not been determined. All class A GPCRs except S1P1 have preserved Cys residues in the first and second extracellular loops and disulfide bridges are formed between two Cys residues in monomers, as shown by structural analysis of several GPCRs, including rhodopsin (5), and this structural motif is presumed to be similar in other GPCRs. P2Y2R is a GPCR activated by ATP/UTP that couples to Gαq/11 (G protein) and is involved in induction of inflammation, hypotension and pain. P2Y2R forms hetero-oligomers or has cross-talk with other class A GPCRs, such as β2-adrenergic receptor (6), CXC chemokine receptor 2 (7), A1R (8) and B2R (9). Given that homodimer formation by disulfide bridges was found in SDS-PAGE, we examined how four extracellular Cys residues in P2Y2R (Cys25, Cys106, Cys183 and Cys278) form bridges in the monomer and dimer. Hillmann et al. proposed Cys25–Cys278 and Cys106–Cys183 disulfide bridges based on the effects of mutation of Cys106 and Cys278 to Ser, homologous residues in rhodopsin, and a similar structure to P2Y1 receptor (10), in which there is a disulfide bond between Cys residues in N terminal domain and third extracellular loop (11). In the current study, we produced point mutants with each of the four Cys residues replaced with Ser. These proteins were used to examine disulfide bridge formation using SDS-PAGE under reducing and nonreducing conditions, and formation of functional homodimers based on UTP-induced activation. Our results show that P2Y2R can function as a monomer or homodimer and that disulfide bonds contribute to homodimer formation. Materials and Methods Cell culture HEK293T and 1321N1 cells were grown in Dulbecco’s Modified Eagle’s Medium with 25 mM glucose supplemented with 10% (v/v) fetal calf serum and kept in a humidified 10% CO2/90% air atmosphere at 37°C. The cells were transiently transfected with plasmids using Lipofectamine 2000 (Invitrogen) for western blotting and Ca2+ imaging. Cell membranes for western blotting were prepared from cells 48 h after transfection. Plasmids and constructs Plasmids encoding human P2Y2R were prepared by inserting P2Y2R cDNA between the EcoRI and BamHI sites of pIRES2-EGFP (Clontech). P2Y2Rs with Ser substitution at Cys25, Cys106, Cys183, Cys278 and Cys25/278 were prepared by PCR. The C-terminal of P2Y2R was fused to the H31R-substituted α donor peptide of a LacZ β-galactosidase reporter enzyme (P2Y2R-α) or Myc epitope. The C-terminal of β-arrestin-2 was fused to the M15 acceptor deletion mutant of β-galactosidase (the ω peptide, arrestin-ω). Plasmids encoding fusion proteins were generated by subcloning PCR products into pAlpha-N1 or pOmega-N1 vectors, which were constructed from pAcGFP-N1 vector (Clontech) (9). Preparation of membrane fractions, SDS-PAGE and western blotting Preparation of membrane fractions (12), SDS-PAGE and western blotting (9) were performed as described previously. β-galactosidase complementation assay The interaction between activated GPCR and β-arrestin (arrestin recruitment) was measured using complementation between the α and ω domains of LacZ β-galactosidase (9, 13). Since P2Y2R was activated with autocrine ATP generated by physical stimulus during cell cultivation in our original method (9), we modified the procedure to avoid autocrine effects by cultivating the cells 24 h before the assay, as described below. HEK293T or 1321N1 cells were cultured in 10 cm φ cell culture dishes to 80% confluence. Cells were washed once with phosphate buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, and 8.1 mM Na2HPO4) and harvested by digestion with 2 mL of 0.05% trypsin/EDTA for 30 s at room temperature. Digestion was stopped by adding 2.5 mL of culture medium. The harvested cells were spun down (700 g, 5 min) and washed with 3 mL of PBS. The pelleted cells were suspended in 36 mL of culture medium and dispensed as 3 mL aliquots. Cell suspensions were mixed with transfection reagents, 0.4 µg of each plasmid DNA coding for GPCR-α and arrestin-ω in 200 µL Opti-MEM, and 1.6 µL of Lipofectamine 2000 (Invitrogen) in 200 µL of Opti-MEM. Then, a 180 µL aliquot of the mixture was seeded on a white tissue culture 96-well plate (Corning 3917) and cultured at 37°C for 24 h. After stimulation of the cells with 20 µL of 10× concentrated UTP for 1 h at 37°C, 150 µL of the supernatant was discarded and 50 µL of Gal Screen (B, Applied Biosystems) was added, followed by incubation at room temperature. Luminescence of the wells was read after about 40 min. Ca2+ imaging Cells cultured on glass-bottom dishes were loaded with the Ca2+ indicator Fura-2 and exposed to stimulants using the bath application method. Images were acquired and analyzed using balanced salt solution (pH 7.3; 130 mM NaCl, 5.4 mM KCl, 20 mM HEPES, 5.5 mM glucose, 0.8 mM MgSO4, and 1.8 mM CaCl2) as the extracellular media (14–16). Statistical analysis Results are presented as means ± SEM. Differences between groups were assessed by two-way ANOVA followed by a Dunnett’s multiple comparison test, using GraphPad software (San Diego, CA). P < 0.05 was considered significant. Results Homodimer formation by P2Y2R and effect of mutation of Cys residues P2Y2R with a C-terminal Myc tag was expressed in HEK293T cells. Western blotting was performed after SDS-PAGE under reducing and non-reducing conditions. Under non-reducing conditions, western blot analysis showed bands at the monomer position and at a position consistent with a dimer (Fig. 1B). Cys to Ser mutants were examined similarly, and all mutants, including the C25S/C278S double mutant, formed bands with molecular masses consistent with the monomer and dimer. However, most dimer bands transferred to the monomer position under reducing conditions (Fig. 1A), which suggests that P2Y2R forms a homodimer with disulfide bonds. The ratios of monomer and dimer forms of wild-type and mutant P2Y2Rs were estimated from Fig. 1B. About 60% of wild-type P2Y2R formed a dimer, while less than 40% of the C278S mutant formed a dimer (Table I). We previously showed that most P2Y2R molecules in COS7 cells are present in lipid rafts (12), and a similar result was found in HEK293T cells, with most wild-type and mutant P2Y2Rs in raft component fractions. Table I. Ratio of monomer and dimer forms of P2Y2R and its Cys to Ser mutantsa P2Y2R  Monomer (%)  Dimer (%)  Wild type  38  62  C25S  48  52  C106S  42  58  C183S  53  47  C278S  69  31  C25S/C278S  62  38  P2Y2R  Monomer (%)  Dimer (%)  Wild type  38  62  C25S  48  52  C106S  42  58  C183S  53  47  C278S  69  31  C25S/C278S  62  38  aRatios were calculated from scanned image of western blot in Fig. 1B. Table I. Ratio of monomer and dimer forms of P2Y2R and its Cys to Ser mutantsa P2Y2R  Monomer (%)  Dimer (%)  Wild type  38  62  C25S  48  52  C106S  42  58  C183S  53  47  C278S  69  31  C25S/C278S  62  38  P2Y2R  Monomer (%)  Dimer (%)  Wild type  38  62  C25S  48  52  C106S  42  58  C183S  53  47  C278S  69  31  C25S/C278S  62  38  aRatios were calculated from scanned image of western blot in Fig. 1B. Fig. 1 View largeDownload slide Wild-type (WT) P2Y2R-Myc and Cys to Ser mutants were expressed in HEK293T cells and detected by Western blotting. Results are shown for detergent-resistant membrane fractions under reducing (A) and nonreducing (B) conditions. Almost no expressed protein was fractioned in the detergent-soluble fraction. Di, dimer; Mon, monomer. Fig. 1 View largeDownload slide Wild-type (WT) P2Y2R-Myc and Cys to Ser mutants were expressed in HEK293T cells and detected by Western blotting. Results are shown for detergent-resistant membrane fractions under reducing (A) and nonreducing (B) conditions. Almost no expressed protein was fractioned in the detergent-soluble fraction. Di, dimer; Mon, monomer. Effect on arrestin recruitment of homodimer formation by wild-type and Cys-mutant P2Y2R Using P2Y2R with the C-terminal attached to the LacZ α-domain, UTP-stimulated arrestin recruitment was determined based on reactivity to the arrestin attached to the LacZ ω-domain. In HEK293T cells, C25S, C278S and C25S/C278S mutants had weaker activity than the wild-type protein but were activated by UTP, whereas C106S and C183S mutants were not activated (Fig. 2A). Since endogenous P2Y2R is expressed in HEK293T cells, the assay was also conducted in 1321N1 cells, which do not have endogenous expression of P2Y2R. In these cells, the activity of the C25S and C25S/C278S mutants disappeared (Fig. 2B). The C278S mutant maintained some activity, but this was weaker than that of wild-type P2Y2R. To confirm the effect of dimers, both wild-type P2Y2R and P2Y2R mutant-α proteins were expressed in 1321N1 cells. Under these conditions, results similar to those observed in HEK293T cells were obtained (Fig. 2C). Combination of wild-type P2Y2R with C25S, C278S or C25S/S278S mutants gave activity of approximately 25% that of the wild-type homodimer. C25S-α was activated in the presence of wild-type or C278S P2Y2R but was not activated in the presence of C25S/C278S mutant (Fig. 2D). This result implies that the activation signal from wild-type or the C278S mutant was transmitted to C25S-α and shows that activation signals from wild-type are not transmitted to C106S-α or C183S-α. Fig. 2 View largeDownload slide Difference in reaction of Cys to Ser mutants with and without wild-type P2Y2R. P2Y2R-α or mutant-α in HEK293T cells with endogenous wild-type P2Y2R (A) and in 1321N1 cells that do not express wild-type P2Y2R (B–D). Wild-type or mutant P2Y2R was coexpressed with arrestin-ω and stimulated by UTP, after which arrestin recruitment activity was determined by an α complementary reaction. Activity is expressed as a multiple of the control activity measured without the reagents. n = 3. ****P < 0.0001; ***P < 0.001; **P < 0. 01; *P < 0. 05. Fig. 2 View largeDownload slide Difference in reaction of Cys to Ser mutants with and without wild-type P2Y2R. P2Y2R-α or mutant-α in HEK293T cells with endogenous wild-type P2Y2R (A) and in 1321N1 cells that do not express wild-type P2Y2R (B–D). Wild-type or mutant P2Y2R was coexpressed with arrestin-ω and stimulated by UTP, after which arrestin recruitment activity was determined by an α complementary reaction. Activity is expressed as a multiple of the control activity measured without the reagents. n = 3. ****P < 0.0001; ***P < 0.001; **P < 0. 01; *P < 0. 05. Examination of G-protein activation The above results show that Cys25, Cys106 and Cys183 are required for recruitment of arrestin. Further assays were conducted to examine whether mutation of these Cys residues inhibited G-protein activity, an upstream reaction in 1321N1 cells, using intracellular Ca2+ imaging. None of the C25S, C106S and C183S mutants caused an increase in Ca2+, in contrast to wild-type P2Y2R (Fig. 3; results for C106S and C183S P2Y2R are not shown but were similar to those for C25S P2Y2R). Expression of the three mutants with pIRES2-EGFP resulted in less expression of EGFP than with wild-type P2Y2R, for a reason that is unclear. Fig. 3 View largeDownload slide Intracellular Ca2+ changes for wild-type and C25S P2Y2R in 1321N1 cells after UTP addition. Wild-type (A) or C25S (B) P2Y2R was expressed in 1321N1 cells and 15 cells each were stimulated by 10 μM UTP, followed by stimulation with 1 μM BK. C106S and C183S mutants gave similar results to those for C25S P2Y2R. (C) Non-transfected 1321N1 cells were stimulated as above and served as controls (n = 21). Fig. 3 View largeDownload slide Intracellular Ca2+ changes for wild-type and C25S P2Y2R in 1321N1 cells after UTP addition. Wild-type (A) or C25S (B) P2Y2R was expressed in 1321N1 cells and 15 cells each were stimulated by 10 μM UTP, followed by stimulation with 1 μM BK. C106S and C183S mutants gave similar results to those for C25S P2Y2R. (C) Non-transfected 1321N1 cells were stimulated as above and served as controls (n = 21). Discussion Extracellular Cys residues in proteins located on the surface of cell membranes maintain protein structure via disulfide bridging. Among GPCRs, rhodopsin has four extracellular Cys residues that form disulfide crosslinks between two central and two terminal residues (cf. Fig. 4A). The activity of GPCRs is regulated by formation of homodimers and heterooligomers, which leads to signaling cross-talk. Many class C GPCRs form homo- or heterodimers, and mGluRs (17) and Ca2+ sensing receptor (18) have disulfide bonds that contribute to dimer formation. In mGluRs, Cys residues in the long N terminal extracellular domain contribute to homodimer formation (19). Several class A GPCRs also form homodimers, including rhodopsin (1), A1R and P2Y2R (3), B2R (2, 4), D2R (20), β2AR (21) and 5-HT2C (22). Many class A GPCRs have Cys residues in N terminal domain and the first, second and third extracellular loops, but the majority view is that homodimer formation depends on interactions between transmembrane domains. Only B2R (4) and P2Y2R in this study have been shown to have disulfide bonds involved in homodimer formation and it is still unclear how these Cys residues are bridged. Fig. 4 View largeDownload slide Proposed disulfide bridges in P2Y2R monomers and homodimers. In monomers, similarly to rhodopsin, Cys25 and Cys278, and Cys106 and Cys183 are likely to be bridged (A). When homodimers are formed, Cys25 is bridged with Cys278 in the other protomer. Consequently, it is possible that bridges between Cys106 and Cys183 occur within one protomer (B) and between two protomers (C). Fig. 4 View largeDownload slide Proposed disulfide bridges in P2Y2R monomers and homodimers. In monomers, similarly to rhodopsin, Cys25 and Cys278, and Cys106 and Cys183 are likely to be bridged (A). When homodimers are formed, Cys25 is bridged with Cys278 in the other protomer. Consequently, it is possible that bridges between Cys106 and Cys183 occur within one protomer (B) and between two protomers (C). Fusion of a GPCR with an α domain to facilitate a LacZ α complementary reaction permits evaluation of signaling cross-talk by GPCR homodimers and heterooligomers (9). In this study, we found that some P2Y2Rs formed dimers under non-reducing conditions (Fig. 1). The dimers were formed with all of the mutants examined. We examined whether extracellular Cys residues at four sites contributed to ‘functional’ dimer formation by evaluating signaling cross-talk of Cys to Ser mutants expressed alone or with wild-type P2Y2R (Fig. 2). All mutants formed dimers with each other or with wild type. The C25S mutant received active signal from wild type revealing arrestin recruitment activity. That is, it formed a ‘functional’ dimer capable of cross-talk. The C106S and C183S mutants were able to form dimers, but they were not activated alone and could not engage in signal cross-talk from wild type. This indicates that these mutants cannot form a ‘functional’ dimer, even though they were able to form a dimer with wild type. The C278S mutant showed somewhat diminished dimer formation but had activity itself and was able to transfer a signal to the C25S mutant. Based on these results, a disulfide bridge pattern can be proposed for P2Y2R (Fig. 4). Cys106 and Cys183 are positioned in the first and second extracellular loops, and mutation of either of these residues to Ser eliminated receptor activity when expressed alone and in coexpression with wild-type P2Y2R. This suggests that a disulfide bond is formed between these two residues, as Hillmann et al. proposed based on homology to rhodopsin (11). Hillmann et al. indicated that C106S P2Y2R was not expressed on the cell surface in 1321N1 cells, as the binding of antibodies to the HA-tag added to the N-terminus is undetectable (11). In our observation, the level of protein expression did not differ between wild-type and mutant proteins in both HEK293T (Fig. 1) and 1321N1 cells (unpublished observation). Thus, C106S mutant protein is expressed but its sorting to the cell surface is prohibited. It is probable that bridging with an appropriate disulfide bond between Cys106 and Cys183 is sufficient for the cell surface expression of the receptor. Cys106 and Cys183 are also probably involved in a disulfide bridge between protomers in homodimer formation (Fig. 4C). Ser mutations at each of these sites formed dimers in SDS-PAGE under nonreducing conditions. In the C25S/C278S double mutant, dimer bands also occurred and were changed to monomers by reduction. Therefore, an inter-protomer disulfide bridge between Cys106 and Cys183 is also involved in homodimer formation (Fig. 4C). Mutation of Cys25 in N terminal domain to Ser did not prevent activation in coexpression with wild-type P2Y2R. If molecules forming homodimers are equivalently expressed, the ratio of homodimer formation with wild-type P2Y2R is approximately half; however, the activity was less than half. Mutation of Cys278 in the third extracellular loop to Ser attenuated activity, but some was maintained. Therefore, Cys278 seems to be less important for monomer and homodimer formation. The C25S/C278S double mutant lost activity, but this was recovered in coexpression with wild-type P2Y2R. These results are likely to be due to homodimer formation and thus support the conclusion that Cys106 and Cys183 are involved in disulfide bridges with wild-type P2Y2R; i.e. they are involved in bridging between molecules. Therefore, a Cys106-Cys183 intra- or interprotomer disulfide bond is essential for receptor expression on the cell surface and activation (Fig. 4). This hypothesis is also supported by the activation of C25S-α in the presence of C278S P2Y2R (Fig. 2D). Signals were transduced from C278S to C25S P2Y2R but not from C25S/C278S P2Y2R (Fig. 2D). This suggest that Cys25 and Cys278 form a disulfide bond, but that this disulfide bond is not essential for homodimer formation, given that signals were transduced from wild-type P2Y2R to the C25S/C278S mutant (Fig. 2A and C). In COS7 cells, most P2Y2R molecules are found in fractions corresponding to lipid raft components (12). The results of this study showed that most P2Y2R molecules also fractionated to lipid raft components in HEK293T cells. Only some of these molecules formed dimers, which shows that dimer formation has no relationship with distribution of P2Y2R to raft fractions. Also, in the presence of wild-type P2Y2R, Cys mutants had no effect on raft distribution. Formation of B2R homodimers with disulfide bonds is promoted by agonist binding (2). However, no agonist was necessary for P2Y2R to form homodimers. P2Y2R shows cross-reaction with B2R (9) and cross-talk with β2-adrenergic receptor (6), CXC chemokine receptor 2 (7), and A1R (8). Thus, P2Y2R may form heteromers with other GPCRs, but it is unknown how homodimer formation is related to the occurrence of heteromers. There were many smear bands up to 100 kDa in non-reducing SDS-PAGE (Fig. 1B). Bands above 100 kDa often appear in non-reducing SDS-PAGE, and some of those seen in Fig. 1B may be homo-oligomers or hetero-oligomers with other GPCRs. Formation of a dimer or oligomer greatly affects the binding kinetics, signal enhancement and termination thereof, as it regulates the density of molecules involved in GPCR signal transduction (1). The monomer and dimer are rapidly interconvertible (23), and involvement of a disulfide bridge is likely to prolong the presence of the dimer. Funding This study was supported by the Strategic Study Base Formation Support Project for Private Universities, Japan. Conflict of Interest None declared. 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Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Biochemistry Oxford University Press

Homodimer formation by the ATP/UTP receptor P2Y2 via disulfide bridges

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Abstract

Abstract Many class C G-protein coupled receptors (GPCRs) function as homo- or heterodimers and several class A GPCRs have also been shown to form a homodimer. We expressed human P2Y2 receptor (P2Y2R) in cultured cells and compared SDS-PAGE patterns under reducing and non-reducing conditions. Under non-reducing conditions, approximately half of the P2Y2Rs were electrophoresed as a dimer. We then produced Cys to Ser mutants at four sites (Cys25, Cys106, Cys183 and Cys278) in the extracellular domains of P2Y2R and examined the effect on dimer formation and receptor activity. All single mutants formed dimers similarly to the wild-type protein, but C25S, C106S and C183S P2Y2R lost activity, while C278S P2Y2R maintained weak activity. Coexpression with wild-type P2Y2R recovered the activity of the C25S mutant. These results show that Cys106 and Cys183 are required for monomer or homodimer activity; Cys25 is required for monomer activity, but it is not needed in one protomer for homodimer activity; and Cys278 can be replaced in the monomer and homodimer. Approximately, half of C25S/C278S double mutants were electrophoresed as a dimer, similarly to the wild-type and single mutants, and dimers with the wild-type protein were active. These results suggest involvement of Cys106 and Cys183 in disulfide bonding between protomers in homodimer formation. α-complementation assay, arrestin recruitment, disulfide bond, GPCR homodimer, GPCR signal cross-talk Some G-protein coupled receptors (GPCRs) form homodimer or heteromers. Among the many class A GPCRs, rhodopsin (1), bradykinin receptor B2 (B2R) (2), purinergic adenosine A1 receptor (A1R) and P2Y2 receptors (P2Y2R) (3) form homodimers. Disulfide bridges are present in B2R (4), but the bridging details are unknown and the quaternary structure has not been determined. All class A GPCRs except S1P1 have preserved Cys residues in the first and second extracellular loops and disulfide bridges are formed between two Cys residues in monomers, as shown by structural analysis of several GPCRs, including rhodopsin (5), and this structural motif is presumed to be similar in other GPCRs. P2Y2R is a GPCR activated by ATP/UTP that couples to Gαq/11 (G protein) and is involved in induction of inflammation, hypotension and pain. P2Y2R forms hetero-oligomers or has cross-talk with other class A GPCRs, such as β2-adrenergic receptor (6), CXC chemokine receptor 2 (7), A1R (8) and B2R (9). Given that homodimer formation by disulfide bridges was found in SDS-PAGE, we examined how four extracellular Cys residues in P2Y2R (Cys25, Cys106, Cys183 and Cys278) form bridges in the monomer and dimer. Hillmann et al. proposed Cys25–Cys278 and Cys106–Cys183 disulfide bridges based on the effects of mutation of Cys106 and Cys278 to Ser, homologous residues in rhodopsin, and a similar structure to P2Y1 receptor (10), in which there is a disulfide bond between Cys residues in N terminal domain and third extracellular loop (11). In the current study, we produced point mutants with each of the four Cys residues replaced with Ser. These proteins were used to examine disulfide bridge formation using SDS-PAGE under reducing and nonreducing conditions, and formation of functional homodimers based on UTP-induced activation. Our results show that P2Y2R can function as a monomer or homodimer and that disulfide bonds contribute to homodimer formation. Materials and Methods Cell culture HEK293T and 1321N1 cells were grown in Dulbecco’s Modified Eagle’s Medium with 25 mM glucose supplemented with 10% (v/v) fetal calf serum and kept in a humidified 10% CO2/90% air atmosphere at 37°C. The cells were transiently transfected with plasmids using Lipofectamine 2000 (Invitrogen) for western blotting and Ca2+ imaging. Cell membranes for western blotting were prepared from cells 48 h after transfection. Plasmids and constructs Plasmids encoding human P2Y2R were prepared by inserting P2Y2R cDNA between the EcoRI and BamHI sites of pIRES2-EGFP (Clontech). P2Y2Rs with Ser substitution at Cys25, Cys106, Cys183, Cys278 and Cys25/278 were prepared by PCR. The C-terminal of P2Y2R was fused to the H31R-substituted α donor peptide of a LacZ β-galactosidase reporter enzyme (P2Y2R-α) or Myc epitope. The C-terminal of β-arrestin-2 was fused to the M15 acceptor deletion mutant of β-galactosidase (the ω peptide, arrestin-ω). Plasmids encoding fusion proteins were generated by subcloning PCR products into pAlpha-N1 or pOmega-N1 vectors, which were constructed from pAcGFP-N1 vector (Clontech) (9). Preparation of membrane fractions, SDS-PAGE and western blotting Preparation of membrane fractions (12), SDS-PAGE and western blotting (9) were performed as described previously. β-galactosidase complementation assay The interaction between activated GPCR and β-arrestin (arrestin recruitment) was measured using complementation between the α and ω domains of LacZ β-galactosidase (9, 13). Since P2Y2R was activated with autocrine ATP generated by physical stimulus during cell cultivation in our original method (9), we modified the procedure to avoid autocrine effects by cultivating the cells 24 h before the assay, as described below. HEK293T or 1321N1 cells were cultured in 10 cm φ cell culture dishes to 80% confluence. Cells were washed once with phosphate buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, and 8.1 mM Na2HPO4) and harvested by digestion with 2 mL of 0.05% trypsin/EDTA for 30 s at room temperature. Digestion was stopped by adding 2.5 mL of culture medium. The harvested cells were spun down (700 g, 5 min) and washed with 3 mL of PBS. The pelleted cells were suspended in 36 mL of culture medium and dispensed as 3 mL aliquots. Cell suspensions were mixed with transfection reagents, 0.4 µg of each plasmid DNA coding for GPCR-α and arrestin-ω in 200 µL Opti-MEM, and 1.6 µL of Lipofectamine 2000 (Invitrogen) in 200 µL of Opti-MEM. Then, a 180 µL aliquot of the mixture was seeded on a white tissue culture 96-well plate (Corning 3917) and cultured at 37°C for 24 h. After stimulation of the cells with 20 µL of 10× concentrated UTP for 1 h at 37°C, 150 µL of the supernatant was discarded and 50 µL of Gal Screen (B, Applied Biosystems) was added, followed by incubation at room temperature. Luminescence of the wells was read after about 40 min. Ca2+ imaging Cells cultured on glass-bottom dishes were loaded with the Ca2+ indicator Fura-2 and exposed to stimulants using the bath application method. Images were acquired and analyzed using balanced salt solution (pH 7.3; 130 mM NaCl, 5.4 mM KCl, 20 mM HEPES, 5.5 mM glucose, 0.8 mM MgSO4, and 1.8 mM CaCl2) as the extracellular media (14–16). Statistical analysis Results are presented as means ± SEM. Differences between groups were assessed by two-way ANOVA followed by a Dunnett’s multiple comparison test, using GraphPad software (San Diego, CA). P < 0.05 was considered significant. Results Homodimer formation by P2Y2R and effect of mutation of Cys residues P2Y2R with a C-terminal Myc tag was expressed in HEK293T cells. Western blotting was performed after SDS-PAGE under reducing and non-reducing conditions. Under non-reducing conditions, western blot analysis showed bands at the monomer position and at a position consistent with a dimer (Fig. 1B). Cys to Ser mutants were examined similarly, and all mutants, including the C25S/C278S double mutant, formed bands with molecular masses consistent with the monomer and dimer. However, most dimer bands transferred to the monomer position under reducing conditions (Fig. 1A), which suggests that P2Y2R forms a homodimer with disulfide bonds. The ratios of monomer and dimer forms of wild-type and mutant P2Y2Rs were estimated from Fig. 1B. About 60% of wild-type P2Y2R formed a dimer, while less than 40% of the C278S mutant formed a dimer (Table I). We previously showed that most P2Y2R molecules in COS7 cells are present in lipid rafts (12), and a similar result was found in HEK293T cells, with most wild-type and mutant P2Y2Rs in raft component fractions. Table I. Ratio of monomer and dimer forms of P2Y2R and its Cys to Ser mutantsa P2Y2R  Monomer (%)  Dimer (%)  Wild type  38  62  C25S  48  52  C106S  42  58  C183S  53  47  C278S  69  31  C25S/C278S  62  38  P2Y2R  Monomer (%)  Dimer (%)  Wild type  38  62  C25S  48  52  C106S  42  58  C183S  53  47  C278S  69  31  C25S/C278S  62  38  aRatios were calculated from scanned image of western blot in Fig. 1B. Table I. Ratio of monomer and dimer forms of P2Y2R and its Cys to Ser mutantsa P2Y2R  Monomer (%)  Dimer (%)  Wild type  38  62  C25S  48  52  C106S  42  58  C183S  53  47  C278S  69  31  C25S/C278S  62  38  P2Y2R  Monomer (%)  Dimer (%)  Wild type  38  62  C25S  48  52  C106S  42  58  C183S  53  47  C278S  69  31  C25S/C278S  62  38  aRatios were calculated from scanned image of western blot in Fig. 1B. Fig. 1 View largeDownload slide Wild-type (WT) P2Y2R-Myc and Cys to Ser mutants were expressed in HEK293T cells and detected by Western blotting. Results are shown for detergent-resistant membrane fractions under reducing (A) and nonreducing (B) conditions. Almost no expressed protein was fractioned in the detergent-soluble fraction. Di, dimer; Mon, monomer. Fig. 1 View largeDownload slide Wild-type (WT) P2Y2R-Myc and Cys to Ser mutants were expressed in HEK293T cells and detected by Western blotting. Results are shown for detergent-resistant membrane fractions under reducing (A) and nonreducing (B) conditions. Almost no expressed protein was fractioned in the detergent-soluble fraction. Di, dimer; Mon, monomer. Effect on arrestin recruitment of homodimer formation by wild-type and Cys-mutant P2Y2R Using P2Y2R with the C-terminal attached to the LacZ α-domain, UTP-stimulated arrestin recruitment was determined based on reactivity to the arrestin attached to the LacZ ω-domain. In HEK293T cells, C25S, C278S and C25S/C278S mutants had weaker activity than the wild-type protein but were activated by UTP, whereas C106S and C183S mutants were not activated (Fig. 2A). Since endogenous P2Y2R is expressed in HEK293T cells, the assay was also conducted in 1321N1 cells, which do not have endogenous expression of P2Y2R. In these cells, the activity of the C25S and C25S/C278S mutants disappeared (Fig. 2B). The C278S mutant maintained some activity, but this was weaker than that of wild-type P2Y2R. To confirm the effect of dimers, both wild-type P2Y2R and P2Y2R mutant-α proteins were expressed in 1321N1 cells. Under these conditions, results similar to those observed in HEK293T cells were obtained (Fig. 2C). Combination of wild-type P2Y2R with C25S, C278S or C25S/S278S mutants gave activity of approximately 25% that of the wild-type homodimer. C25S-α was activated in the presence of wild-type or C278S P2Y2R but was not activated in the presence of C25S/C278S mutant (Fig. 2D). This result implies that the activation signal from wild-type or the C278S mutant was transmitted to C25S-α and shows that activation signals from wild-type are not transmitted to C106S-α or C183S-α. Fig. 2 View largeDownload slide Difference in reaction of Cys to Ser mutants with and without wild-type P2Y2R. P2Y2R-α or mutant-α in HEK293T cells with endogenous wild-type P2Y2R (A) and in 1321N1 cells that do not express wild-type P2Y2R (B–D). Wild-type or mutant P2Y2R was coexpressed with arrestin-ω and stimulated by UTP, after which arrestin recruitment activity was determined by an α complementary reaction. Activity is expressed as a multiple of the control activity measured without the reagents. n = 3. ****P < 0.0001; ***P < 0.001; **P < 0. 01; *P < 0. 05. Fig. 2 View largeDownload slide Difference in reaction of Cys to Ser mutants with and without wild-type P2Y2R. P2Y2R-α or mutant-α in HEK293T cells with endogenous wild-type P2Y2R (A) and in 1321N1 cells that do not express wild-type P2Y2R (B–D). Wild-type or mutant P2Y2R was coexpressed with arrestin-ω and stimulated by UTP, after which arrestin recruitment activity was determined by an α complementary reaction. Activity is expressed as a multiple of the control activity measured without the reagents. n = 3. ****P < 0.0001; ***P < 0.001; **P < 0. 01; *P < 0. 05. Examination of G-protein activation The above results show that Cys25, Cys106 and Cys183 are required for recruitment of arrestin. Further assays were conducted to examine whether mutation of these Cys residues inhibited G-protein activity, an upstream reaction in 1321N1 cells, using intracellular Ca2+ imaging. None of the C25S, C106S and C183S mutants caused an increase in Ca2+, in contrast to wild-type P2Y2R (Fig. 3; results for C106S and C183S P2Y2R are not shown but were similar to those for C25S P2Y2R). Expression of the three mutants with pIRES2-EGFP resulted in less expression of EGFP than with wild-type P2Y2R, for a reason that is unclear. Fig. 3 View largeDownload slide Intracellular Ca2+ changes for wild-type and C25S P2Y2R in 1321N1 cells after UTP addition. Wild-type (A) or C25S (B) P2Y2R was expressed in 1321N1 cells and 15 cells each were stimulated by 10 μM UTP, followed by stimulation with 1 μM BK. C106S and C183S mutants gave similar results to those for C25S P2Y2R. (C) Non-transfected 1321N1 cells were stimulated as above and served as controls (n = 21). Fig. 3 View largeDownload slide Intracellular Ca2+ changes for wild-type and C25S P2Y2R in 1321N1 cells after UTP addition. Wild-type (A) or C25S (B) P2Y2R was expressed in 1321N1 cells and 15 cells each were stimulated by 10 μM UTP, followed by stimulation with 1 μM BK. C106S and C183S mutants gave similar results to those for C25S P2Y2R. (C) Non-transfected 1321N1 cells were stimulated as above and served as controls (n = 21). Discussion Extracellular Cys residues in proteins located on the surface of cell membranes maintain protein structure via disulfide bridging. Among GPCRs, rhodopsin has four extracellular Cys residues that form disulfide crosslinks between two central and two terminal residues (cf. Fig. 4A). The activity of GPCRs is regulated by formation of homodimers and heterooligomers, which leads to signaling cross-talk. Many class C GPCRs form homo- or heterodimers, and mGluRs (17) and Ca2+ sensing receptor (18) have disulfide bonds that contribute to dimer formation. In mGluRs, Cys residues in the long N terminal extracellular domain contribute to homodimer formation (19). Several class A GPCRs also form homodimers, including rhodopsin (1), A1R and P2Y2R (3), B2R (2, 4), D2R (20), β2AR (21) and 5-HT2C (22). Many class A GPCRs have Cys residues in N terminal domain and the first, second and third extracellular loops, but the majority view is that homodimer formation depends on interactions between transmembrane domains. Only B2R (4) and P2Y2R in this study have been shown to have disulfide bonds involved in homodimer formation and it is still unclear how these Cys residues are bridged. Fig. 4 View largeDownload slide Proposed disulfide bridges in P2Y2R monomers and homodimers. In monomers, similarly to rhodopsin, Cys25 and Cys278, and Cys106 and Cys183 are likely to be bridged (A). When homodimers are formed, Cys25 is bridged with Cys278 in the other protomer. Consequently, it is possible that bridges between Cys106 and Cys183 occur within one protomer (B) and between two protomers (C). Fig. 4 View largeDownload slide Proposed disulfide bridges in P2Y2R monomers and homodimers. In monomers, similarly to rhodopsin, Cys25 and Cys278, and Cys106 and Cys183 are likely to be bridged (A). When homodimers are formed, Cys25 is bridged with Cys278 in the other protomer. Consequently, it is possible that bridges between Cys106 and Cys183 occur within one protomer (B) and between two protomers (C). Fusion of a GPCR with an α domain to facilitate a LacZ α complementary reaction permits evaluation of signaling cross-talk by GPCR homodimers and heterooligomers (9). In this study, we found that some P2Y2Rs formed dimers under non-reducing conditions (Fig. 1). The dimers were formed with all of the mutants examined. We examined whether extracellular Cys residues at four sites contributed to ‘functional’ dimer formation by evaluating signaling cross-talk of Cys to Ser mutants expressed alone or with wild-type P2Y2R (Fig. 2). All mutants formed dimers with each other or with wild type. The C25S mutant received active signal from wild type revealing arrestin recruitment activity. That is, it formed a ‘functional’ dimer capable of cross-talk. The C106S and C183S mutants were able to form dimers, but they were not activated alone and could not engage in signal cross-talk from wild type. This indicates that these mutants cannot form a ‘functional’ dimer, even though they were able to form a dimer with wild type. The C278S mutant showed somewhat diminished dimer formation but had activity itself and was able to transfer a signal to the C25S mutant. Based on these results, a disulfide bridge pattern can be proposed for P2Y2R (Fig. 4). Cys106 and Cys183 are positioned in the first and second extracellular loops, and mutation of either of these residues to Ser eliminated receptor activity when expressed alone and in coexpression with wild-type P2Y2R. This suggests that a disulfide bond is formed between these two residues, as Hillmann et al. proposed based on homology to rhodopsin (11). Hillmann et al. indicated that C106S P2Y2R was not expressed on the cell surface in 1321N1 cells, as the binding of antibodies to the HA-tag added to the N-terminus is undetectable (11). In our observation, the level of protein expression did not differ between wild-type and mutant proteins in both HEK293T (Fig. 1) and 1321N1 cells (unpublished observation). Thus, C106S mutant protein is expressed but its sorting to the cell surface is prohibited. It is probable that bridging with an appropriate disulfide bond between Cys106 and Cys183 is sufficient for the cell surface expression of the receptor. Cys106 and Cys183 are also probably involved in a disulfide bridge between protomers in homodimer formation (Fig. 4C). Ser mutations at each of these sites formed dimers in SDS-PAGE under nonreducing conditions. In the C25S/C278S double mutant, dimer bands also occurred and were changed to monomers by reduction. Therefore, an inter-protomer disulfide bridge between Cys106 and Cys183 is also involved in homodimer formation (Fig. 4C). Mutation of Cys25 in N terminal domain to Ser did not prevent activation in coexpression with wild-type P2Y2R. If molecules forming homodimers are equivalently expressed, the ratio of homodimer formation with wild-type P2Y2R is approximately half; however, the activity was less than half. Mutation of Cys278 in the third extracellular loop to Ser attenuated activity, but some was maintained. Therefore, Cys278 seems to be less important for monomer and homodimer formation. The C25S/C278S double mutant lost activity, but this was recovered in coexpression with wild-type P2Y2R. These results are likely to be due to homodimer formation and thus support the conclusion that Cys106 and Cys183 are involved in disulfide bridges with wild-type P2Y2R; i.e. they are involved in bridging between molecules. Therefore, a Cys106-Cys183 intra- or interprotomer disulfide bond is essential for receptor expression on the cell surface and activation (Fig. 4). This hypothesis is also supported by the activation of C25S-α in the presence of C278S P2Y2R (Fig. 2D). Signals were transduced from C278S to C25S P2Y2R but not from C25S/C278S P2Y2R (Fig. 2D). This suggest that Cys25 and Cys278 form a disulfide bond, but that this disulfide bond is not essential for homodimer formation, given that signals were transduced from wild-type P2Y2R to the C25S/C278S mutant (Fig. 2A and C). In COS7 cells, most P2Y2R molecules are found in fractions corresponding to lipid raft components (12). The results of this study showed that most P2Y2R molecules also fractionated to lipid raft components in HEK293T cells. Only some of these molecules formed dimers, which shows that dimer formation has no relationship with distribution of P2Y2R to raft fractions. Also, in the presence of wild-type P2Y2R, Cys mutants had no effect on raft distribution. Formation of B2R homodimers with disulfide bonds is promoted by agonist binding (2). However, no agonist was necessary for P2Y2R to form homodimers. P2Y2R shows cross-reaction with B2R (9) and cross-talk with β2-adrenergic receptor (6), CXC chemokine receptor 2 (7), and A1R (8). Thus, P2Y2R may form heteromers with other GPCRs, but it is unknown how homodimer formation is related to the occurrence of heteromers. There were many smear bands up to 100 kDa in non-reducing SDS-PAGE (Fig. 1B). Bands above 100 kDa often appear in non-reducing SDS-PAGE, and some of those seen in Fig. 1B may be homo-oligomers or hetero-oligomers with other GPCRs. Formation of a dimer or oligomer greatly affects the binding kinetics, signal enhancement and termination thereof, as it regulates the density of molecules involved in GPCR signal transduction (1). The monomer and dimer are rapidly interconvertible (23), and involvement of a disulfide bridge is likely to prolong the presence of the dimer. Funding This study was supported by the Strategic Study Base Formation Support Project for Private Universities, Japan. Conflict of Interest None declared. 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Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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The Journal of BiochemistryOxford University Press

Published: Jan 18, 2018

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