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J Biol Chem, Vol. 275, Issue 10, 7021-7029, March 10, 2000

Dissecting G Protein-coupled Receptor Signaling Pathways with Membrane-permeable Blocking Peptides
ENDOGENOUS 5-HT_2C RECEPTORS IN CHOROID PLEXUS EPITHELIAL CELLS^*

Mike Chang, Lianshan Zhang^§¶, James P. Tam^§, and Elaine Sanders-Bush

From the  Department of Pharmacology and Center for Molecular Neuroscience and the ^§ Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

To determine the intracellular signaling mechanism of the 5-HT_2C receptor endogenously expressed in choroid plexus epithelial cells, we implemented a strategy of targeted disruption of protein-protein interactions. This strategy entails the delivery of conjugated membrane-permeable peptides that disrupt domain interaction at specific steps in the signaling cascade. As proof of concept, two peptides targeted against receptor-G protein interaction domains were examined. Only G_qCT, which targets the receptor-G_q protein interacting domain, disrupted 5-HT_2C receptor-mediated phosphatidylinositide hydrolysis. G_sCT, targeting the receptor-G_s protein, disrupted 2 adrenergic receptor-mediated activation of cAMP but not 5-HT_2C receptor-mediated phosphatidylinositide hydrolysis. The peptide MPS-PLC1M, mimicking the domain of phospholipase C1 (PLC1) interacting with active G_q, also blocked 5-HT_2C receptor activation. In contrast, peptides PLC2M and Phos that bind to and sequester free G subunits were ineffective at blocking 5-HT_2C receptor-mediated phosphoinositol turnover. However, both peptides disrupted G-mediated _2A adrenergic receptor activation of mitogen-activated protein kinase. These results provide the first direct demonstration that active G_q subunits mediate endogenous 5-HT_2C receptor activation of PLC and that G subunits released from G_q heterotrimeric proteins are not involved. Comparable results were obtained with metabotropic glutamate receptor 5 expressed in astrocytes. Thus, conjugated, membrane-permeable peptides are effective tools for the dissection of intracellular signals.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The 5-HT₂ receptor family consists of three members, 5-HT_2A, 5-HT_2B, and 5-HT_2C. All three receptors belong to the G protein-coupled serpentine receptor superfamily. Their pharmacological profiles are very similar, leading to difficulty in defining their functional roles. 5-HT₂ receptors have been implicated in behaviors such as sleep, feeding, aggression, pain, and anxiety and are thought to play a role in a number of central nervous system disorders including affective disease, schizophrenia, and epilepsy (1). In addition, 5-HT₂ receptors may play a major role in mediating the actions of hallucinogenic drugs (2) as well as antipsychotic drugs (3, 4). Mice expressing nonfunctional 5-HT_2C receptors exhibit epileptic and obese phenotypes (5, 6), suggesting that these receptors play a crucial role in moderating central nervous system function.

Expression of the 5-HT_2C receptor is exceptionally high in the choroid plexus (7, 8), where it plays a role in the regulation of production and composition of cerebrospinal fluid (9-11). Initial studies of 5-HT_2C receptor signaling showed that these receptors activate the downstream intracellular effector, phospholipase C (PLC)¹ resulting in the hydrolysis of phosphatidylinositol-4,5-bisphosphate into inositol-1,4,5-triphosphate and diacylglycerol (12). In addition, activation of the 5-HT_2C receptor has been observed to release arachidonic acid (13), increase cyclic GMP (14), and regulate potassium channels and Ca²⁺-activated chloride channels (15-18). These observations suggest that 5-HT_2C receptor activation results in the induction of multiple signaling pathways. However, it is unclear how each individual intracellular signaling pathway contributes to modulation of the cell as a whole. In this paper, we employed a novel strategy to dissect intracellular signaling pathways, which combines a newly developed peptide synthesis technology with the application of targeted disruption of protein-protein interactions. This strategy is applied to examine PI hydrolysis signaling, a well defined pathway associated with 5-HT_2C receptor activation.

The current model of 5-HT_2C receptor signaling suggests that G_q/11 heterotrimers are the immediate G protein mediators of receptor signaling based on two indirect observations. First, activation of PLC predominantly occurs through the G_q family. Second, 5-HT_2C receptor-mediated PI hydrolysis is largely pertussis toxin (PTX) -insensitive, which suggests that G_i/o heterotrimers, which activate PLC via their subunits, are not involved (19). However, previous studies that directly examined the identity of the G protein-mediating 5-HT_2C receptor signaling were all conducted in artificial systems (20-22) where the receptors may have promiscuous interactions with various heterotrimeric G proteins. To address this question in a native environment, we examined the G protein mediator of 5-HT_2C receptor signaling in primary cultures of choroid plexus epithelial cells and further assessed the role of G and G subunits.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Most peptides used were synthesized in our laboratory; G_sCT, G_qCT, G_oCT, and PLC2M were also synthesized by Genosys (The Woodlands, Texas). Antibodies against PLC isozymes and G_i/o/z were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). G_q antibodies were a kind gift of Dr. Tom Martin (University of Wisconsin-Madison, WI).

Peptide Design-- The sequence of the various peptides and their proposed targets are presented in Table I. The membrane-permeable sequence (MPS) peptide was based on a hydrophobic membrane-permeable sequence described previously (23-25). Lysine and serine residues, as a pseudo-dipeptide, were added at the carboxyl terminus to serve as a linker and a masked aldehyde to facilitate conjugation. The lysine was attached to the carboxyl terminus of the MPS sequence by its primary amine, and then serine was attached at the side chain amine (Fig. 1).

Phospholipase C1-mimicking peptide (PLC1M) was derived from amino acids 1053-1084 of the PLC1 enzyme. This design stems from the observation that loss of the last 10 kDa from the carboxyl terminus of PLC1 results in the loss of interaction with active G_q (26). Additional work, using a series of deletion mutants, defined region 1030-1142 as the domain required for interaction with G subunits (27). A specific segment within this region (amino acids 1053-1084) was observed to dose dependently inhibit GTPS-dependent activation of PLC using either purified PLC1 or a crude membrane assay.

Phospholipase C2 mimicking peptide (PLC2M) is based on residues 564-583. The domain of PLC2 interacting with G subunits has been determined utilizing a peptide fragment strategy. Two twenty amino acid segments of PLC2 (564-583 and 574-593) were defined as the domains binding to G (28). The segment with the optimal interaction with G subunits was observed to span amino acids 564-583. Synthetic peptide of this region exhibited specific binding to G subunits as well as specific inhibition of G- effector interactions.

Phosducin-like peptide (Phos) was derived from carboxyl-terminal residues 168-195 of phosducin-like protein (PhLP). PhLP isolated from rat brain (29) was determined to be an ubiquitous inhibitor of G-mediated signaling. The region of PhLP conferring interactions with G subunits was delineated to be in the carboxyl-terminal domain (30). Expression of glutathione S-transferase fusion proteins including residues 168-195 of PhLP had inhibitory effects on G_o GTPase activity, demonstrating the ability to bind G. In addition, carboxyl-terminal peptides of PhLP (including amino acids 168-195) inhibited G-enhanced rhodopsin phosphorylation by ARK.

G carboxyl-terminal peptides (G_qCT and G_sCT) were designed based on the last 10 amino acids of the carboxyl terminus, a region of G subunits that has been recognized as a site of interaction between G proteins and receptors (31, 32). Peptides corresponding to the last 11 residues of G_i/o/s proteins have been demonstrated to be effective in the specific inhibition of receptor-G protein interaction (33). For the purpose of this paper, the corresponding carboxyl-terminal peptides were derived from the following residues: G_s (amino acids 385-394) and G_q/11 (amino acids 350-359).

Peptide Synthesis-- Peptides were synthesized using solid-phase chemistry on a CS-Bio Peptide Synthesizer (fluroenylmethyloxycarbonyl chemistry (Fmoc)) or an ABI 430A Peptide Synthesizer (tert-butyoxycarbonyl chemistry). Peptides derived from Fmoc synthesis were cleaved from the resin using trifluoroacetic acid/thioanisole/triisopropylsilane/1,2-ethanedithiol/water/phenol (81.5:5:1:2.5:5:5 v/v/v/v/v/v/weight) at 20 °C for 4 h. Peptides synthesized by tert-butyoxycarbonyl chemistry were cleaved from the resin using HF/anisole (9:1, v/v) at 4 °C for 1 h. Crude peptides were purified by preparative reverse phase-high performance liquid chromatography (HPLC) using a C-18 column (Vydac, Holland, MI) and a linear acetonitrile gradient. The gradient was established using two buffers (A: H₂O, 0.05% trifluoroacetic acid; B: 80% acetonitrile in H₂O, 0.039% trifluoroacetic acid) ranging from 10-100% buffer B over a 25 min period at a flow rate of 10 ml/min. Purified peptides was verified using matrix-assisted laser desorption ionization mass spectrometry (Voyager Elite, Perseptive Biosystems, Framingham, MA). Sample purity was assessed using analytical HPLC with an analytical C-18 column (Vydac) under a 25-min linear gradient of 10-90% buffer B at a flow rate of 1 ml/min.

For chemical conjugation of the MPS and the cargo peptide, two requirements of these peptides were necessary. First, cargo peptide contained an amino-terminal cysteine residue (Fig. 1). Second, the carboxyl-terminal domain of the signal sequence (MPS) contained a masked aldehyde moiety. For this purpose, a pseudo-dipeptide Lys(Ser) was attached to the carboxyl-terminal of the MPS sequence (Fig. 1). The peptide is synthesized with a lysine carboxyl terminus, and then a serine residue was chemically added to the -amine moeity of the lysine residue. To make the MPS peptide chemically reactive, NaIO₄ treatment was used to oxidize the serine residue to an aldehyde moiety. The oxidized MPS was purified by preparative reverse phase-HPLC and the aldehyde product was again verified using matrix-assisted laser desorption ionization-mass spectrometry. Conjugation of the MPS with cargo peptide was accomplished by dissolving both peptides in 0.5 ml of dimethylformamide and by adding 0.5 ml of 0.2 M sodium acetate buffer, pH 5.4, to the mixture. After agitation for 16-20 h, the conjugate, as a thiazolidine bond formed between the amino-terminal cysteine of the cargo peptide and the MPS aldehyde, was purified using preparative reverse phase-HPLC and the product characterized by matrix-assisted laser desorption ionization-mass spectrometry (Fig. 2). Several peptides had limited solubility (Table I), and these were used in our experiments at the maximum soluble concentration.

View larger version (21K): [in this window] [in a new window]   Fig. 1.   A representative scheme for functionalizing MPS and subsequent conjugation with the cargo peptide PLC2M to yield the membrane-permeable product MPS-PLC2M.

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Example of sample preparation. A, products from the conjugation of PLC1M with MPS were separated by preparative HPLC. B, matrix-assisted laser desorption ionization-mass spectrometry was performed on the fractions collected in A. Results shown are from the fraction in A denoted by an asterisk. C, the fraction identified to be the MPS-PLC1M peptide (denoted by * in A) is checked with analytical HPLC to verify purity.

Cell Culture-- Primary cultures of choroid plexus epithelial (CPE) cells were prepared as described previously (34). Briefly, choroid plexi, removed from 20-day-old Harlan Sprague-Dawley rats, were treated with Pronase (333 µg/ml) containing DNase (7.5 µg/ml) in Hanks' balanced salt solution (HBSS) (Ca²⁺/Mg²⁺) for 10 min at 37 °C to dissociate cells. Dissociated cells were resuspended in 10% dialyzed calf serum or 10% fetal bovine serum in Dulbecco's modified Eagle's medium (DMEM) + D-Val (Life Technologies, Inc.). Cells were plated in 48-well plates for 3-5 days.

Primary cultures of astrocytes were prepared as described previously (35). Brains were removed from postnatal day 2-5 rats, the meninges were carefully removed, and the cerebral cortex was dissected using visual landmarks. Cells were dissociated in horse serum by mechanical trituration, collected by centrifugation, and resuspended in 1 ml of fetal bovine serum. Dissociated cells were cultured in DMEM containing 10% fetal bovine serum in 75-cm² culture flasks coated with poly-D-lysine. After 7 days, cells were shaken in an orbital shaker at 37 °C overnight to remove nonastrocytic cells. The cells were then replated in 24- or 48-well plates for functional assays.

Human embryonic kidney-293 cells stably expressing the rat adrenergic _2A receptor (HEK-_2A) were a generous gift from Dr. Lee Limbird (Vanderbilt University). Cells were cultured in DMEM containing 10% fetal bovine serum (100 units penicillin/ml, 100 mg of streptomycin/ml) under 5% CO₂ at 37 °C.

Phosphoinositide (PI) Hydrolysis Assay-- CPE cells plated in 48-well plates were incubated for 16-20 h with 2 µCi/ml myo-[³H]inositol (20-25 Ci/mmol, NEN Life Science Products) in serum-free, inositol-free DMEM to label phospholipid pools. Labeling medium was aspirated, and the cells washed twice with HBSS containing 1 mM Ca²⁺ and 1 mM Mg²⁺. Cells were treated with peptides solubilized in HBSS (+Ca²⁺/Mg²⁺) at 37 °C for 30 min. Subsequently, 10 mM lithium chloride and 10 µM pargyline were added to the cells for 10 min prior to agonist activation for 30 min at 37 °C. The reaction was stopped by aspirating the solution and fixing with 25 µl of methanol/well. [³H] Inositol monophosphates were isolated as described previously (36).

HEK-_2A cells were plated in 24-well plates for PI hydrolysis assay and assayed as described above for CPE cells, except pargyline was not added. Cells were incubated with thrombin receptor-activating peptide for 30 min.

Primary cultured astrocytes were plated in 48-well plates and assayed for PI hydrolysis as described for HEK-_2A cells above. Metabotropic glutamate receptors were activated with 100 µM (R,S)-3,5-dihydrophenylglycine.

ADP-Ribosylation Assay-- ADP ribosylation was performed as described previously (37). Briefly, CPE cells were plated in 100-mm plates and cultured for 3-4 days in DMEM. Cells were incubated with 500 ng/ml PTX for 16 h in the absence of serum. Membranes, suspended in 50 mM Tris, pH 8.0, containing 5 mM MgCl₂ and 1 mM EDTA buffer, were subjected to ADP ribosylation at 30 °C for 1 h in a 50-µl reaction containing 100 µg of membrane protein, 1 mM ATP, 20 mM arginine, 20 mM thymidine, 100 mM NaCl, 0.25% Lubrol, 5 mM dithiothreitol, 1 µg/ml PTX, and 2.5 µCi of [³²P]nicotinamide adenine dinucleotide. The reaction was terminated by adding 1.2 ml of 20 mM HEPES, pH 8.0. Membranes were pelleted and separated by polyacrylamide gel electrophoresis, and ribosylated proteins were visualized using a Molecular Dynamics PhosphorImager system.

Western Blot-- Protein extraction from cells and separation on acrylamide gel was done as described previously (38). The molecular masses were determined using Sigma high molecular weight markers.

For the MAP kinase assay, HEK-_2A cells in 24-well plates, containing serum-free medium, were treated with appropriate peptides for 30 min at 37 °C prior to activation with 100 µM epinephrine for 2 min. Supernatant was aspirated, and cells were solubilized with 1X sample buffer (62.5 mM Tris, 2% SDS, pH 6.8, containing 10% (v/v) glycerol). Proteins were separated in 12% SDS-polyacrylamide gels. Active MAP kinase was detected using the Promega anti-phospho-MAP kinase antibody at 1:1000 dilution with an overnight incubation at 4 °C. Total MAP kinase was detected using NEB total MAP kinase antibodies at 1:500 dilution with an overnight incubation at 4 °C. Secondary peroxidase-conjugated donkey anti-rabbit antibodies were used at 1:2000 dilution with incubation at room temperature for 30 min. Immunoreactive protein bands were visualized by treatment of blots with NEB ECL reagent and subsequent exposure to Kodak Biomax film.

For the detection of PLC isozymes and G protein isoforms, CPE were removed from Spargue-Dawley rats and solubilized in Tris buffer containing 10 mM CHAPS (39). The CHAPS-soluble fraction was fractionated in 7.5% SDS-polyacrylamide gels. Detection of PLC isozyme was performed using PLC isozyme-specific antibodies as per the manufacturer recommendations (Santa Cruz Biotechnology). G_q/11 detection was achieved using affinity purified polyclonal antibodies provided by Dr. Tom Martin.

cAMP Assay-- Primary cultured astrocytes plated in 48-well plates were labeled for 16-20 h with 2 µCi/ml [³H]adenosine in serum-free DMEM. Peptides solubilized in HBSS (+Ca²⁺/Mg²⁺) were added to cells and incubated at 37 °C for 30 min prior to initiation of the assay. Agonist was added and the incubation continued for 30 min at 4 °C in presence of 1 mM isobutylmethylxanthine. The reaction was stopped with 10% trichloroacetic acid containing 2 mM ATP and 2 mM cAMP. Accumulated [³H]cAMP was separated on alumina columns as described previously (40).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

PLC Signaling Machinery in CPE Cells-- Immunoblots were utilized to evaluate signaling molecules including the PLC isozymes, which have differential specificity for activation either by G_q or G subunits. Three isoforms of PLC were detected in the choroid plexus (Fig. 3A). Using anti-PLC1 antibodies two bands at approximately 140 and 100 kDa were detected; the latter is an expected degradation product of PLC1 (Santa Cruz antibody protocol). PLC2 and PLC3 were detected with apparent masses of approximately 100 and 140 kDa, respectively. However, PLC4 was not present at a detectable level. We also probed CPE extracts with anti-G_i/o as well as anti-G_q antibodies to verify the potential for G- and G_q-mediated signaling (Fig. 3B). These results demonstrated that G_i/o as well as G_q are expressed in CPE.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Identification of potential signaling machinery in CPE cells. Western analysis of (A) PLC isozymes and (B) G protein expression in CPEs. Extracts of rat choroid plexi were separated by 7.5% and 12.5% SDS-polyacrylamide gel electrophoresis for PLC isozymes and G proteins, respectively. C, effects of PTX on 5-HT2C receptor-mediated PI hydrolysis stimulated by serotonin in CPE cells. Graph is representative of six independent experiments; each point represents an average of triplicate determinations. D, ADP-ribosylation assay of CPE cells. Lane 1, control-untreated CPE cells. Lane 2, CPE cells were pretreated overnight using 500 ng/ml PTX.

G Protein Mediators of Endogenous 5-HT_2C Receptor Signal in CPE Cells-- Given that both PLC2 and G_i/o exist in CPE cells, the possibility that activation of PLC in CPE cells is mediated by G subunits released from G_i/o heterotrimers was examined using an indirect method based on PTX sensitivity. The 5-HT_2C receptor PI hydrolysis response in CPE cells is predominantly insensitive to PTX (Fig. 3C), which ADP ribosylates G_i/o heterotrimers leading to their inactivation. As a control for PTX activity, CPE cells were pretreated overnight with PTX and then subjected to an in vitro ADP-ribosylation assay. Cells treated overnight with PTX were not ADP-ribosylated by PTX added in vitro (Fig. 3D), whereas non-PTX-treated cells were ADP-ribosylated. These results suggest that G_i/o heterotrimers are predominantly not involved in the endogenous 5-HT_2C receptor PI signal.

To determine directly the heterotrimeric G protein mediator of endogenous 5-HT_2C receptor signaling, we introduced the membrane-permeable MPS-G_qCT peptide to cultured CPE cells. This peptide is designed to disrupt receptor coupling to G_q/11 heterotrimeric protein (Fig. 4A). MPS-G_qCT, at 5 µM, was able to block PI hydrolysis resulting from treatment of CPE cells with 100 nM serotonin (Fig. 4B). A peptide designed to disrupt receptor coupling to G_s heterotrimer (MPS-G_sCT) was ineffective in perturbing endogenous 5-HT_2C receptor-mediated PI hydrolysis. Signal sequence (MPS) peptide alone was also ineffective demonstrating that the domain conferring permeability does not attenuate the observed PI signal. Additionally, membrane-impermeable, nonconjugated G_qCT peptide was unable to disrupt 5-HT_2C receptor signaling, which is consistent with the idea that without membrane permeability the peptide is not functional. This is also an indication that the effect of the G_qCT peptide is not due to general toxicity during the course of the experiment. The observation that neither MPS nor the G_qCT peptide alone was an effective inhibitor of receptor-G_q coupling validates the MPS-importing strategy.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Effect of Gq carboxyl-terminal (MPS-GqCT) Peptide. Primary cultured CPE cells were stimulated with 100 nM 5-HT and assayed for PI hydrolysis in the absence or presence of blocking peptides. Peptides were applied at the following concentrations: 5 µM MPS-GqCT, 50 µM MPS-GsCT, 300 µM MPS, and 100 µM GqCT. A, schematic illustrating the designated point of blockade by MPS-GqCT peptide. MPS-GqCT inhibits PI hydrolysis in cultured CPE cells as compared with the untreated control. Signal sequence and nonpermeable GqCT alone have no significant effect. Peptides designed to block receptor-Gs (MPS-GsCT) interaction were also ineffective. Graph illustrates average results from three independent experiments with each experiment performed in triplicate. B, schematic illustrating 2 adrenergic receptor activation of cAMP signaling cascade and the point of disruption by the peptide MPS-GsCT. 2 adrenergic receptor signal is antagonized by 10 µM propranolol. MPS-GsCT peptide blocks 2 adrenergic receptor mediated by Gs heterotrimers in primary cultured astrocytes. The graph illustrates data from two independent experiments with triplicate determinations. Individual responses were normalized to the average control value corresponding to that particular experiment and are plotted as mean ± S.E. Statistical analyses were performed using one-way analysis of variance (ANOVA) with a nonparametric TUKEY test.

As an additional proof-of-concept for the use of membrane-permeable peptides designed from the carboxyl terminus of G subunits, we examined the functional effect of MPS-G_sCT on endogenous 2 adrenergic receptor signaling in cultured astrocytes. As seen in Fig. 4B, MPS-G_sCT is effective in blocking, to almost basal levels, 2 adrenergic receptor-mediated activation of adenylate cyclase. In contrast, the peptide MPS-G_qCT was not functionally disruptive in this system. Furthermore, at the same concentration, MPS-G_sCT was ineffective in blocking 5-HT_2C receptor-mediated PI hydrolysis in CPE (Fig. 4B). These results contribute collectively to demonstrate peptide specificity and their lack of toxicity.

Demonstration of Function and Specificity of Membrane-permeable Peptides Targeting G Protein Subunits Using HEK-_2A Cells-- To assess directly whether the activation of PLC is mediated by active G_q subunits or free G subunits, we designed the following peptides: MPS-PLC1M targeted against the disruption of G_q-PLC interaction; and MPS-PLC2M and MPS-Phos, both designed to bind and sequester free G subunits thereby preventing subsequent activation of PLC. Because CPE cells lack the appropriate receptor-signaling pathways to determine peptide function, specificity and toxicity, we exploited HEK cells stably expressing _2A-adrenergic receptors (HEK-_2A) for this purpose. HEK-_2A cells endogenously express thrombin receptors as well as transfected _2A adrenergic receptors, which signal through the G_i heterotrimeric proteins leading to G-mediated activation of MAP kinase (41); this serves as a suitable model to evaluate the effects of MPS-PLC2M and MPS-Phos peptides on free G-mediated signaling (Fig. 5A). Thrombin receptors have been observed to activate a PTX-insensitive PI signal postulated to be through the G_q heterotrimeric proteins (Fig. 5B). These two receptor systems provide divergent signaling pathways to test the aforementioned functional peptides.

View larger version (27K): [in this window] [in a new window]   Fig. 5.   Effects and specificity of peptides on 2A and thrombin receptors in HEK-293 cells. A, schematic of 2A adrenergic receptor signaling in HEK-2A cells illustrating G-mediated activation of MAP kinase and designated point of blockade by sequestering peptides. HEK-2A cells treated with 100 µM epinephrine in the above indicated conditions were analyzed for the activation of MAP kinase. Concentrations of peptides used were as follows: 6.7 µM MPS-PLC2M, 100 µM PLC2M, 34 µM MPS-Phos, 100 µM Phos, 100 µM MPS-PLC1M, and 100 µM PLC1M. For the PTX lane, cells were treated overnight with 500 ng/ml of PTX in serum-free medium. B, the schematic of thrombin receptor signaling in HEK-2A cells illustrates point of blockade by MPS-PLC1M peptides. PI hydrolysis assay using 100 µM thrombin receptor-activating peptide in HEK-2A cells in the presence of 300 µM MPS-PLC1M or 100 µM MPS-PLC2M.

Using antibodies directed against the phosphorylated, active form of MAP kinase or against a region of MAP kinase away from the phosphorylation site, we could discern active versus inactive forms of MAP kinase and visualize total MAP kinase. Activation _2A-adrenergic receptors in HEK-_2A cells with 100 µM epinephrine (Fig. 5A, control lanes) results in an increase in the level of active MAP kinase as compared with the basal levels. Total MAP kinase labeling of the same blot indicates that the levels of MAP kinase are equal or even higher in basal versus control lanes. Pretreatment of cells with MPS-PLC2M and MPS-Phos peptides disrupted activation of MAP kinase by 100 µM epinephrine through the _2A receptors (Fig. 5A). However, the nonconjugated, membrane impermeant forms of Phos or PLC2M did not inhibit MAP kinase activation. MPS alone had no functional effect on MAP kinase activation. In addition, PTX pretreatment abrogated subsequent MAP kinase activation confirming that MAP kinase activation is through G subunits released from the G_i/o protein. When tested in the thrombin receptor PI hydrolysis pathway, the MPS-Phos and MPS-PLC2M peptides were shown to have no inhibitory effects (Fig. 5B) demonstrating the specificity and lack of toxicity of these peptides. These results validate the interpretation that MPS-Phos and MPS-PLC2M are functional and specific to target the sequestration and disruption of signaling by free G subunits.

Pretreatment of HEK-_2A cells with 100 µM MPS-PLC1M produced no disruptive effect on _2A receptor-mediated activation of MAP kinase, indicating that this peptide is apparently not toxic to the cells and does not nonspecifically disrupt G-mediated signaling (Fig. 5A). However, signaling of endogenous thrombin receptors in HEK-_2A cells was disrupted by MPS-PLC1M (Fig. 5B), demonstrating that in the same cells PI hydrolysis blockade can be achieved.

Role of Active G_q and Free G Subunits in Mediating Endogenous 5-HT_2C Receptor Signaling-- To examine the direct contribution of active G_q subunits mediating 5-HT_2C receptor signals, MPS-PLC1M, designed to disrupt the G_q-PLC interaction, was applied to cultured CPE cells. This peptide blocked, down to basal levels, serotonin-induced PI hydrolysis (Fig. 6A). However, in the same assay, the G-sequestering peptides, MPS-PLC2M and MPS-Phos, at levels that blocked MAP kinase activation, did not significantly inhibit 5-HT_2C receptor-mediated PI signaling. Because evidence suggests that the 5-HT_2C receptor in CPE is the sole mediator of serotonin stimulation (12), these results indicate that active G_q subunits are involved in 5-HT_2C receptor signaling. The effect of MPS-PLC1M was dose-dependent, as seen in Fig. 6B, with an IC₅₀ of 55 µM. Additionally, concentration response studies showed that MPS-PLC1M decreased the maximal signal produced by 5-HT without altering the EC₅₀ (Fig. 6C). Although this type of effect may be attributed to general toxicity, results observed in earlier experiments do not support this conclusion.

View larger version (15K): [in this window] [in a new window]   Fig. 6.   Effect of peptides on 5-HT2C receptor signaling in CPE cells. A, PI hydrolysis assay of CPE cells pretreated for 30 min with various peptides before 30 min activation with 100 nM 5-HT. Concentrations of peptides used are those previously demonstrated to be maximally inhibiting: 100 µM MPS-PLC1M, 40 µM MPS-Phos, and 10 µM MPS-PLC2M. Statistical analysis was a one-way ANOVA using a nonparametric TUKEY test (n = 5-9). B, dose response of MPS-PLC1M peptide on 5-HT2C receptor PI signaling in CPE cells (IC50 = 55 µM). Graph is representative of three independent experiments. C, effects of MPS-PLC1M peptide on 5-HT dose response in CPE cells. Cells were untreated (solid line) or treated with 60 µM peptide (dashed line). EC50 values for Control and MPS-PLC1M were 124 and 67 nM, respectively. Emax for control and MPS-PLC1M is 1602 and 990 cpm, respectively. The values plotted are means of triplicate determinations and are representative of three independent experiments each point with three replicates.

Role of Membrane-permeable Peptides in Endogenous Receptors Expressed in Primary Cultures Astrocytes-- To further demonstrate that the effects of these peptides are not receptor- or cell type-specific, we analyzed their effects on metabotropic glutamate receptor (mGluR) signaling in primary cultures of astrocytes. Type I mGluR, consisting of mGluR1 and mGluR5, activate PLC (42). Overexpression of G_q augments PI hydrolysis of mGluR1a transfected into HEK-293 cells, suggesting that this receptor couples through G_q heterotrimeric protein (43). Furthermore, in astrocytes, the activation of mGluR5 signaling appears to be PTX-insensitive (44-46). However, the exact identity of the G protein mediating endogenous mGluR5 signal remains unclear. We addressed this question by analyzing primary cultures of astrocytes, which express mGluR5 but not mGluR1 (47). (R,S)-3,5-Dihydrophenylglycine, a mGluR1- and mGluR5-specific agonist, was used to activate mGluR5 in these cells. In the presence of MPS-PLC1M (300 µM), mGluR5-mediated PI hydrolysis was significantly inhibited. In contrast, treatment with MPS-PLC2M (100 µM) or MPS-Phos (100 µM) peptides did not decrease signaling relative to controls (Fig. 7). These results suggest that G subunits are not involved in mediating PLC activation of mGluR5, affirming the observed lack of PTX sensitivity and presumed lack of involvement of G_i/o heterotrimers. These results indicate that signaling of mGluR5 receptors in astrocytes is mediated by active G_q subunits, which is consistent with the current consensus of the involvement of G_q heterotrimers.

View larger version (14K): [in this window] [in a new window]   Fig. 7.   Effects and specificity of peptides on mGluR5 in primary astrocytes. mGluR5-mediated PI hydrolysis was measured as described under "Experimental Procedures" subsequent to a 30-min pretreatment with the indicated peptides. Concentration of peptides is as follows: 300 µM MPS-PLC1M, 100 µM MPS-PLC2M, 100 µM MPS-GsCT, 100 µM MPS-Phos, 100 µM MPS. Higher solubility of MPS-PLC2M, MPS-Phos, and MPS-GsCT was achieved by solubilizing first in Me2SO then in HBSS buffer containing 100 µM bovine serum albumin. These results are the average of three independent experiments in triplicate. Significance determined using one-way ANOVA with TUKEY nonparametric test. DHPG, (R,S)-3,5-dihydrophenylglycine.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Most studies of receptor function involve the use of agonists and antagonists to modulate receptor activity in an attempt to understand how these receptors regulate cell physiology. Many G protein-coupled receptors activate multiple independent intracellular signaling cascades, and it is equally important to understand how these various signals contribute to the physiological actions of drugs and to whole cell function. Current tools available for studying multicomponent intracellular signaling pathways are limited in comparison to the plethora of available receptor ligands. Contemporary methods for assessing the contribution of specific signaling molecules generally involve molecular strategies such as antisense oligonucleotides to knockdown expression of a specific protein or transfection experiments to overexpress the protein of interest. However, these approaches disrupt the stoichiometry of the protein within the signal transduction system and are not always applicable to native cell systems. For signaling proteins with enzymatic activity, chemical inhibitors or toxins may be available to provide a more temporally controlled dissection of signaling. The drawbacks of these compounds are that many of them have specificity problems and may be toxic. Currently, there is no systematic broadly applicable approach to the dissection of intracellular signaling events in native cells or tissues.

In this paper, we describe a strategy that involves the use of functional peptides coupled to membrane-permeable peptides for dissecting intracellular signaling pathways. Signaling in many cases requires an activated protein to contact directly with its immediate downstream mediator. Thus, it is possible to disrupt protein-protein interactions of specific coupling domains by introducing into cells only the binding domain. Specificity is intrinsic to the amino acids encoding the peptide as well as their inherent binding properties. The blocking peptide strategy to disrupt protein-protein interactions has been applied sporadically by other investigators in signaling studies. Hamm and Rarick (31) as well as Taylor and Neubig (32) reviewed the use of peptides as probes for G protein signal transduction. Hamm and Rarick (31) analyzed the use of peptides to study receptor-G protein interaction and G protein-effector interaction and pointed out that, with a blocking peptide strategy, signaling pathways can be disrupted at any level, provided that protein-protein interactions exist. However, a limitation of this strategy lies in the intracellular import of peptides. Because most peptides do not readily penetrate the cell membrane, their use in disrupting intracellular signaling has been primarily limited to in vitro analyses, a major impediment to general use of this methodology. To alleviate this problem, we have adopted a noninvasive approach to introduce peptides into cells, the addition of a MPS to the blocking peptides. The feasibility of this approach has been demonstrated in experiments to prevent the inducible nuclear import of transcription factors in human monocytic, endothelial, and T lymphocyte cell lines (23, 25). We have also implemented a recently developed approach of modular peptide synthesis, which involves separate synthesis of the MPS and functional peptides with subsequent chemical ligation of the two peptides under mild aqueous conditions (24). The biological activity of the resulting modular peptides has been extensively documented by comparing modular peptides with those synthesized conventionally (23, 25, 48, 49). This modular approach not only provides versatility in preparing multiple MPS-coupled functional peptides for structure and function studies, but it also serves to increase yield and reduce the cost of synthesis.

The conjugation of MPS with blocking peptide has great potential as a versatile tool for studying intracellular signaling. This approach was validated in recombinant cell lines and then applied to cultured CPE cells for direct analysis of endogenous 5-HT_2C receptor signaling at the receptor- G protein level and at the G protein-effector level. A decapeptide mimicking the carboxyl terminus of G_q subunits (MPS-G_qCT), a domain conferring specific interaction with receptors, profoundly disrupted 5-HT_2C receptor-mediated PI hydrolysis in CPE cells. A peptide targeting the disruption of receptor-G_s interaction (MPS-G_sCT) was ineffective, although the same G_s peptide was able to block 2-AR-mediated cAMP formation in astrocytes. The MPS domain alone was inactive, suggesting that the carrier peptide made no significant contribution to the blockade of any signal cascades tested. Nonconjugated peptides also did not have a significant effect, consistent with the premise that, without attachment of the MPS sequences, the blocking peptides are unable to penetrate the plasma membrane. These results indicate that the G_q heterotrimer mediates 5-HT_2C receptor signaling in choroid plexus, consistent with previous observations in reconstituted systems (20), and also demonstrate the specificity of the G protein-targeted approach.

It is well documented that G subunits released from G_i/o heterotrimers have the ability to activate effectors, including PLC2 (50-53). More recently, effector activation by G released from G_s heterotrimers has been reported (54). The possibility also exists for signaling mediated by G subunits released from G_q heterotrimers. For example, studies in Xenopus oocytyes have shown that the M3 muscarinic receptor, which is G_q/11-coupled, activates PLC mainly through G subunits (55). However, transfected (COS) cells expressing the G_q/11-coupled parathyroid hormone or calcitonin receptor failed to show augmented PI hydrolysis when cotransfected with G subunits (56). Studies such as these in artificial systems may not represent the in vivo situation, therefore, we developed specific membrane-permeable peptides to evaluate the contribution of G protein subunits to the PI hydrolysis signal mediated by endogenous 5-HT_2C receptors. To elucidate the function and specificity of the newly developed conjugated G_q and G peptides, we used an HEK-_2A stable cell line expressing the cloned _2A adrenergic receptor as well as endogenous thrombin receptors. _2A adrenergic receptor activation of MAP kinase is mediated by G subunits released by G_i/o heterotrimer (41), whereas thrombin receptor signaling, in these cells, has been observed to be mediated by G_q protein (57). The addition of MPS-PLC1M peptide into the HEK-_2A cell line blocked the subsequent activation of PLC by thrombin receptor-activating peptide, consistent with the expected role for G_q. The specificity of MPS-PLC1M peptide was confirmed, because it had no effect on _2A-adrenergic receptor-mediated activation of MAP kinase, a G-dependent response. In contrast, G-sequestering peptides, MPS-Phos and MPS-PLC2M, were both effective in disrupting _2A-adrenergic receptor activation of MAP kinase. The consistent results of both G-sequestering peptides, which have different size and amino acid composition, provide converging support for their functionality and specificity. When the G-sequestering peptides were tested on thrombin receptors in HEK-_2A cells, they failed to block receptor-activated PI hydrolysis, providing evidence for the specificity of these constructs.

The current studies of 5-HT_2C receptors in CPE cells suggest that G does not contribute to the G_q/11 heterotrimer-mediated PI hydrolysis signal in this endogenous receptor system. This conclusion is based on results obtained with the specific peptides that block active G_q and free G subunits released from activated G_q/11 heterotrimers. MPS-PLC1M blocked 5-HT-mediated PI hydrolysis in CPE cells, whereas both G-sequestering peptides, MPS-Phos and MPS-PLC2M, had no effect at concentrations that eliminated _2A-adrenergic receptor-mediated MAP kinase activation. Analysis of mGluR5 in astrocytes, another putative endogenously expressed G_q/11-coupled receptor, using the same membrane-permeable peptides showed similar results. MPS-PLC1M markedly decreased mGluR5-mediated PI hydrolysis, whereas both MPS-PLC2M and MPS-Phos did not attenuate signaling in this pathway. The consistency of these results obtained with two different endogenous receptors in different native environments suggests that, unlike in artificial systems (55), endogenous G subunits released from G_q/11 heterotrimers may not contribute to the activation of PLC in native systems. However, these results do not rule out a role of G subunits released from G_q/11 heterotrimers in other receptor signaling cascades. For example, 5-HT_2C receptors also induce the release of arachidonic acid via phospholipase A₂ activation (58, 59) as well as an increase in the intracellular level of cGMP (14). 5-HT_2C receptor activation also modulates various potassium channels (17, 18). Currently, it is not known which intracellular pathways are involved in the activation of these various effectors. The availability of cell-permeant peptides that block at the level of receptor-G protein and G protein-effector should allow a more precise dissection of these various downstream signals.

In conclusion, we have directly demonstrated that PLC signaling of endogenously expressed 5-HT_2C receptors in CPE cells is mediated by G_q/11 heterotrimeric protein and specifically by the G_q subunit. In addition, the G_q subunit is responsible for the metabotropic GluR5 receptor activation of PLC in primary cultures of astrocytes. These studies serve as the first direct demonstration that active G_q subunit released from G_q/11 heterotrimers mediates the downstream activation of PLC in native systems. We have also provided evidence that subsequent to receptor-G_q/11 activation, G subunits released from G_q/11 heterotrimers do not contribute to the activation of PI hydrolysis signal in natural systems where the stoichiometry of signaling molecules is undisturbed. These studies demonstrate that membrane-permeable peptides, generated by a chemical ligation strategy, are effective tools in the dissection of multiple intracellular signals of G protein-coupled receptors in their native environment.

	ACKNOWLEDGEMENTS

We thank Dr. Tom Martin for providing antibodies against G_q, Dr. Lee Limbird for providing the HEK-_2A cells, and Richard Peavy and Dr. Jeff Conn for their assistance in establishing primary cultured astrocytes. We also thank Dr. Yi-an Lu and Cheng-Wei Wu for their technical assistance and expertise in peptide synthesis and purification and Dr. Viet Nguyen (Mass Spectrometry Center, Vanderbilt University Medical Center) for his technical assistance with mass spectrometric analysis of peptides. The assistance of Antoinette Poindexter, Ray Price, and Dr. Paul Gresch in preparing cultured CPE cells is greatfully acknowledged. We also thank Dr. Jon Backstrom for his assistance with Western blots and constructive discussions.

	FOOTNOTES

^* This work was supported by National Institutes of Health research Grants MH34007 (to E. S. B.) and CA36544 (to J. P. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Present address: Sphinx Pharmaceuticals/A Division of Eli Lilly and Company, 840 Memorial Dr., Cambridge, MA 02139.

To whom correspondence should be addressed. Tel.: 615-936-1685; Fax: 615-343-6532; E-mail: Elaine.bush@mcmail.vanderbilt.edu.

	ABBREVIATIONS

The abbreviations used are: PLC, phospholipase C; PI, phosphoinositide; PTX, pertussis toxin; MPS, membrane-permeable sequence; PLC1M, phospholipase C1-mimicking peptide; Phos, phosducin-like peptide; PhLP, phosducin-like protein; Fmoc, fluroenylmethyloxycarbonyl; HPLC, high performance liquid chromatography; CPE, choroid plexus epithelial; HBSS, Hanks' balanced salt solution; DMEM, Dulbecco's modified Eagle's medium; MAP, mitogen-activated protein; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; mGluR, metabotropic glutamate receptor.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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