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Department of Behavioral Neuroscience, Oregon Health & Science University, Portland, Oregon (Y.L., D.C.B., K.A.N.); and Portland Veterans Affairs Medical Center, Portland, Oregon (D.C.B, K.A.N.)
Received for publication January 4, 2008.
Accepted for publication April 28, 2008.
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). The dopamine receptor family is composed of D1-like (D1 and D5) and D2-like (D2L, D2S, D3, and D4) receptors (Neve and Neve, 1997). The dopamine D2 receptor belongs to a subfamily of seven-transmembrane domain G protein-coupled receptors that interact with the G proteins G
i and Go to modulate several effectors, including adenylate cyclase, potassium channels, and mitogen-activated protein kinases (Neve et al., 2004).
Protein-protein interactions are central to most important cellular processes, including DNA replication, transcription, translation, cell cycle control, and signal transduction. The yeast two-hybrid assay is a powerful method for identifying and characterizing protein-protein interactions (Fields and Song, 1989), but it is a tedious procedure, limited by the basic biology of the yeast. Yeast grows slowly, is difficult to transform efficiently, and requires unique reagents and techniques. The bacterial two-hybrid (B2H) system has the following advantages: fast growth rate, high transformation efficiency, and manipulations that are routine in most molecular biology laboratories (Joung et al., 2000). The purpose of this study was to use the B2H system to identify additional proteins that bind to and regulate the function of the D2 receptor.
S100 proteins comprise an extremely diverse and highly specialized family of approximately 21 Ca2+-binding proteins (Donato, 1999; Zimmer et al., 2003; Marenholz et al., 2004). An S100 protein is typically a low molecular mass protein (molecular mass between 9 and 13 kDa) characterized by the presence of two Ca2+-binding sites of the EF-hand type (Donato, 2003). S100 proteins have been implicated in the regulation of protein phosphorylation, Ca2+ homeostasis, enzyme activity, gene transcription, cell growth and differentiation, and the inflammatory response (Schäfer and Heizmann, 1996; Donato, 1999). Alterations of S100 function have been implicated in many diseases, including cancer, Down's syndrome, Alzheimer's disease, cardiomyopathy, psoriasis, cystic fibrosis, amyotrophic lateral sclerosis, and epilepsy (Heizmann, 2002; Heizmann et al., 2002). Thus, S100 proteins may be important diagnostic markers and therapeutic targets. The results of clinical studies on the S100 protein S100B in schizophrenia suggest that patients suffering from schizophrenia have increased S100B serum concentrations in the acutely psychotic stage of disease (Rothermundt et al., 2004). S100B is an acidic protein with a molecular mass of 21 kDa as a homodimer, and it is perhaps the best characterized of the S100 proteins (McClintock and Shaw, 2000). S100B has no known enzymatic function and exerts its intracellular effects by interacting with and modulating the activity of other proteins. In vitro, S100B interacts with more than 20 substrates in a Ca2+-sensitive manner (Donato, 1999).
We now describe a novel interaction between S100B and the dopamine D2 receptor, identified using the B2H system. We confirmed the novel interaction using coimmunoprecipitation in human embryonic kidney (HEK) 293 cells and in rat neostriatum. We identified S100B immunoreactivity in D2 receptor-expressing neostriatal neurons. We determined that the third intracellular loop of the D2 receptor (D2-IC3) is a contact point for the interaction with S100B by a histidine-tagged pull-down assay. S100B bound to IC3 of both D2L and D2S but not D3. We also proposed a putative binding motif for the interaction by sequence alignment. Finally, we found that coexpression of the D2 receptor and S100B significantly increased D2 receptor stimulation of extracellular signal-regulated kinases (ERKs) and inhibition of adenylate cyclase in HEK293 cells.
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-cI antibody (1/1000 dilution; Stratagene), rabbit anti-myc (1/1000 dilution; Bethyl, Montgomery, TX), mouse anti-myc (1/1000 dilution; Millipore), rabbit anti-dually phosphorylated (i.e., activated) ERKs (1/1000 dilution; Invitrogen), mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1/50,000 dilution; Millipore), and rabbit anti-microtubule-associated protein-2 (MAP2) (1/1000 dilution; Abcam, Cambridge, MA). Alexa Fluor 568-labeled goat anti-mouse IgG antibody, Alexa Fluor-486 goat anti-rabbit IgG antibody, and Prolong anti-fade kit were obtained from Invitrogen. S100B was from US Biological (Swampscott, MA). The cAMP enzyme immunoassay kit was from Cayman Chemical (Ann Arbor, MI). The BCA protein assay kit, secondary antibodies for immunoblot analysis, and the Super-Signal West Pico chemiluminescent kit were from Pierce Biotechnology (Rockford, IL). Protease inhibitor cocktail (set III) was from EMD Biosciences (San Diego, CA). HEK293 cells, a transformed cell line from human embryonic kidney, were purchased from American Type Culture Collection (Manassas, VA).
DNA Constructs for Bacterial Two-Hybrid Assay. The sequence encoding D2-IC3, amino acids 206 to 375 (leucine to methionine), was amplified by PCR and subcloned in-frame with the -cI DNA-binding domain into pBT (B2H System; Stratagene) to generate pBT-D2-IC3 as "bait." The construct was verified by DNA sequencing, and the presence of -cI-tagged D2-IC3 with the expected molecular size was also verified by immunoblot using anti--cI antibody. A B2H rat brain cDNA library (as a "target") was purchased from Stratagene. It contains pooled rat brain tissues (Sprague-Dawley, male, 10 weeks). The vector is pTRG, and the average insert size is approximately 1.8 kilobases.
Bacterial Two-Hybrid Screening. The B2H System reporter strain competent cells (Stratagene) were transformed with pBT-D2-IC3 and the BacterioMatch II Rat Brain Library (Stratagene) according to the manufacture's protocol (Stratagene). Detection of protein-protein interaction is based on transcriptional activation of the HIS3 reporter gene, which allows growth in the presence of 3-AT (5-20 mM), a competitive inhibitor of the His3 enzyme. Positives are verified using the aadA gene, which confers streptomycin resistance, as a secondary reporter. All positive clones were analyzed by DNA sequencing. To validate the putative protein-protein interactions, we then retransformed the reporter strain with the isolated target plasmid plus bait plasmid as described by the manufacturer (Stratagene).
Cell Culture, Transfection, and Selection. Cells were maintained in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum and 5% calf bovine serum, penicillin-streptomycin, appropriate selection antibiotics (G418 sulfate 600 µg/ml; puromycin 2 µg/ml), and grown in a humidified incubator at 37°C in the presence of 10% CO2.
The creation of a cell line stably expressing a c-myc-tagged D2L dopamine receptor (referred to hereinafter as the D2 receptor) was described in a previous report (Liu et al., 2007). Because HEK293 cells have endogenously expressed S100B detectable via immunoblotting using anti-S100B antibody, a cell line stably coexpressing c-myc-tagged D2 dopamine receptor and FLAG-tagged S100B (myc-D2/FLAG-S100B-HEK293) was generated as follows: cDNA encoding rat brain S100B was amplified using the polymerase chain reaction, digested with EcoRI-BamHI, and subcloned into the pcDNA-DNA3 expression vector, placing the FLAG-tag at the NH2 terminus of S100B. The FLAG-S100B construct was transfected into myc-D2-HEK cells using Lipofectamine 2000 transfection reagent and selection with puromycin (2 µg/ml) and G418 (600 µg/ml). Cell lines expressing the myc-tagged D2 receptor and FLAG-tagged S100B were isolated by screening via radioligand binding using [3H]spiperone and via immunoblot analysis using a mouse anti-myc and a mouse anti-FLAG antibody. The binding of [3H]spiperone was assessed as described previously (Liu et al., 2006), and the c-myc-tagged D2 receptor with coexpression of FLAG-tagged S100B had similar affinity for [3H]spiperone as reported previously for the c-myc-tagged D2 receptor (Liu et al., 2007). The molar ratio of D2 to S100B in the myc-D2/FLAG-S100B-HEK293 cell line was approximately 1:0.9 (data not shown).
Neostriatal Neuronal Cultures. Rat striatal neurons were cultured as follows: the cells were removed from liquid nitrogen and placed in a 37°C water bath for 2 to 3 min and then gently into a 15-ml centrifuge tube, to which was added prewarmed Primary Neuron Growth Medium (Lonza Walkersville) drop-wise into the cells while rotating the tube by hand. The cell suspension was mixed by inverting the tube twice. Cells were plated on 18-mm diameter poly(D-lysine)-coated glass coverslips at a density of 75,000 cells per coverslip and placed in a humidified 5% CO2 incubator at 37°C. After 4 h, the medium was replaced with fresh, prewarmed medium. After 4 days, the medium was changed, and cells were ready for use after 6 to 8 days in culture. Just fewer than 90% of the cells in the cultures were neurons, as determined by the presence of MAP2 immunoreactivity.
Coimmunoprecipitation of the myc-Tagged D2 Receptor and S100B, and Endogenous D2 Receptor and S100B. myc-D2/FLAG-S100B-HEK293 cells from confluent 10-cm2 plates were washed and incubated twice for 3 min each time with calcium- and magnesium-free phosphate-buffered saline (58 mM Na2HPO4, 17 mM NaH2PO4, and 68 mM NaCl, pH 7.4). Cells were released from the plate using trypsin, triturated, and centrifuged at 600g. The cells were resuspended in phosphate-buffered saline with 1.0% n-dodecylmaltoside and protease inhibitor cocktail and solubilized on ice for 2 h with gentle shaking. Sprague-Dawley rats (12 weeks old, female) were killed by decapitation, and the heads of the animals were immediately immersed in liquid nitrogen for 6 s. The brains were then removed, and the striatum was rapidly (20 s) dissected out on an ice-cold surface. The tissue was then triturated using fire-polished Pasteur pipettes in phosphate-buffered saline with 1.0% n-dodecylmaltoside and protease inhibitor cocktail and solubilized on ice for 8 h with gentle shaking. The insoluble material from the two tissue preparations was removed by centrifugation at 25,000g for 30 min. The protein concentration of the supernatant was analyzed by BCA protein assay reagent. An aliquot with 1.5 mg of protein was incubated with 2 µg of rabbit anti-myc antibody (for coIP with myc-D2/FLAG-S100B-HEK293 cells) or rabbit anti-D2 antibody (for coIP with neostriatal lysates) at 4°C for 2 h and further incubated with 20 µl of a 50% slurry of Protein G Plus beads overnight at 4°C. Beads were washed, and samples were eluted according to the manufacturer's instructions, separated by SDS-PAGE, and immunoblotted using mouse anti-FLAG antibody for myc-D2/FLAG-S100B-HEK293 cells or using mouse anti-S100B antibody for endogenous D2 receptor and S100B.
In Vitro Histidine-Tagged Dopamine Receptor-IC3 Pull-Down Assay. For the construction of the histidine-tagged fusion proteins, D2S-IC3 (amino acids 206 to 346), D2L receptor (D2L-IC3; amino acids 206 to 375), and D3 receptor (D3-IC3; amino acids 206-376) were PCR-amplified. The PCR products were cut as BamHI-SalI fragments and subcloned into pET-24a (+) (Novagen, Madison, WI) and then transformed into BL21(DE3)-competent cells (Novagen). Transformants were screened by induction with 0.5 mM isopropyl β-D-thiogalactoside and immunoblot analysis using a rabbit anti-His antibody. For larger-scale purification, the His-tagged dopamine receptor-IC3 clones were grown in Luria broth containing kanamycin (50 µg/ml) at 37°C to A600 = 0.5 and induced with 0.5 mM isopropyl β-D-thiogalactoside for 4 h at 23°C. Bacteria were pelleted and washed with phosphate-buffered saline. Pellets were resuspended in B-PER II bacterial protein extraction reagent (Pierce Biotechnology) with 0.5 mg/ml lysozyme (Fermentas, Hanover, MD) and protease inhibitor and incubated for 20 min with gentle rotation at room temperature. The bacterial cell lysates containing the same amount of His-tagged dopamine receptor-IC3 fusion proteins or a hexa-His peptide without insert as control were clarified by centrifugation, and the supernatants were applied to nickel-nitrilotriacetic acid agarose (QIAGEN, Valencia, CA). Prebound, washed beads were incubated with 500 ng of purified S100B overnight at 4°C, followed by wash and elution steps. The eluates were separated by SDS-PAGE, and bound proteins were analyzed by immunoblotting with rabbit anti-S100B antibody.
Confocal Immunofluorescence Imaging. Neostriatal neurons grown on glass coverslips were fixed in 4% paraformaldehyde in phosphate-buffered saline (58 mM Na2HPO4, 17 mM NaH2PO4, and 68 mM NaCl, pH 7.4) for 15 min, permeabilized with 0.5% Triton X-100 for 15 min, and then blocked with 5% goat serum for 1 h at room temperature. Neurons were incubated with rabbit anti-MAP2 or rabbit anti-D2L/S and mouse anti-S100B at 4°C overnight and then incubated for 1 h with Alexa Fluor 486 goat anti-rabbit IgG (1/1000) and Alexa Fluor-568-tagged goat anti-mouse IgG (1/1000) and followed by five 10-min washes with phosphate-buffered saline. The coverslips were then mounted onto a slide with the ProLong antifade kit, dried in the dark, and scanned alternating between 486 and 568 nm using a Leica TCS SP confocal laser scanning microscope (Leica, Wetzlar, Germany). System settings were held constant for all imaging.
Immunoblotting. Proteins were separated by SDS-PAGE through a 4 to 20% or 10% polyacrylamide gel and transferred to polyvinyl membranes (Millipore). The membranes were blocked for 1 h at room temperature with 5% nonfat milk with 0.05% Tween 20 in Tris-buffered saline (TBS), pH 7.4, at 4°C, washed twice for 5 min, followed by two 10-min washes with TBS, and incubated with primary antibody at room temperature for 2 h or overnight at 4°C. The membranes were washed twice for 5 min, followed by two 10-min washes with TBS, and then incubated with secondary antibody (horseradish peroxidase-conjugated goat anti-mouse IgG or anti rabbit IgG) at room temperature for 1 h. In addition to measuring protein concentrations to ensure equal loading, in many experiments in which phosphorylated forms of ERKs were detected, the membranes were then stripped with Restore Western blot-stripping buffer (Pierce) for 20 min at room temperature, followed by two 5-min washes with TBS, blocked for 1 h at room temperature with 5% nonfat milk in TBS, and then incubated with mouse anti-GAPDH antibody, a housekeeping gene product, to further ensure that equal amounts of proteins were loaded, followed by goat anti-mouse IgG. Immunodetection was accomplished using a SuperSignal West Pico chemiluminescent kit. The intensity of bands was quantified using Gel Doc EQ System (Bio-Rad Laboratories, Hercules, CA). A one-way analysis of variance and Bonferroni post hoc comparison was used to analyze data.
Cell Stimulation for Immunodetection of ERKs. Cells expressing myc-D2 or myc-D2/FLAG-S100B were grown in 12-well plates to 80 to 85% confluence. The cells were starved in serum-free Dulbecco's modified Eagle's medium overnight and then incubated with the D2-like receptor agonist quinpirole or epidermal growth factor at the indicated concentrations for 5 min at 37°C. Incubation was terminated by placing the tissue culture cluster on ice and rapidly aspirating the medium, followed by the addition of ice-cold radioimmunoprecipitation assay buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 1 mM NaVO3, protease inhibitors, and phosphatase inhibitor) and incubation for 20 min with shaking. After centrifugation (14,000g at 4°C for 15 min), the supernatant was collected, and the protein concentration was measured and adjusted using radioimmunoprecipitation assay buffer. The cell lysates (20 µl) with equal amounts of protein mixed with Laemmli loading buffer were denatured at 70°C for 10 min and separated by SDS-PAGE for immunodetection as described.
cAMP Accumulation Assay. The ability of the D2 receptor agonist 7-OH-DPAT to inhibit 30 µM forskolin-stimulated cAMP accumulation was measured in intact myc-D2-HEK293 cells and myc-D2/FLAG-S100B-HEK293 cells. Cells were plated between 100,000 and 150,000 cells/well in 48-well tissue culture plates and used in experiments 2 to 3 days later. Before the assay, cells were preincubated with Earle's balanced salt solution with 0.2% ascorbic acid, 500 µM 3-isobutyl-1-methylxanthine (a phosphodiesterase inhibitor), and 2% fetal bovine serum, pH 7.4, for 20 min at 37°C. The cells were placed on ice for the addition of 7-OH DPAT and 30 µM forskolin and then incubated at 37°C. The assay was terminated after 20 min by decanting the medium, and the cells were lysed with 100 µl of 3% trichloroacetic acid. Lysates were stored at 4°C at least 2 h before quantification of cAMP. The amount of cAMP in each well was measured using a cAMP enzyme immunoassay kit (Cayman Chemical).
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Coimmunoprecipitation of the myc-Tagged D2 Receptor and FLAG-S100B, and Endogenous D2 Receptor and S100B. To confirm a direct interaction between full-length D2 receptor and S100B, we expressed both FLAG epitope-tagged S100B and c-myc-D2L receptor in HEK293 cells. Immunoprecipitation of the D2 receptor with anti-myc resulted in the precipitation of FLAG-S100B, as indicated by immunoblotting with anti-FLAG antibody (Fig. 2A). FLAG-S100B immunoreactivity was not detected in control cells in which the immunoprecipitation was performed with an irrelevant antibody to a hemagglutinin epitope. The amount of S100B immunoreactivity in eluates from cells treated with the D2-like receptor agonist quinpirole (10 µM) for 5 min or 2 h was similar to that of cells treated with vehicle. Immunoprecipitation of the endogenous D2 receptor from rat neostriatum also resulted in the precipitation of S100B immunoreactivity; S100B immunoreactivity was not detected in control samples in which the immunoprecipitation was performed with rabbit normal IgG (Fig. 2B). Therefore, the D2 dopamine receptor specifically and constitutively bound S100B in HEK293 cells and in rat neostriatum.
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). To determine whether S100B is also expressed in neostriatal neurons, which express the D2 receptor, the colocalization of immunoreactivity for S100B and the neuronal marker MAP2 (Fig. 3, A-C) and for S100B and D2 receptor (Fig. 3, D-F) was determined in neostriatal neuronal cultures. In 12 visual fields from 3 independently prepared cultures, all cells that expressed immunoreactivity for MAP2 (green; 85 neurons) also expressed S100B (red). In two additional experiments, we assessed the colocalization of immunoreactivity for S100B and the D2 receptor. In 8 visual fields with 83 cells exhibiting a neuronal morphology, 
75% of the cells expressed both S100B (red) and the D2 receptor (green) (Fig. 3).
Putative S100B Binding Motif. The identification of an S100B binding "epitope" from bacteriophage studies provides a useful probe to search for a binding motif. Ivanenkov et al. (1995) screened a bacteriophage random peptide display library and identified the consensus sequence +OXO*XOO (+, basic; O, hydrophobic; *, hydrophilic; X, variable) as the "epitope" binding region for S100B. The peptide TRTKIDWNKILS (TRTK-12), a 12-residue peptide containing the consensus sequence, successfully competes with other S100B binding proteins such as glial fibrillary acidic protein and CapZ for calcium-sensitive S100B binding (Ivanenkov et al., 1995; Bianchi et al., 1996). This consensus sequence for S100B binding has been identified in 25 proteins found to interact in vitro with S100B (McClintock and Shaw, 2000). We scanned the D2 receptor for this motif and identified a potential S100B binding site at amino acid residues 233 to 240 close to the amino terminus of the third intracellular loop and adjacent to the alternatively spliced region of the receptor (Fig. 4). We did not find this motif in the third intracellular loop of the D3 receptor or in the D4 receptor.
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To determine whether S100B enhanced activation of ERKs at a step downstream from the D2 receptor, we assessed the effect of S100B expression on epidermal growth factor (EGF)-induced activation of ERKs. Coexpression of S100B had no significant effect on the ERK response to EGF (10 ng/ml) (Fig. 5A). To confirm that the lack of effect of S100B on the response to EGF was not because ERK was already maximally activated, we also tested lower concentrations of EGF (100 and 200 pg/ml). Although there was a tendency for EGF-induced activation of ERK to be enhanced in cells expressing S100B, the effect was not statistically significant, in contrast to the robust enhancement of quinpirole-induced activation of ERK in cells coexpressing S100B (Fig. 5B).
To verify that EGF-induced signaling was independent of D2 receptor activation of Gi/o, we used pertussis toxin (PTX) to inactivate this class of G proteins. Treatment of HEK293 cells with PTX (50 ng/ml overnight) abolished D2 receptor activation of ERKs without altering EGF-induced activation (Fig. 6). Overall, these results imply that the increased ERK activation with coexpression of S100B is primarily due to the interaction of D2 and S100B rather than to binding of S100B to downstream elements that are shared between signaling pathways of the EGF and D2 receptors.
Increased Inhibition of Forskolin-Stimulated cAMP Accumulation in Cells Expressing S100B. We also characterized the effect of coexpression of S100B on D2L receptor inhibition of adenylate cyclase. The experiment shown in Fig. 7, representative of three independent experiments, demonstrates that the potency of the agonist is unchanged by the coexpression of S100B, but maximal inhibition of forskolin-stimulated cAMP accumulation is significantly increased. The average EC50 values for the D2 and the D2/S100B cell lines were 21 and 15 nM, respectively. The maximal inhibition of forskolin-stimulated cAMP accumulation for the D2 and D2/S100B cell lines was 57 ± 12% and 66 ± 9%, respectively (p < 0.05, by paired t test, n = 3), respectively. This is qualitatively similar to the effect of S100B coexpression on quinpirole-induced activation of ERK.
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). It has been postulated that modulation, rather than direct blockade, of the D2 receptor might offer the therapeutic benefit without the adverse effects of most antipsychotic drugs (Dixon et al., 1999). One way to achieve this would be to inhibit the binding of dopamine receptor-interacting proteins that enhance D2 receptor signaling.
Because the third cytoplasmic loop is the primary contact site between G protein-coupled receptors and G proteins, interactions that have been identified between the third cytoplasmic loop of D2-like receptors and a number of other proteins are likely to influence D2-like receptor signaling. For example, D2 and D3 receptors but not D1 or D4 receptors bind the actin-binding protein filamin A, or ABP-280, at a segment in the carboxyl terminus of the third cytoplasmic loop, where both D2 and D3 receptors have a potential site of phosphorylation by protein kinase C. D2 and D3 receptors expressed in cells that lack ABP-280 have diminished ability to inhibit adenylate cyclase (Li et al., 2000, 2002). Calmodulin (CaM) modulates D2 receptor signaling by binding to the amino terminal end of the D2 receptor third cytoplasmic loop (Bofill-Cardona et al., 2000; Liu et al., 2007). Understanding fully how the D2 receptor functions will clearly require determining the full complement of binding partners for the receptor.
The purpose of this study was to identify and characterize novel binding partners of the dopamine D2 receptor that might modulate receptor signaling. The B2H system is an efficient Escherichia coli-based method for detecting protein-protein interactions in vivo. In this system, detection of protein-protein interactions is based on transcriptional activation of the HIS3 reporter gene, which allows growth in the presence of 3-AT, a competitive inhibitor of His3 enzyme. Positives are verified using the aadA gene, which confers streptomycin resistance, as a secondary reporter. The B2H system offers the ability to screen for binding partners with little background, and using E. coli for two-hybrid screening instead of a eukaryotic cell reduces the chance that the host harbors a homolog of one of the interacting protein partners (Joung et al., 2000).
Using the B2H system to screen a rat brain cDNA library, we identified a novel interaction between S100B and the D2 receptor. The S100 protein family is a highly conserved group of Ca2+ binding proteins with molecular masses from 9 to 13 kDa. S100B, a particularly well-characterized member of the S100 family, was first discovered as a major constituent of glia (Moore, 1965); however, it is now known to be expressed in tissues and cell lines including C6 glioma cells, cardiomyocytes, renal tumors, and melanomas (Donato, 1991; Suzushima et al., 1994; Takashi et al., 1994; Zimmer et al., 1997). S100B is a homodimer of 21 kDa, and each S100B subunit contains two EF-hand calcium-binding domains (Zimmer et al., 1997). Although the precise mechanisms for intra- and extracellular functions of S100B are not well understood, processes such as neurite extension, Ca2+ flux, cell growth, apoptosis, energy metabolism, and protein phosphorylation are all believed to be modulated in some manner by S100B (Kligman and Hilt, 1988; Donato, 1991; Schäfer and Heizmann, 1996). Most significantly for the proposed interaction with the D2 receptor, S100B has also been identified in neurons (Ellis et al., 2007). The general model for S100-target protein interactions is similar to that of other Ca2+-binding proteins such as CaM and troponin C; S100B undergoes a conformational change upon binding Ca2+ that promotes its interaction with a variety of target proteins (Kligman and Hilt, 1988; Chaudhuri et al., 1997; Drohat et al., 1997).
For further evaluation of the interaction between the D2 receptor and S100B, we used confocal microscopy to assess the colocalization of endogenous S100B with the neuronal marker MAP2 or with endogenous D2 receptor in neostriatal neurons. We observed extensive coexpression of S100B and MAP2 as well as S100B and D2 receptor in our neuronal cultures. We verified the interaction by coimmunoprecipitation of the D2 receptor with FLAG-S100B from HEK293 cell homogenates and with endogenous S100B from rat neostriatal homogenates. We demonstrated that the third intracellular loop of the D2L and D2S receptors but not D3 is a contact point for the interaction with S100B using an in vitro histidine-tagged pull-down assay. We also identified an S100B binding motif located at residues 233 to 240 of the D2 receptor, toward the amino terminus of D2-IC3 and immediately upstream of the alternatively spliced region, a motif that is not found in the D3 receptor.
The first signaling pathway identified for D2-like receptors was inhibition of cAMP accumulation. Another important effector in the D2 receptor signaling pathway that is potentially regulated by the interaction between the D2 receptor and S100B is ERK. ERKs belong to the family of mitogen-activated protein kinases, components of parallel protein kinase cascades that transmit signals from a variety of extracellular stimuli to the cell nucleus, thus participating in cell proliferation, differentiation, and survival (Gutkind, 1998). Although the pathway from D2-like receptors to ERK has not been thoroughly elucidated and may differ depending on cell type and receptor subtype, D2-like receptor activation of ERK is frequently mediated by pertussis toxin-sensitive G proteins (Welsh et al., 1998; Choi et al., 1999; Wang et al., 2005). Both of these signaling responses to D2 receptor stimulation were enhanced in cells coexpressing S100B. The mechanism by which S100B enhanced D2 receptor signaling is unknown. Receptor-interacting proteins frequently alter the trafficking of receptors to the membrane, but preliminary results using cell-surface fluorescence and inhibition of radioligand binding by the membrane-impermeant ligand sulpiride suggest that the abundance of cell-surface receptors was similar in myc-D2L-HEK293 cells and myc-D2L/S100B-HEK293 cells (data not shown). Because S100B is a homodimer, another possibility is that it binds to both the D2 receptor and to another protein involved in signaling, bringing the two proteins together, but it is not known what other protein might be involved.
Another Ca2+-binding protein, CaM, is important for the activation of ERK by several G protein-coupled receptors (Melien et al., 2002). CaM mediates the activation of ERK by the µ-opioid receptor through a pathway involving the transactivation of the EGF receptor (Belcheva et al., 2001) and by the serotonin 5-HT1A receptor through a process involving agonist-induced receptor internalization (Della Rocca et al., 1999). Our previous work suggests that binding of CaM to the D2 receptor enhances receptor signaling (Liu et al., 2007). The data presented here suggest that binding of S100B, too, enhances D2 receptor signaling to both ERKs and cAMP. To our knowledge, this is the first report regarding the interaction of S100B and the D2 receptor and its role in dopamine D2 receptor signaling.
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ABBREVIATIONS: B2H, Bacteriomatch Two-Hybrid; 3-AT, 3-amino-1,2,4-triazole; CaM, calmodulin; EGF, epidermal growth factor; ERK, extracellular signal-regulated kinase; G protein, heterotrimeric GTP-binding protein; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HEK, human embryonic kidney; D2-IC3, the third intracellular loop of the D2 receptor; MAP2, microtubule-associated protein-2; PTX, pertussis toxin; TBS, Tris-buffered saline; PAGE, polyacrylamide gel electrophoresis; coIP, coimmunoprecipitation; PCR, polymerase chain reaction; 7-OH DPAT, 7-hydroxy-2-dipropylaminotetralin.
Address correspondence to: Dr. Kim A. Neve, 3710 SW US Veterans Hospital Road, Portland, OR 97239-2999. E-mail: nevek{at}ohsu.edu
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