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Keywords:

  • Akt;
  • Extracellular signal-regulated kinase;
  • Mitogen-activated protein kinase;
  • Serotonin 5-HT1B receptors;
  • Serotonin

Abstract

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. RESULTS
  5. 5-HT and sumatriptan stimulate phosphorylation of ERK in transfected BE(2)-C neuroblastoma cells
  6. RGS4 inhibits activation of ERK and Akt
  7. DISCUSSION
  8. Acknowledgements

Abstract: Regulator of G protein signaling (RGS) proteins are GTPase-activating proteins for heterotrimeric G proteins. One of the best-studied RGS proteins, RGS4, accelerates the rate of GTP hydrolysis by all Gi and Gqα subunits yet has been shown to exhibit receptor selectivity. Although RGS4 is expressed primarily in brain, its effect on modulating the activity of serotonergic receptors has not yet been reported. In the present study, transfected BE(2)-C human neuroblastoma cells expressing human 5-HT1B receptors were used to demonstrate that RGS4 can inhibit the coupling of 5-HT1B receptors to cellular signals. Serotonin and sumatriptan were found to stimulate activation of extracellular signal-regulated kinase. This activation was attenuated, but not completely inhibited, by RGS4. Similar inhibition by RGS4 of the protein kinase Akt was also observed. As RGS4 is expressed at high levels in brain, these results suggest that it may play a role in regulating serotonergic pathways.

Medications that modulate serotonergic pathways are commonly used to treat such diverse disorders as depression, schizophrenia, and anxiety. These medications elicit actions at one or more of the at least 16 reported types of mammalian receptors (for review, see Hoyer et al., 1994; Scalzitti and Hensler, 1996) for serotonin (5-HT). Receptors within the 5-HT1 family (5-HT1A, 5-HT1B, 5-HT1D, 5-HT1E, and 5-HT1F) all appear to couple to G proteins of the Gi class and inhibit adenylyl cyclase. With respect to 5-HT1B receptors, most studies of cellular signaling have used rodent receptors as models for the human receptor. Rodent receptors have been shown to inhibit the activity of adenylyl cyclase (Unsworth and Molinoff, 1992; Berg et al., 1994; Giles et al., 1996) and in some cases to evoke small increases in intracellular Ca2+ (Dickenson and Hill, 1995; Giles et al., 1996). We have additionally found that the receptors activate p70 S6 kinase and the mitogen-activated protein kinase (MAP kinase) extracellular signal-regulated kinase-2 (ERK2) (Pullarkat et al., 1998; Mendez et al., 1999).

The human receptor (previously classified as 5-HT1Dβ) has been less extensively studied. Although the amino acid sequences of the rat and human receptors are 94% homologous, the two receptors exhibit very different pharmacological profiles (Veldman and Bienkowski, 1992). For example, the human receptor has a higher affinity for sumatriptan and a lesser affinity for pindolol and propranolol than does the rodent receptor. The differences in pharmacology suggest the possibility that the human and rodent receptors may exhibit differences in coupling to cellular signals. However, it has been reported that human receptors, like rodent receptors, do couple negatively to adenylyl cyclase (Veldman and Bienkowski, 1992). In the present studies, we demonstrate that the human receptor is also similar to the rodent receptor in activating MAP kinase. Additionally, we show that the human receptor activates the protein kinase Akt.

Regulator of G protein signaling (RGS) proteins are negative regulators of heterotrimeric G protein-mediated signaling. Specific RGS proteins target particular G proteins. RGS proteins have been found for Gαi, Gαo, Gαt, Gαq, and Gα12/13 (for review, see De Vries and Farquhar, 1999). RGS4, one of the best-studied RGS proteins, has been shown to attenuate the activity of both Gi- and Gq-mediated signals (Druey et al., 1996; Huang et al., 1997). It is expressed primarily in brain (Druey et al., 1996).

Significantly, although RGS4 appears to attenuate the activity of all Gi and Gq types of G proteins in vitro, it exhibits differences in inhibitory activity at various receptors. Xu et al. (1999) demonstrated that the increases in intracellular Ca2+ stimulated by carbachol are much more sensitive to inhibition by RGS4 than those stimulated by bombesin and cholecystokinin. Significantly, this selectivity was shown to reflect an interaction of the N-terminal domain of RGS4 with receptor complexes and not preferential coupling to different subtypes of Gq (Zeng et al., 1998; Xu et al., 1999). Additionally, RGS4 has been reported not to inhibit the actions of Gi-coupled μ-opioid receptors in a model Xenopus laevis dermal melanophore system (Potenza et al., 1999).

In the present study, we demonstrate that RGS4 does inhibit the coupling of Gi-coupled 5-HT1B receptors to two cellular signals: ERK and Akt. ERK1 and ERK2 are serine/threonine protein kinases that are classified as MAP kinases. They have been shown to phosphorylate a number of transcription factors and appear to regulate translation of mRNA (for review, see Denton and Tavare, 1995). Activation requires phosphorylation at Thr202/Tyr204 by MAP kinase kinase. Akt (also referred to as protein kinase B) is a serine/threonine protein kinase that regulates the activity of a number of transcription factors. It is activated by phosphorylation at Thr308/Ser473 by phosphatidylinositol 3,4,5-trisphosphate-dependent protein kinases 1 and 2 (Alessi et al., 1996, 1997). We found that RGS4 causes partial, but incomplete, inhibition of the activation by 5-HT1B receptors of both ERK and Akt. It may therefore represent a protein responsible for regulating the function of 5-HT1B receptors (and possibly other 5-HT1 receptors) in brain (Druey et al., 1996), where it is highly expressed.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. RESULTS
  5. 5-HT and sumatriptan stimulate phosphorylation of ERK in transfected BE(2)-C neuroblastoma cells
  6. RGS4 inhibits activation of ERK and Akt
  7. DISCUSSION
  8. Acknowledgements

Materials

Sumatriptan succinate was kindly provided by Glaxo Wellcome (Hertfordshire, U.K.). Tranylcypromine, SB 224289 hydrochloride, and 5-HT were purchased from Sigma (St. Louis, MO, U.S.A.).

Cell culture

Human BE(2)-C neuroblastoma cells and cDNA for the human 5-HT1B receptor in p3CLNeo were obtained from the American Type Culture Collection (Rockville, MD, U.S.A.). A stable cell line expressing 5-HT1B receptors was obtained by transfecting (using calcium phosphate precipitation) BE(2)-C cells with cDNA for the human 5-HT1B receptor and selecting for resistance to geneticin (400 μg/ml). These cells express receptors at a density of 500 fmol/mg of membrane as determined in binding studies using the radioligand [3H]5-HT (Veldman and Bienkowski, 1992), obtained from NEN Life Sciences (Boston, MA, U.S.A.). Assays contained 50-100 μg of membrane protein and 1-40 nM [3H]5-HT in a total volume of 100 μl. Specific binding was defined with 10 μM sumatriptan. Inclusion of the monoamine oxidase inhibitor tranylcypromine (10 μM) did not affect binding results. Transfected cells were routinely cultured in Ham's F-12 nutrient mixture with L-glutamine and Eagle's minimal essential medium with nonessential amino acids (1:1), 10% dialyzed fetal bovine serum (dialyzed in membranes with 1,000-Da molecular mass cutoffs against a 100-fold greater volume of 150 mM NaCl to remove endogenous 5-HT), 100 U of penicillin/100 U of streptomycin/ml, and 400 μg/ml geneticin at 37°C (95% air/5% CO2).

Transient transfections of cells

A cDNA encoding a FLAG epitope-tagged human RGS4, kindly provided by Dr. John Kehrl (NIAID, NIH, Bethesda, MD, U.S.A.), was expressed under the CMV promoter, following cloning into the Kpn1/EcoRV sites of a mammalian expression vector, denoted pTEX (GenBank accession no. AF191496). Transient transfections with 4 μg of plasmid cDNA per 100-mm cell culture dish were performed under serum-free conditions 48 h prior to cellular studies with Lipofectamine Plus according to the manufacturer's suggestions (GibcoBRL, Rockville, MD, U.S.A.). Cells transfected with empty vector (pTEX) were used as controls in studies of transfected RGS4.

Immunoblots

Monoclonal antiphospho-ERK1/ERK2 (Thr202/Tyr204, no. 9106) and rabbit polyclonal antiphospho-Akt (Ser473, no. 9271) were obtained from New England Biolabs (Beverly, MA, U.S.A.). Goat polyclonal anti-RGS4 (no. C17), rabbit polyclonal total ERK1/ERK2 (no. C14), goat polyclonal total Akt1 (no. C20), and horseradish peroxidase-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.). The day prior to use, cells were washed with phosphate-buffered saline and cultured overnight under serum- and geneticin-free conditions. Cells were stimulated with the specified concentrations of agonists and lysed with a 26-gauge needle in 25 mM HEPES (pH 7.4), 50 mM NaF, 5 mM EDTA, 1 mM sodium orthovanadate, 100 μM phenylmethylsulfonyl fluoride, 0.1% aprotinin, and 10 μg/ml leupeptin. Proteins were separated on 12% resolving gels (Bio-Rad Laboratories, Hercules, CA, U.S.A.) and transferred to 0.45-μm Immobolin-P polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA, U.S.A.). Membranes were blocked overnight with 3% powdered milk before incubation with primary and secondary antibodies. Bound antibodies were visualized using Enhanced Luminol Chemiluminescence Reagent (NEN Life Sciences) and exposure to a Kodak Image Station 440CF with a cooled full frame capture CCD camera (Kodak). Net intensity of bands was calculated using Kodak Digital Science 1D Image Analysis Software (version 3.0.2) on defined regions of interest.

5-HT and sumatriptan stimulate phosphorylation of ERK in transfected BE(2)-C neuroblastoma cells

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. RESULTS
  5. 5-HT and sumatriptan stimulate phosphorylation of ERK in transfected BE(2)-C neuroblastoma cells
  6. RGS4 inhibits activation of ERK and Akt
  7. DISCUSSION
  8. Acknowledgements

Studies of the coupling of human 5-HT1B receptors to the MAP kinases ERK1 and ERK2 were performed in a stable transfected BE(2)-C human neuroblastoma cell line (referred to herein as BE2-1B). BE2-1B cells express receptor at a density of 500 fmol/mg of membrane protein and a KD for the radioligand [3H]5-HT of 6 nM. Activation of ERK1 and ERK2 requires phosphorylation of both Thr202 and Tyr204 by MAP kinase kinase (Cobb and Goldsmith, 1995). Therefore, detection of phosphorylation can be used as a means for assaying activation of MAP kinase. Immunoblots of cell lysate from BE2-1B cells treated with 5-HT or the selective human 5-HT1B/1D receptor agonist sumatriptan revealed a large increase in the level of double-phosphorylated ERK (Fig. 1A). In contrast, 5-HT and sumatriptan did not stimulate increased phosphorylation of ERK in nontransfected (wild) cells.

image

Figure 1. Activation of ERK by 5-HT and sumatriptan (sum) in cells expressing human 5-HT1B receptors. A: Nontransfected (wild) BE(2)-C neuroblastoma cells (lanes 1-3) and transfected BE2-1B cells (lanes 4-6) were incubated with 1 μM 5-HT or 1 μM sumatriptan for 5 min and then lysed. B: BE2-1B cells were incubated with 1 μM sumatriptan for the specified times and then lysed. Total lysate (10 μg/lane) was analyzed by immunoblotting with antibody to phospho-ERK1/ERK2 (p-ERK). Membranes were then stripped and analyzed with antibody to total ERK. All immunoblots are representative of three separate experiments performed in duplicate.

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The activation of ERK by 5-HT1B receptors was rapid but transient (Fig. 1B). Stimulated activity was seen within 2 min and was maximal at 5 min. After 30 min of incubation with sumatriptan, the amount of phosphorylated ERK approached that seen at baseline but remained somewhat elevated. Although both sumatriptan and 5-HT stimulated phosphorylation of ERK, 5-HT was a more potent agonist (Fig. 2). The EC50 for 5-HT, as calculated by nonlinear regression analysis of the net intensities of bands, was 2.4 nM. In contrast, the EC50 for sumatriptan was 0.6 μM. Significantly, the activation of ERK stimulated by both 5-HT and sumatriptan was completely inhibited by the selective 5-HT1B receptor antagonist SB 224289 (Fig. 2C).

image

Figure 2. 5-HT is a more potent agonist than is sumatriptan (sum). BE2-1B cells were incubated for 5 min with the indicated concentrations of 5-HT (A) or sumatriptan (B) and then lysed. BE2-1B cells (C) were treated for 5 min with 0.1 μM 5-HT or 0.1 μM sumatriptan in the presence or absence of 10 μM SB 224289, an antagonist selective for 5-HT1B receptors. Cells not treated with SB 224289 were exposed to an equal volume of vehicle (dimethyl sulfoxide). Total lysate (10 μg/lane) was analyzed by immunoblotting with antibody to phospho-ERK1/ERK2 (p-ERK). All immunoblots are representative of three separate experiments performed in duplicate.

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RGS4 inhibits activation of ERK and Akt

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. RESULTS
  5. 5-HT and sumatriptan stimulate phosphorylation of ERK in transfected BE(2)-C neuroblastoma cells
  6. RGS4 inhibits activation of ERK and Akt
  7. DISCUSSION
  8. Acknowledgements

The inhibitory effect of RGS4 on 5-HT1B receptor-stimulated MAP kinase activity was studied in BE2-1B cells transiently transfected with cDNA for human RGS4. As shown in Fig. 3, BE(2)-C cells do not express detectable levels of endogenous RGS4. Expression of RGS4 in transfected cells was found to inhibit receptor-mediated stimulation of phosphorylation of ERK. This inhibition by RGS4 was greatest when lower concentrations of sumatriptan were used (Fig. 3). The phosphorylation of ERK stimulated by 100 nM sumatriptan was inhibited by 60% (Fig. 4A). In contrast, RGS4 caused only a 15% inhibition of the phosphorylation stimulated by 1 μM sumatriptan, a finding that was not statistically significant.

image

Figure 3. Inhibition of 5-HT1B receptor-stimulated ERK activity by RGS4. BE2-1B cells were transfected 48 h prior to use with cDNA for human RGS4 (lanes 6-10) or an equal amount of empty control plasmid (pTEX) (lanes 1-5). Cells were incubated for 5 min with the specified concentrations of sumatriptan and then lysed. Total lysate (10 μg/lane) was analyzed by immunoblotting with antibody to phospho-ERK1/ERK2 (p-ERK). Membranes were then stripped and analyzed with antibody to total ERK1/ERK2 (Total ERK), phospho-Akt (p-Akt), total Akt 1 (Total Akt), and RGS4. Immunoblots are representative of three separate experiments performed in duplicate.

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image

Figure 4. Inhibition of ERK and pAkt by RGS4 is greatest at submaximal concentrations of sumatriptan. BE2-1B cells were transfected 48 h prior to use with cDNA for human RGS4 or an equal amount of empty control plasmid (pTEX). Cells were incubated with 0.1 or 1 μM sumatriptan for 5 min and then lysed. Total lysate (10 μg/lane) was analyzed by immunoblotting with antibody to phospho-ERK1/ERK2 (p-ERK) (A) or phospho-Akt (p-Akt) (B). Net intensities of bands were calculated from five separate experiments, and the means ± SE were expressed as -fold basal net intensity of p-ERK (A) and -fold basal net intensity of p-Akt (B). *p < 0.05 vs. absence of RGS4, two-sided paired Student's t calculated for each concentration of sumatriptan.

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To determine whether the inhibition by RGS4 was specific to activation of ERK, we also studied the effect of RGS4 on activation of the protein kinase Akt. Similar to ERK, Akt is activated by phosphorylation, though through a different pathway. Sumatriptan was found to stimulate phosphorylation of Akt in BE2-1B cells (Fig. 3) but not in nontransfected BE(2)-C cells (not shown). Expression of RGS4 was found to inhibit this phosphorylation. The activation of Akt stimulated by 100 nM sumatriptan in cells transfected with RGS4 was inhibited by an average of 50% relative to that seen in cells transfected with control empty vector (Fig. 4). In contrast, the activation of Akt stimulated by 1 μM sumatriptan in cells transfected with RGS4 was inhibited by 32%, a difference that did not reach statistical significance. Therefore, as was found with activation of ERK, RGS4 caused a partial inhibition of 5-HT1B receptor-stimulated activation of Akt that was greatest at submaximal concentrations of sumatriptan. Significantly, the activation of both ERK and Akt was completely inhibited by pertussis toxin (not shown), demonstrating a requirement for G proteins of the Gi/o family. Therefore, RGS4, which can interact with both Gαi/o and Gαq, interferes with the coupling of 5-HT1B receptors to both signals by inhibiting the activity of Gαi/o.

DISCUSSION

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. RESULTS
  5. 5-HT and sumatriptan stimulate phosphorylation of ERK in transfected BE(2)-C neuroblastoma cells
  6. RGS4 inhibits activation of ERK and Akt
  7. DISCUSSION
  8. Acknowledgements

Both sumatriptan and 5-HT stimulated activation of ERK in cells expressing human 5-HT1B receptors. However, sumatriptan was a less potent agonist. Differences in agonist affinity for the 5-HT1B receptor may be at least partially responsible. Veldman and Bienkowski (1992) reported a Ki of 1 nM for 5-HT and a Ki of 9.4 nM for sumatriptan. Significantly, we found that the selective antagonist SB 224289 completely inhibited the actions of both 5-HT and sumatriptan. Therefore, both agonists acted only through transfected 5-HT1B receptors and not through additional endogenous receptors.

Our finding that RGS4 caused a 60% inhibition of sumatriptan-stimulated phosphorylation of ERK represents a magnitude of inhibition almost identical to that reported for the Gi-coupled interleukin-8 receptor (Druey and Kehrl, 1997). Our finding that the inhibition by RGS4 could be overcome at higher concentrations of sumatriptan is similar to that seen for dopamine D2 receptors where the inhibition by RGS4 was attenuated at higher concentrations of agonist. Yan et al. (1997) reported that RGS4 caused a 50% inhibition of the ERK activity stimulated by 10 nM quinpirole but only an approximate 20% inhibition of the activity stimulated at 1 μM.

Significantly, although RGS4 has been previously demonstrated to inhibit the coupling of these two other Gi-coupled receptors to MAP kinase, it cannot be assumed that RGS4 inhibits coupling to all such receptors. RGS4 has been shown to differentially couple to specific receptors that couple to Gq (Zeng et al., 1998; Xu et al., 1999). Additionally, RGS4 has been reported not to inhibit the actions of Gi-coupled μ-opioid receptors (Potenza et al., 1999).

Our finding that RGS4 attenuates the coupling of 5-HT1B receptors to activation of ERK and Akt suggests a possible role for RGS4 in modulating the activity of serotonergic pathways. That RGS4 is expressed primarily in brain (Druey et al., 1996) makes this an attractive hypothesis. If future studies bear this out, abnormal regulation of the expression of RGS4 could represent a contributing factor in the etiology of depression, anxiety, and schizophrenia—disorders treated with medications that modulate serotonergic pathways. Interestingly, stress is known to exacerbate these disorders, and RGS4 has been shown to be regulated in a region-specific manner by both stress and glucocorticoids (Ni et al., 1999). Future clinical and animal studies addressing the role of RGS4 in psychiatric pathology may therefore prove productive. In fact, there is evidence that RGS2 and RGS3 may mediate some aspect of amphetamine-induced tolerance (Burchett et al., 1999).

Acknowledgements

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. RESULTS
  5. 5-HT and sumatriptan stimulate phosphorylation of ERK in transfected BE(2)-C neuroblastoma cells
  6. RGS4 inhibits activation of ERK and Akt
  7. DISCUSSION
  8. Acknowledgements

These studies were supported by an NARSAD Young Investigator Award, a grant from the Foundation of the University of Medicine and Dentistry of New Jersey, and by NIMH grant MH60100 to D.S.C. We thank Dr. John Kehrl (NIAID, NIH) for kindly providing the cDNA for human RGS4.

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