From the
¶ Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106,
Department of Neurosciences, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106,
|| Department of Psychiatry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106
Received for publication, February 24, 2003
, and in revised form, April 3, 2003.
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ABSTRACT |
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INTRODUCTION |
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The 5-HT2A receptor, the most abundant serotonin receptor in the cerebral cortex, is enriched in dendritic shafts and, to a lesser extent, asymmetric synapses and dendritic spines of pyramidal neurons primarily from Layers IV and V (913). The last four amino acids (VSCV) of the carboxyl terminus of the 5-HT2A receptor constitute a canonical Type I PDZ-binding domain (X-Ser/Thr-X-) (14). The PDZ-binding domain is located at the carboxyl terminus of a variety of proteins including many GPCRs and is known to associate with PSD-95/Discs-large/ZO-1 (PDZ) domain-containing proteins of which postsynaptic density 95 (PSD-95, also known as synapse-associated protein 90 or SAP-90, is a prototypic member (15, 16). These multi-domain molecules not only target and provide scaffolds for protein-protein interactions but can also modulate the functions of ion channels and receptors with which they associate (1620). The disruption of the association between PDZ proteins and their targets contributes to the pathogenesis of a number of human diseases (16, 21, 22), most probably because of the failure of PDZ proteins to appropriately target and modulate the actions of the associated proteins.
Na+/H+ exchanger regulatory factor (NHE-RF) was the first PDZ domain-containing protein that was reported to modulate the function of a GPCR (e.g. the 2-adrenergic receptor (
2-AR)) (23). Subsequently, PSD-95 was found to associate with and inhibit the internalization of
1-ARs and to facilitate the interaction between the
1-AR and the N-methyl-D-aspartate ionotropic glutamate receptor (24, 25). Among the 5-HT2 class of serotonin receptors, a multi-PDZ domain-containing protein, MUPP1, associates with 5-HT2C receptors in vitro and in vivo (26). MUPP1 has also been predicted to interact with both the 5-HT2A and the 5-HT2B receptor based on its ability to bind to purified COOH-terminal fusion proteins of both receptors in vitro (26). Furthermore, a proteomic approach has recently identified PSD-95 as a component of a multi-protein complex that associates with the 5-HT2C receptor in vivo (27). PSD-95, similar to the 5-HT2A receptor, is enriched in asymmetric synapses and dendritic spines of cortical pyramidal neurons (28, 29) and is thus predicted to interact with 5-HT2A receptors. The functional significance of any association between PDZ proteins and 5-HT receptors is unknown because none has been previously reported.
Therefore, we set out to investigate whether PSD-95 interacts with and regulates 5-HT2A receptors. A combination of biochemical and immunocytochemical approaches demonstrated that PSD-95 directly interacts with the 5-HT2A receptor in vitro. We also discovered that PSD-95 enhanced 5-HT2A receptor-mediated signaling, inhibited agonist-induced 5-HT2A receptor internalization, and promoted 5-HT2A receptor clustering on the plasma membrane. Thus, this work demonstrates a novel role for PSD-95 in modulating the activity of a GPCR. These findings have important implications for elucidating the roles of PDZ domain-containing proteins in regulating the functions of the 5-HT family of receptors and possibly other GPCRs.
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EXPERIMENTAL PROCEDURES |
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5-Hydroxytryptamine creatinine sulfate (5-HT), quipazine, clozapine, chlorpromazine, (±)-2,5-dimethoxy-4-iodoamphetamine hydrochloride, CHAPS, and sodium orthovanadate were acquired from Sigma. [3H]Ketanserin and myo-[3H]inositol were purchased from PerkinElmer Life Sciences.
Transfection of HEK-293 CellsHuman embryonic kidney 293 (HEK-293) cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1 mM sodium pyruvate, penicillin (100 units/ml), and streptomycin (100 mg/ml) (Invitrogen) at 37 °C and 5% CO2. Transient transfection with FuGENE 6 (Roche Applied Sciences) using a FuGENE 6 to DNA ratio of 5:1 was performed according to the manufacturer's recommendations. A total of 6 µg of DNA was used in each co-transfection, 4 µg of which were vector, 5-HT2A receptor, or mutant 5-HT2A receptor DNA and 2 µg of which were DNA encoding either PSD-95-GFP or GFP.
Co-immunoprecipitation and ImmunoblotAt 24 h after co-transfection, HEK-293 cells were split into 6-well plates and cultured first in DMEM supplemented with 5% dialyzed FBS for 24 h and then in DMEM without serum for an additional 24 h. Cells were washed at 4 °C twice with PBS and once with Buffer A (50 mM HEPES, 150 mM NaCl, 1mM EDTA, 10 mM Na4P2O7,and2mM sodium orthovanadate, pH 7.5). Cells were lysed by incubation on a rocker platform at 4 °C for 15 min in Buffer A containing 1.5% CHAPS and EDTA-free Complete protease inhibitor tablets (Roche Applied Sciences). A portion of the cell lysates was saved for subsequent determination of total protein expression levels, and the remainder was incubated with Sepharose beads conjugated to a monoclonal FLAG antibody (Sigma) at 4 °C for 2 h. The beads were washed at 4 °C for three times in Buffer B (50 mM HEPES, 150 mM NaCl, 0.3% Triton X-100, and 10% glycerol, pH 7.5). Immunoprecipitated proteins were then eluted from the beads by resuspension in 2x SDS sample buffer. Samples were heated for 5 min at 67 °C, and proteins were resolved by SDS-PAGE and transferred to nitrocellulose membranes. Immunoblotting was performed according to standard methods (30, 34). A polyclonal GFP antibody (1:2000) (Abcam, Cambridge, United Kingdom) was used to detect PSD-95-GFP or GFP. A rabbit polyclonal FLAG antibody (1:1500) was used to detect FLAG-tagged WT and mutant 5-HT2A receptors. The constitutively active Gq mutant and the endogenous WT G
q in cell lysates were detected by a rabbit polyclonal G
q antibody (1:2000) (Santa Cruz Biotechnology, Santa Cruz, CA). The levels of PSD-95-GFP expression from immunoprecipitates and cell lysates were quantified using a chemiluminescence imaging system (Kodak Scientific Imaging System, Eastman Kodak Co.). The levels of PSD-95-GFP expression from immunoprecipitates were subsequently normalized to corresponding levels in cell lysates.
Agonist-mediated InternalizationAt 24 h after co-transfection, HEK-293 cells were split into 24-well plates containing poly-L-lysine-coated coverslips and maintained in DMEM supplemented with 5% dialyzed FBS for 24 h and then in DMEM without serum for an additional 24 h. Cells were treated for 15 min at 37 °C with 5-HT at a final concentration of 10 µM. Treatment was terminated by placing cells at 4 °C, removing media, and fixing with 4% paraformaldehyde. Immunocytochemistry was performed, confocal images were acquired (see "Immunocytochemistry, Confocal Microscopy, and Image Quantification"), and internalization was quantified using the MetaView imaging software (Universal Imaging, Downingtown, PA).
Phosphoinositide Hydrolysis, Radioligand Binding, and Desensitization AssaysPhosphoinositide (PI) hydrolysis and saturation radioligand binding assays were performed on cells from the same transfection. At 24 h following transfection, HEK-293 cells were either 1) split into poly-L-lysine-coated 24-well plates and maintained in DMEM supplemented with 5% dialyzed FBS for 24 h and then in serum-free and inositol-free DMEM for an additional 24 h before performing PI hydrolysis assay or 2) kept in DMEM supplemented with 5% dialyzed FBS for 24 h and then in serum-free DMEM for additional 24 h before harvesting cell membranes for binding assay. As described previously, agonist efficacy (Emax) and potency (EC50) were determined from dose-response PI hydrolysis assays and kinetic binding parameters (Bmax and Kd) were determined from saturation binding assays with [3H]ketanserin (35). The resulting Emax values were then normalized to Bmax values with results replicated in at least three separate experiments. PI hydrolysis data were analyzed by nonlinear regression using the GraphPad Prism software (San Diego, CA), and saturation binding data were analyzed using GraphPad Prism or the weighted nonlinear least-squares curve-fitting program LIGAND (36). Agonist-dependent desensitization of 5-HT2A receptor-mediated accumulation of inositol phosphate (IP) was performed as described previously (34, 35).
Immunocytochemistry, Confocal Microscopy, and Image QuantificationDual-labeling immunocytochemistry was performed essentially as described previously (30). HEK-293 cells were fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton X-100 at 4 °C, blocked with 5% milk, incubated with primary antibodies overnight at 4 °C and then with secondary antibodies, conjugated to the appropriate Alexa Fluor dyes (Molecular Probes, Eugene, OR), and finally mounted on glass slides. FLAG-tagged constructs were visualized using a monoclonal FLAG antibody (1:1500) (Stratagene). GFP-tagged constructs were visualized with a rabbit polyclonal GFP antibody (1:2000) (Abcam, Cambridge, United Kingdom). Images of cells were acquired digitally using a Zeiss 410 confocal microscope (Oberkochen, Germany) without saturation of pixel intensities and were subsequently analyzed using MetaView (Universal Imaging) as previously detailed (30, 34, 35, 37, 38). For each cell in which 5-HT2A receptor internalization was quantified, two measurements were made: 1) total cellular immunofluorescence and 2) intracellular immunofluorescence. The extent of 5-HT2A receptor internalization was then defined as the percentage of the total 5-HT2A receptor immunofluorescence, which was intracellular. For each sample group, at least 50 cells were quantified in a blinded fashion. In prior studies (30, 37), we have demonstrated that this method provides quantitative estimates of internalization that are essentially equivalent to results obtained by biochemical techniques such as surface biotinylation.
Statistical AnalysisData from co-immunoprecipitation and internalization studies were analyzed using the unpaired Student's t test with statistical significance defined as p < 0.05. Statistical significance of the PI hydrolysis data was analyzed by GraphPad Prism using the two-tailed paired Student's t test and was defined as p < 0.05.
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RESULTS |
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To identify the responsible binding motif(s) on the 5-HT2A receptor, we constructed a mutant 5-HT2A receptor (5-HT2A469471) that lacks the PDZ-binding domain. Ablating the PDZ-binding domain attenuated the interaction between FLAG-5-HT2A and PSD-95-GFP to control levels (Fig. 1, A, lanes 11 and 12 versus lanes 7 and 8, and E), demonstrating that the PDZ-binding domain of the 5-HT2A receptor (residues 469471, SCV) mediates the association with PSD-95.
To further establish the specificity of the interaction between the 5-HT2A receptor and PSD-95, we performed a number of controls. First, we showed that cells co-transfected with PSD-95-GFP and an empty vector (Fig. 1, A, lanes 7 and 8, and E) yielded minimal pull-down of PSD-95. Second, no PSD-95 was immunoprecipitated from nontransfected cells (Fig. 1A, lanes 1 and 2) or from cells co-expressing 5-HT2A receptors (WT or mutant) and GFP (Fig. 1A, lane 36). These findings demonstrated the specificity of PSD-95 association with 5-HT2A receptors. Finally, the significantly higher levels of PSD-95 that co-immunoprecipitated with WT 5-HT2A receptors (Fig. 1A, lanes 9 and 10) resulted neither from variations in the expression of WT or mutant 5-HT2A receptors in immunoprecipitates (Fig. 1B, lanes 36 and lanes 912) or in cell lysates (Fig. 1D, lanes 36 and lanes 912) nor from variations in PSD-95 expression (Fig. 1C, lanes 712; see Fig. 1E for quantification). Therefore, we conclude that PSD-95 specifically associates with 5-HT2A receptors by interacting with the PDZ-binding domain of the 5-HT2A receptor.
PSD-95 Potentiates 5-HT2A Receptor-mediated Signal TransductionWe next examined whether the association between PSD-95 and the 5-HT2A receptor has functional consequences on 5-HT2A receptor signal transduction. Because the 5-HT2A receptor is a Gq-coupled receptor that activates phospholipase C (PLC) (39), we measured inositol phosphate (IP) accumulation resulting from 5-HT2A receptor-mediated PI hydrolysis (Fig. 2) and determined in parallel the expression of 5-HT2A receptors by immunoblot analysis (Fig. 1, B and D) and saturation binding assays. We then normalized the functional activity of the 5-HT2A receptor (i.e. IP accumulation) to the expression level of the 5-HT2A receptor (i.e. Bmax), thus accounting for minor variations in 5-HT2A receptor activation that might have resulted from variations in receptor expression. Assays were also performed under the conditions of minimal receptor reserve (35).
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To investigate the possibility that PSD-95 augments the PLC signaling pathway by associating with downstream effectors (i.e. Gq and PLC), we examined whether PSD-95 increased IP accumulation in cells expressing a constitutively active form of G
q (Q229L and G
q*), which activates PLC directly (33). In the absence of 5-HT2A receptor expression, PSD-95 neither altered the G
q*-stimulated IP accumulation when compared with GFP alone (Fig. 3C) nor affected the relative expression of G
q* (Fig. 3, A and B). These findings strongly suggest that PSD-95 enhances intracellular signaling at the level of receptor-effector coupling and not via a receptor-independent augmentation of G
q signaling.
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PSD-95 Does Not Affect the Agonist-dependent Desensitization of 5-HT2A ReceptorsTo determine the potential cellular mechanisms subserving the PSD-95-mediated augmentation of 5-HT2A receptor signaling, we examined whether PSD-95 alters the time course of 5-HT2A receptor desensitization. We have previously reported that exposure to agonists inhibits the activation of the 5-HT2A receptor by the process of desensitization whereby receptors on the cell surface no longer respond to agonists and fail to efficiently signal downstream pathways (41). PSD-95 did not attenuate agonist-dependent 5-HT2A receptor desensitization when compared with the base line (Fig. 4, WT 5-HT2A + PSD-95 versus WT 5-HT2A + GFP). Likewise, disrupting the association between PSD-95 and 5-HT2A receptors had no effect on agonist-dependent desensitization of 5-HT2A receptors (Fig. 4, 5-HT2A469471 + PSD-95). Hence, PSD-95 does not enhance 5-HT2A receptor signaling by attenuating the desensitization of 5-HT2A receptors.
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PSD-95 Inhibits the Agonist-induced Internalization and Promotes the Cell Surface Clustering of 5-HT2A ReceptorsWe next investigated whether PSD-95 affects the targeting and trafficking of 5-HT2A receptors. We have previously demonstrated that acute exposure to agonists facilitates the redistribution of 5-HT2A receptors from the plasma membrane to the intracellular pool via receptor internalization (30). As expected, 5-HT induced the rapid internalization of WT 5-HT2A receptors (Fig. 5, AC versus DF; see Fig. 7 for quantification). Interestingly, PSD-95 significantly inhibited the agonist-induced 5-HT2A receptor internalization (Figs. 5, GI versus JL, and 7) and promoted the co-clustering (i.e. formation of puncta containing both proteins) of 5-HT2A receptors with PSD-95 on the cell surface (Figs. 5, GI (cross-section view), and 8, AC (top view)).
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As an essential control, we found that disrupting the PDZ-binding domain of the 5-HT2A receptor blocked the ability of PSD-95 to inhibit 5-HT2A receptor internalization (Figs. 6, AC versus DF, and 7). Furthermore, disrupting the PDZ-binding domain attenuated 5-HT2A receptor clustering on the cell surface (Figs. 6, AC (cross-section view), and 8, DF (top view)). Interestingly, whereas WT 5-HT2A receptors appeared to co-cluster with the membrane-bound PSD-95, mutant 5-HT2A469471 receptors did not facilitate the formation of PSD-95-immunoreactive puncta on the cell surface (Figs. 5, GI versus JL, and 6, AC versus DF). Taken together, these findings suggest that PSD-95 augments 5-HT2A receptor-mediated signaling at least in part by inhibiting the internalization and promoting the cell surface clustering of 5-HT2A receptors.
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DISCUSSION |
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PSD-95 is known to associate with a variety of target proteins, including the NMDA receptor (14), 1-AR (24), neuroligin (42), cysteine-rich interactor of PDZ three (43), and citron (44). Superficially, the association of PSD-95 with the 5-HT2A receptor is reminiscent of PSD-95 interactions with the
1-AR. Most notably, the extreme carboxyl terminus of the
1-AR (Ser-Lys-Val) and the 5-HT2A receptor (SCV) shares the same Type I PDZ-binding motif (X-Ser/Thr-X-
) and could conceivably interact with the same PDZ domain on PSD-95. Moreover, PSD-95 inhibits the agonist-induced internalization of both the
1-AR (24) and the 5-HT2A receptor. On the other hand, we have discovered significant differences. In particular, PSD-95 augments 5-HT2A receptor signaling but has no effect on
1-AR signaling (24).
The precise mechanism by which PSD-95 augments 5-HT2A-mediated signaling is unknown but is probably because of a direct physical interaction. Thus, PSD-95 potentiates the signaling of wild-type 5-HT2A receptors but not mutant 5-HT2A receptors lacking the PDZ-binding domain. Additionally, PSD-95 does not augment basal (constitutive) 5-HT2A receptor signaling, alter agonist potency, perturb the kinetics of agonist-dependent 5-HT2A receptor desensitization, or affect the ability of a constitutively active Gq (G
q-Q229L) to activate PLC. Taken together, these results are consistent with the notion that PSD-95 augments receptor-effector signaling via a direct physical interaction between 5-HT2A receptors and PSD-95. Based on the present finding that PSD-95 augments 5-HT2A receptor signaling, it is reasonable to speculate that PSD-95 provides scaffolding for Gq and PLC, thereby facilitating the interaction between the 5-HT2A receptor and these down-stream signaling molecules. Studies to test this hypothesis are in progress.
We have also found that PSD-95 inhibits the agonist-induced 5-HT2A receptor internalization. A possible mechanism by which PSD-95 inhibits 5-HT2A receptor internalization is that PSD-95 recruits and anchors multiple proteins including the 5-HT2A receptor to the plasma membrane, assembling a complex that in turn prevents the internalization of component molecules. For example, PSD-95 is known to assemble 1-AR and NMDA receptor in the same complex (24). Consistent with reports of cell surface co-localization between MUPP1 and 5-HT2C receptor (26) and between PSD-95 and
1-AR (24), 5-HT2A receptors also co-cluster with PSD-95 as the distribution of PSD-95 on the plasma membrane appears more diffuse in the absence of its association with the 5-HT2A receptor. The co-clustering between 5-HT2A receptors and PSD-95 may reflect the assembly of a multi-protein signaling complex on the cell surface that does not easily internalize following agonist exposure. The net effect in neurons would most probably be an enhancement of 5-HT2A-mediated signaling in regions where 5-HT2A receptors and PSD-95 are co-localized.
It is worth noting that PSD-95 inhibits the internalization of the 5-HT2A receptor but does not affect the agonist-dependent desensitization, considering that these regulatory mechanisms for GPCRs (i.e. internalization, desensitization, and resensitization) are frequently linked. We have previously demonstrated that both the desensitization and the resensitization of the 5-HT2A receptor are not affected by various well characterized inhibitors of clathrin-mediated endocytosis in HEK-293 cells (35, 41). In addition, it is unlikely that the PDZ-binding domain is involved in agonist-dependent GPCR desensitization because disruption of the PDZ-binding domain does not prevent 5-HT2C receptors from undergoing desensitization (45).
Another potential mechanism for PSD-95-mediated inhibition of 5-HT2A receptor internalization could involve competition for the same binding motif with regulatory molecules that facilitate 5-HT2A receptor internalization. Studies of PSD-95/Kir2.3 and NHE-RF/2-AR associations suggest that phosphorylation of the serine or threonine residue at the 2 position of the canonical PDZ-binding domain by G protein-coupled receptor kinases (GRK) may be the regulatory mechanism with which PSD-95 competes (23, 4648). One such counter-regulatory molecule is GRK5. Upon agonist stimulation, GRK5 phosphorylates the serine residue of the PDZ-binding domain of both
1-AR and
2-AR, thereby diminishing receptor association with PDZ proteins (PSD-95 and NHE-RF, respectively) and facilitating receptor internalization (47, 48). Neither GRK5 nor GRK2 is likely to compete with PSD-95 for binding sites on the 5-HT2A receptor, however, since we have demonstrated that 5-HT2A receptors are regulated by GRK2- and GRK5-independent processes (35).
Because PSD-95 becomes membrane-bound following palmitoylation (49), the preferential localization of PSD-95 to the plasma membrane makes it unlikely that PSD-95 could enhance 5-HT2A receptor recycling (50), a possibility that might otherwise confound the interpretation of the present observation. It would be interesting to examine in the future whether an alternatively spliced isoform of PSD-95 (PSD-95) that lacks palmitoylation sites in the amino terminus and is not membrane-bound (51) could inhibit 5-HT2A receptor internalization.
In summary, we have discovered a novel role for PSD-95 for regulating the activity of GPCRs. PSD-95 augments 5-HT2A receptor-mediated signaling in a way that requires a direct physical interaction between 5-HT2A receptors and PSD-95. This study demonstrates that PSD-95, a component of the neuronal protein sorting apparatus, regulates 5-HT2A receptor signaling at least in part by anchoring 5-HT2A receptors to the cell surface in clusters that are not easily internalized by agonists and possibly by facilitating 5-HT2A receptor interaction with downstream signaling molecules. Because 5-HT2A receptors are responsible for the actions of atypical antipsychotic drugs (4, 5, 37, 52) and most (1, 5) but not all (2, 3) hallucinogens, elucidating the cellular machinery responsible for targeting and regulating 5-HT2A receptors will enhance our understanding of how diverse classes of drugs exert their profound effects on human perception, emotion, and cognition.
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FOOTNOTES |
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Supported in part by Medical Scientist Training Program Grant GM07250 and Neuroscience Training Grant AG00271.
** To whom correspondence should be addressed: Dept. of Biochemistry, Rm. W438, Case Western Reserve University School of Medicine, 10900 Euclid Ave., Cleveland, OH 44106-4935. Tel.: 216-368-2730; Fax: 216-368-3419; E-mail: roth{at}biocserver.cwru.edu.
1 The abbreviations used are: 5-HT2A, serotonin (5-hydroxytryptamine) 2A receptor; GPCR, G protein-coupled receptor; PSD, postsynaptic density 95; PDZ, PSD-95/Discs-large/ZO-1; NHE-RF, Na+/H+ exchanger regulatory factor; AR, adrenergic receptor; WT, wild type; GFP, green fluorescent protein; HEK, human embryonic kidney; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; Emax, efficacy value; GRK, G protein-coupled receptor kinase; IP, inositol phosphate; PI, phosphoinositide.
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ACKNOWLEDGMENTS |
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REFERENCES |
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