2022 年 45 巻 6 号 p. 691-697
G-protein-coupled receptors (GPCRs) trigger various physiological functions. GPCR-mediated effects largely depend on the receptor-associated G-protein subtypes. However, compelling evidence suggests that single receptor proteins activate multiple G-protein subtypes to induce diverse physiological responses. This study compared responses mediated by three different Gq-binding uridine nucleotide receptors, P2Y2, P2Y4, and P2Y6, by measuring Ca2+ signaling and interleukin (IL)-8 production. In 1321N1 human astrocytoma cells stably expressing these receptors, agonist stimulation evoked concentration-dependent intracellular Ca2+ elevation to a similar extent. In contrast, agonist-induced IL-8 production was prominent in P2Y6-expressing cells, but not in P2Y2- and P2Y4-expressing cells. In addition to inhibition of Gq signaling, G12 signal blockade attenuated uridine 5′-diphosphate (UDP)-induced IL-8 production, suggesting the involvement of a small G-protein pathway. The Rac inhibitor EHop-16 prevented UDP-induced IL-8 release. The P2Y6-triggered IL-8 production was also inhibited by extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and protein kinase B (Akt) inhibitors. These results suggest that P2Y6 receptor-induced IL-8 production requires Gq-mediated Ca2+ signaling as well as G12-mediated activation of Rac. The results also suggest the importance of considering the involvement of multiple G proteins in understanding GPCR-mediated functions.
G protein-coupled receptors (GPCRs) transmit extracellular signals into target cells by activating different G proteins that are classified into four families according to their α subunits: Gi, Gs, Gq, and G12/13.1) Among them, the Gs and Gi families regulate adenylyl cyclase activity to control the cAMP signaling system,2) whereas Gq activates phospholipase Cβ to trigger inositol-1,4,5 trisphosphate-mediated Ca2+ signaling and diacylglycerol-mediated protein kinase C activation.3) G12/13 can activate small guanosine 5′-triphosphatase (GTPase) families.4) Biological reactions triggered by the activation of various GPCRs converge depending on the types of G proteins that are primarily coupled to the receptors.5,6) For example, regardless of the receptor type, activation of Gs-coupled receptors on smooth muscle cells induce relaxation via the cAMP signaling pathway, whereas activation of Gq-coupled receptors commonly induces smooth muscle contraction via Ca2+ and protein kinase C-mediated pathways.7,8) Recent studies, however, have provided evidence that a single receptor signal can be transmitted via multiple G protein families, particularly in pathophysiological responses.9,10) For example, stimulation of AT1 receptor induces vasoconstriction via the Gq/11 protein,11) whereas receptor activation also stimulates vascular remodeling and cardiac hypertrophy via the G12/13 protein.12) The AT1 receptor activation is also known to stimulate G protein-independent signaling pathways such as the β-arrestin-mediated mitogen-activated protein (MAP) kinase activation.13) Therefore, it is important to evaluate and compare the agonist-induced different responses in order to understand the signaling mechanism induced by GPCRs, even those that are coupled to the same G protein subtype.
Extracellular nucleotides, such as ATP and uridine 5′-triphosphate (UTP), regulate physiological responses through various purinergic receptors. Purinergic receptors include the P1 receptor for adenosine, ionotropic P2X receptors that exclusively respond to ATP, and GPCR-type P2Y receptors. The P2Y receptors include P2Y1,2,4,6, and 11–14, which recognize various nucleotides such as ATP, ADP, UTP, and uridine 5′-diphosphate (UDP).14) Among them, P2Y2, P2Y4, and P2Y6 receptors, that recognize uridine nucleotides, are classified into the same category as the Gq-coupled receptors. These receptors are ubiquitously expressed and are involved in a variety of physiological functions.15) In general, one cell population expresses multiple P2Y receptor subtypes, making it difficult to evaluate individual receptor function. The human astrocytoma 1321N1 cells do not express endogenous functional P2 receptors, and are suitable for analyzing recombinant purinergic receptor functions.16) In this study, we established stable recombinant P2Y2, P2Y4, and P2Y6 receptor-expressing 1321N1 cells, and investigated the individual Gq-coupled receptor function by measuring [Ca2+]i and chemokine interleukin (IL)-8 production.
ATP, ADP, UTP, UDP, and fluo-4-acetoxymethylester (AM) were purchased from Sigma-Aldrich (Tokyo, Japan). Fura-2-AM and carbachol were obtained from Wako (Osaka, Japan). A fluorometric imaging plate reader (FLIPR) Calcium 4 Kit was purchased from Molecular Devices (Sunnyvale, CA, U.S.A., Bulk Kit R8141). The pcDNA3.1 expression vector encoding human P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, and P2Y14 receptors were obtained from the UMR cDNA Resource Centre (U.S.A.). Anti-phospho-p42/44 mitogen-activated protein kinase (MAPK) (Thr202/Tyr204, #9101), anti-phospho p38 (Thr180/Tyr182, #9211), anti-phospho-c-Jun N-terminal kinase (JNK) (Thr183/Tyr185, #9251), anti-phospho-Akt (S473, #9271), and HRP-conjugated anti-rabbit immunoglobulin G (IgG) antibodies (#7074) were purchased from Cell Signaling (Danvers, MA, U.S.A.). All other chemicals were of reagent grade or were of the highest quality available.
Cell Culture1321N1 human astrocytoma cells (1321N1 cells), donated by Prof. Norimichi Nakahata of the Tohoku University, were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% (v/v) heat-inactivated fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin. Cells were maintained in 100-mm dishes and incubated at 37 °C in a humidified atmosphere of 95% air and 5% CO2. The 1321N1 cells were transfected with a pcDNA3.1 expression vector encoding human P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, and P2Y14 receptors. To obtain stable P2Y receptor-expressing cells, the transfected cells were selected using 0.7 mg/mL geneticin. The transfected 1321N1 cells were cultured in DMEM supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 µg/mL streptomycin, and 0.7 mg/mL geneticin.
Measurement of Intercellular Ca2+ Concentration ([Ca2+]i) Using a Fluorescence DetectorVarious P2Y receptor-expressing 1321N1 cells were loaded with 1 µM Fura-2-AM at 37 °C for 20 min, washed twice with KRH–BSA buffer, and adjusted to 1–2 × 105 cells/mL. Changes in Fura 2 fluorescence at 510 nm with dual excitation wavelengths at 340 and 380 nm were monitored as previously described,17) and intercellular Ca2+ concentrations ([Ca2+]i) were calculated using the FL Solution 4.2 (Hitachi, Tokyo, Japan) software, with a Kd value of 224 nM for the Fura-2/Ca2+ equilibrium. In the other case, the day before any assay, cells were seeded into black-walled, clear-bottomed 384-well plates at a density of 15000 cells/well. Following media aspiration, the cells were incubated at 37 °C for 50 min with Hank’s buffered salt solution (HBSS) and 20 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES) buffer (pH 7.4) containing the cytoplasmic calcium indicator, reconstituted FLIPR calcium 4 assay reagent (component A), and 2.5 mM probenecid. The loaded cells were then placed in an FDSS7000 real-time fluorescence detector (Hamamatsu Photonics, Hamamatsu, Japan). Cells were pre-incubated with the test compound for 5 min and then stimulated with agonists at room temperature. Fluorescence was detected by excitation and emission at wavelengths of 488 and 540 nm, respectively.18)
IL-8 MeasurementP2Y receptor-expressing 1321N1 cells were seeded in a 48-well plate and grown to confluence. The cells were washed twice with DMEM and incubated under various conditions at 37 °C for 6 h. Then, the culture medium was subjected to the IL-8 assay using a human IL-8 enzyme-linked immunosorbent assay (ELISA) kit (Endogen, U.S.A.), according to the manufacturer’s instructions.
Western BlottingCells were seeded onto a 6-well plate and grown to confluence.19) On the day of the experiment, the cells were washed with KRH buffer and stimulated with the same buffer containing various drugs at 37 °C for 10 min, dissolved in a sample buffer (25% glycerine, 1% sodium dodecyl sulfate (SDS), 62.5 mM Tris–Cl, and 10 mM β-mercaptoethanol), and heated at 98 °C for 10 min. Proteins in the same amount of cell lysate were separated by 11% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to Immobilon-P polyvinylidene fluoride membranes (Merck Millipore, Tokyo, Japan). The membranes were incubated with primary antibodies overnight at 4 °C and with secondary antibodies for 2 h at 25 °C. The primary antibodies were used at 1000-fold dilutions, and horseradish peroxidase-conjugated secondary antibodies were used at a 10000-fold dilution. Immunoreactive proteins were detected by enhanced chemiluminescence (GE Healthcare Bio-sciences, Tokyo, Japan) using an Image Reader LAS-3000 (FUJIFILM, Tokyo, Japan).
Production of AdenovirusRecombinant adenoviruses of Gαq-ct (peptide coding carboxyl terminal of Gαq), Gα12-ct (peptide coding carboxyl terminal of Gα12), and Gα13-ct (peptide coding carboxyl terminal of Gα13) were produced as described by He et al.,20) Kurose and colleagues,21) and Nishida et al.,22) with slight modifications.
StatisticsAll values were expressed as mean ± standard error of the mean (S.E.M.). Data were analyzed using Mann–Whitney U tests for two data comparisons and one-way ANOVA, and with Dunnett’s two-tailed test for multiple data comparison. Statistical significance was set at p < 0.05.
The 1321N1 cells used in this study did not respond to nucleotide stimuli, indicating that the 1321N1 cells do not functionally express purinergic receptors that affect intracellular Ca2+ concentration ([Ca2+]i) levels (Fig. 1A). Therefore, we prepared 1321N1 cells that stably expressed the uridine receptors (P2Y2, P2Y4, and P2Y6) coupled to the Gq protein, and analyzed their functions. First, we confirmed that the receptors were functionally expressed in the cells using the [Ca2+]i assay (Fig. 1A). Stimulation of the P2Y receptor-expressing cells with uridine nucleotides, which have a high affinity for each receptor, increased in [Ca2+]i in a concentration-dependent manner (Fig. 1B). As a reference, stimulation with carbachol of muscarinic receptors that are expressed endogenously in 1321N1 cells also evoked a concentration-dependent increase in [Ca2+]i (Fig. 1).
Parental and human P2Y2, P2Y4, and P2Y6 receptor-expressing 1321N1 cells were loaded with a fluorescent Ca2+ indicator and stimulated with appropriate receptor agonists. (A) Typical traces of changes in intracellular Ca2+ concentration ([Ca2+]i). Cells were loaded with Fura 2-AM at 37 °C for 30 min and stimulated with a receptor agonist, as indicated in the figure. Changes in Fura 2 fluorescence at 510 nm with dual excitation wavelengths of 340 and 380 nm were monitored. (B) Concentration-dependent effects of receptor agonists on [Ca2+]i. Parental and human P2Y2, P2Y4, and P2Y6 receptor-expressing 1321N1 cells were seeded onto 384-well plates. The calcium indicator-loaded cells were stimulated with receptor agonists, as indicated in the figure, at room temperature. Fluorescence was monitored by excitation and emission at wavelengths of 488 and 540 nm, respectively. Values are shown as mean ± S.E.M. of at least three independent experiments and calculated for the maximum response of the receptor agonist. * p < 0.05. Carb: carbachol.
Stimulation of the uridine nucleotide receptor has been reported to promote the production of IL-8, a chemokine.23) Therefore, P2Y receptor-expressing cells were used to investigate the effect of agonist stimulation on IL-8 production. Significant differences were observed in IL-8 secretions in each P2Y receptor-expressing cell when stimulated with an agonist at a concentration that caused a sufficient increase in [Ca2+]i (Fig. 2). The release of IL-8 increased after the agonist stimulation in cells expressing the P2Y6 receptor, but not in the P2Y2 and P2Y4 receptor-expressing cells.
Parental and human P2Y2, P2Y4, and P2Y6 receptor-expressing 1321N1 cells were seeded onto a 48-well plate and grown to confluence. The cells were washed twice with DMEM medium and stimulated for 6 h with the following receptor agonists: UTP for P2Y2 receptor, UTP for P2Y4 receptor, and UDP for P2Y6 receptor. The supernatant was then collected. IL-8 concentration in the supernatant was measured using enzyme-linked immunosorbent assay (ELISA). Values are shown as the mean ± S,.E.M. of at least three independent experiments and normalized as indicated. ** p < 0.01, significantly different from none.
Next, we focused on the mechanism underlying the UDP-induced IL-8 production in P2Y6 receptor-expressing cells. Parental 1321N1 cells did not respond to UDP (Fig. 3A), but stimulation of P2Y6 receptor-expressing cells with UDP induced IL-8 release in a concentration-dependent manner (Fig. 3B). Similar to the agonist stimulation in P2Y2 and P2Y4 receptor-expressing cells, no IL-8 release was observed upon stimulation of the endogenous muscarinic receptor with carbachol (Fig. 3C).
Parental and P2Y6 receptor-expressing cells (P2Y6-1321N1) were seeded onto a 48-well plate and stimulated with UDP or carbachol (Carb). After 6 h, the supernatant was collected to measure the concentration of IL-8 using ELISA. (A) Effect of UDP (100 µM) on IL-8 release in parental 1321N1 cells. (B) Concentration-dependent effects of UDP on IL-8 release in P2Y6-1321N1 cells. (C) Effect of Carb on IL-8 release in P2Y6-1321N1 cells. Values are shown as the mean ± S.E.M. of at least three independent experiments. ** p < 0.01, significantly different from none.
The effects of the selective antagonists of P2Y6 receptors on UDP-induced IL-8 production in P2Y6 receptor-expressing cells were inhibited by MRS2578, a P2Y6 receptor-selective inhibitor (Fig. 4). We previously identified a novel P2Y6 receptor antagonist TIM-38.18) UDP-induced IL-8 release was also suppressed by TIM-38 in a concentration-dependent manner (Fig. 4).
P2Y6-1321N1 cells were seeded onto a 48-well plate and stimulated with UDP in the presence or absence of P2Y6 receptor antagonists TIM-38 and MRS2578. After 6 h, the supernatant was collected to measure the concentration of IL-8 using ELISA. Values are shown as the mean ± S.E.M. of at least three independent experiments. ** p < 0.01, significantly different from UDP alone.
In addition to Gq protein, the P2Y6 receptor has been shown to be coupled to G12/13 proteins, which link to guanine nucleotide exchange factors for the Rho small GTPases.24) Therefore, we examined the possible involvement of the G12/13 protein in addition to Gq in the P2Y6 receptor-mediated IL-8 release, using an adenovirus vector-mediated expression of inhibitory peptides for these G-proteins. The UDP-induced IL-8 release was inhibited by the expression of inhibitory peptides of Gq and G12 proteins, but not by the expression of the G13 protein (Fig. 5A). Pharmacological inhibition with the Gq protein-specific inhibitor YM-254890 (YM254) also suppressed UDP-induced IL-8 release (Fig. 5B). We next examined the involvement of the small G protein signaling in P2Y6 receptor-mediated IL-8 production using fluvastatin, an hydroxymethylglutaryl-CoA (HMG-CoA) reductase inhibitor, which depresses cellular isoprenoids such as farnesyl pyrophosphate and geranylgeranyl pyrophosphate, resulting in the prevention of lipid modification of the small G proteins.
(A) P2Y6-1321N1 cells were seeded onto a 48-well plate and transfected with various adenoviruses which express G-protein dominant negative proteins. After 48 h of adenovirus transfection, the cells were washed twice with DMEM medium and stimulated with UDP (100 µM) for 6 h. The IL-8 concentration in the supernatant was measured using ELISA. Values are mean ± S.E.M. (n = 3). * p < 0.05, ** p < 0.01, significantly different from UDP alone. (B) P2Y6-1321N1 cells were pre-incubated in the presence or absence of the Gq-protein selective antagonist, YM-254890 (YM-254, 10 µM) for 30 min, and then stimulated with UDP (10 µM) for 6 h. The IL-8 concentration in the supernatant was normalized with non-treated control. Values are mean ± S.E.M. (n = 3). P2Y6-1321N1 cells were pre-incubated in the presence or absence of fluvastatin (Flv, 10 µM) (C) or Rac inhibitor, EHop-16 (D) for 15 min and then stimulated with UDP (10 µM) for 6 h. The IL-8 concentration in the supernatant was measured by ELISA and normalized with UDP alone. Values are mean ± S.E.M. (n = 3). ** p < 0.01, significantly different from UDP alone.
Pre-treatment with fluvastatin inhibited UDP-induced IL-8 production (Fig. 5C). In addition, treatment with EHop-16, an inhibitor of Rac, markedly suppressed the release of IL-8 induced by UDP (Fig. 5D).
Stimulation of the P2Y6 receptor by UDP caused activation of various intracellular kinases, ERK, p38, JNK, and Akt, and this activation was suppressed by the selective inhibitors of each kinase (Fig. 6A). Carbachol stimulation of the 1321N1 cells also causes activation of these kinases to a similar extent (Supplementary figure). UDP-induced IL-8 production was significantly inhibited by the MEK inhibitor U0126, the JNK inhibitor SP60025, and the PI3-kinase inhibitor LY294002, and strongly inhibited by the PCK inhibitor Gӧ6983 but not the inhibitor of the transcription factor nuclear factor-kappaB (NF-κB) (Fig. 6B).
UDP stimulation of the P2Y6 receptor induces activation of intracellular kinases (A). P2Y6-1321N1 cells were seeded onto a 6-well plate and stimulated with UDP (100 µM) for 10 min at 37 °C in the presence or absence of the following kinase inhibitors: U0126 (U, 10 µM) for MEK (MAPK/extracellular regulated kinase), SB203580 (SB, 10 µM) for p38MAPK, SP60025 (SP, 10 µM) for JNK, and LY294002 (LY, 10 µM) for PI-3 kinase. Same amount of whole cell lysates were separated by SDS-PAGE, followed by immunoblotting using anti-phospho-ERK, p38, JNK, and Akt antibodies. (B) P2Y6-1321N1 cells were pre-incubated in the presence or absence of several inhibitors including Gӧ6983 (Gӧ, 10 µM) for PKC, or NF-κBI (10 µM) for NF-κB for 15 min, and then stimulated with UDP (10 µM) for 6 h. The IL-8 concentration in the supernatant was measured by ELISA and normalized to that of UDP alone. Values are presented as the mean ± S.E.M. (n = 3). ** p < 0.01, significantly different from UDP alone.
Finally, we examined the effects of the stimulation of endogenous Gq-coupled receptors expressed in the 1321N1 cells on changes in [Ca2+]i and IL-8 production. Although the stimulation of 1321N1 cells with histamine, STA2, thrombin or lysophosphatidic acid, the agonists for histamine H1, prostanoid TP, and proteinase-activated and lysophospholipid LPA receptors, respectively, caused an increase in [Ca2+]i (Fig. 7A), IL-8 production was enhanced only by prostanoid TP receptor stimulation with STA2, stable thromboxane A2 analogue. In addition, the Ca2+ ionophore, ionomycin caused a strong elevation in [Ca2+]i without affecting IL-8 production (Fig. 7B).
Parental 1321N1 cells were stimulated with following various agonists such as carbachol (Carb, 100 µM), histamine (His, 100 µM), stable thromboxane A2 analogue (STA2, 1 µM), thrombin (Thr, 1 unit/mL), lysophosphatidic acid (LPA, 10 µM), and ionomycin (Iono, 3 µM). (A) Typical traces of changes in intracellular Ca2+ concentration ([Ca2+]i). Cells were loaded with Fura 2-AM at 37 °C for 30 min and stimulated with these receptor agonists. (B) The IL-8 concentration in the supernatant obtained after 6 h of stimulation with these receptor agonists was measured by ELISA and normalized to the control. Values are presented as the mean ± S.E.M. (n = 3). ** p < 0.01, significantly different from control.
In this study, we established cell lines that stably expressed the three types of Gq-coupled uridine nucleotide receptors, P2Y2, P2Y4, and P2Y6, in the 1321N1 cells lacking endogenous functional P2 receptors.25) Stimulation of these receptors with UTP or UDP induced similar [Ca2+]i elevations in a concentration-dependent manner. However, there was a large difference in the receptor-stimulated IL-8 production. Specifically, the P2Y6 receptor stimulation evoked a remarkable IL-8 production, but the stimulation of P2Y2 and P2Y4 receptors had little effect on IL-8 production. Stimulation of endogenous Gq-coupled muscarinic receptors with carbachol resulted in a marked increase in [Ca2+]i without stimulating IL-8 production. As the UDP-induced IL-8 production in P2Y6 receptor-expressing cells was suppressed by P2Y6 receptor antagonists, it was considered to be a receptor-mediated response. The crucial role of Gq-dependent signaling in P2Y6 receptor-mediated IL-8 production was suggested by direct inhibition of Gq function by adenovirus vector-mediated expression of a Gq-specific inhibitory peptide or by the pharmacological approach using the Gq protein-specific inhibitor YM-254890. Among the Gq protein downstream signaling pathways, the potent inhibitory effect of Gӧ6983 suggested a role for PKC in IL-8 production. Nevertheless, the lack of an IL-8 producing effect in the stimulation of recombinant P2Y2 and P2Y4 receptors and endogenous histamine H1 receptors suggests that P2Y6 receptor-mediated IL-8 production may involve another signaling mechanism in addition to the Gq-mediated signal.
P2Y6 receptors are implicated in inflammatory responses and pathophysiological tissue remodeling via activation of various signaling mechanisms, including small G protein activation.23,24,26) In this study, we demonstrated that the P2Y6 receptor-mediated IL-8 production was inhibited by introducing a G12-specific inhibitory peptide. G12 is known to stimulate the Rho-guanine nucleotide exchange factor to transmit GPCR signaling to the small G-protein pathway. In line with this, interference of the small G protein lipid modification by the HMG-CoA reductase inhibitor (fluvastatin) inhibited the P2Y6 receptor-mediated IL-8 production. In addition,27–29) Rac inhibitor EHop-16 effectively prevented P2Y6 receptor-mediated IL-8 production. These results are consistent with those of a previous study that found that in cardiomyocytes, the P2Y6 receptor couples with the G12 protein to stimulate reactive oxygen production via Rac activation.12,30,31) Therefore, it is suggested that P2Y6 receptor-mediated IL-8 production involves the G12 and Rac signaling pathways in addition to the Gq-mediated signaling.
Measurement of changes in intracellular kinase activity after UDP stimulation demonstrated the activation of p42/44 ERK, p38, JNK, and Akt. P2Y6 receptor-mediated IL-8 production was significantly suppressed by p42/44ERK, JNK, and Akt inhibitors. These kinase activation processes seemed to be the downstream events of Gq-mediated signaling, as stimulation of endogenous muscarinic M3 receptor with calbachol, which was unable to induce IL-8 production, also stimulated the activation of p42/44 ERK, p38, JNK, and Akt in a similar time-dependent manner (Supplementary figure). Therefore, it is suggested that activation of these intracellular kinases is required, but insufficient for P2Y6 receptor-mediated IL-8 production. In the present study, NF-κB inhibitor did not affect the P2Y6 receptor-mediated IL-8 production. On the other hand, P2Y6 receptor was reported to activate NF-κB signaling pathway and increased survival of Osteoclasts.32) In1321N1 cells, we observed that NF-κB signaling pathway was activated by TNF-α, but not by STA2, (unpublished data). It is therefore suggested that IL-8 production induced by P2Y6 and endogenous TP prostanoid receptor stimulation may utilize a transcription factor different from NF-κB in 1321N1 cells.
The present results showed that P2Y2 and P2Y4 receptor stimulation per se induced phospholipase C activation, but nil or only weak IL-8 production. Little is known about the role of P2Y4 receptor stimulation on IL-8 production, whereas P2Y2 receptor-mediated IL-8 production has been demonstrated in different cell types.33–35) These differences may be because the P2Y2 receptor signaling enhances IL-8 production in the presence of another signal input. Indeed, different GPCR co-stimulations have been reported to induce synergistic production of cytokines and chemokines.36) In addition, as a feature of purinergic signaling, different receptor stimuli such as T-cell receptors and Toll-like receptors induce nucleotide release from activated cells and coordinate with other receptor signals to enhance cytokines and chemokines production.35,37) The data in this study represent the characteristics of individual receptor stimuli.
Our data suggest that P2Y6 receptor-mediated IL-8 production is a highly integrated response orchestrated by Gq- and other G-protein families such as G12 in P2Y6 receptor-expressing 1321N1 cells. These orchestrated signaling pathways may explain the differential effects of Gq-coupled receptor stimulation on IL-8 production. These inferences are further supported by the results shown in Fig. 7, indicating that stimulation of several Gq-coupled receptors endogenously expressed in 1321N1 cells resulted in differences in IL-8 production. A significant effect was observed only with prostanoid TP receptor stimulation, which has been reported to couple with G12.38,39) In GPCR studies, it was found that the response to the ligand was influenced by the dominant type of G protein coupled to the receptor. Further studies on the differential IL-8 producing response shown in this study should reveal a regulatory mechanism by which ligand stimulation selectively activates specific G protein signaling. In fact, recent studies have shown that differences in ligand chemical structure cause changes in receptor structure that can control the type of downstream signaling molecules.40–42) Elucidation of the mechanism by which receptor structure affects G protein selectivity may lead to the design of biased ligands with fewer side effects.
This work was supported by a Grant-in-Aid (20K16010) to M.I.
The authors declare no conflict of interest.
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