Binding Protein S100B: Role in D2 Receptor Function

S100B is a calcium-binding protein with both extracellular and intracellular regulatory activities in the mammalian brain. We have identified a novel interaction between S100B and the dopamine D2 receptor. Our results also suggest that the binding of S100B to the dopamine D2 receptor enhances receptor signaling. This conclusion is based on the following observations: 1) S100B and the third cytoplasmic loop of the dopamine D2 receptor interact in a bacterial two-hybrid system and in a poly-histidine pull-down assay; 2) immunoprecipitation of the D2 receptor also precipitates FLAG-S100B from human embryonic kidney 293 cell homogenates and endogenous S100B from rat neostriatal homogenates; 3) S100B immunoreactivity was detected in cultured neostriatal neurons expressing the D2 receptor; 4) a putative S100B binding motif is located at residues 233 to 240 of the D2 receptor, toward the amino terminus of the third cytoplasmic loop. D3-IC3, which does not bind S100B, does not contain this motif; and 5) coexpression of S100B in D2 receptor-expressing 293 cells selectively increased D2 receptor stimulation of extracellular signal-regulated kinases and inhibition of adenylate cyclase. Interest in dopamine receptor research has been fueled by studies of brain diseases such as Parkinson’s disease and schizophrenia, showing that dopamine has a role in either the pathogenesis or symptoms of the diseases and that substances acting at the receptors act as therapeutic agents (Strange, 1992). 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 G o 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 Ca -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 Ca -binding sites of the EF-hand type (Donato, 2003). S100 proteins have been implicated in the regulation of protein phosphorylation, Ca homeostasis, enzyme activity, gene transcription, cell growth and differentiation, and the inflammatory response (Schäfer and Heizmann, 1996; Donato, 1999). Alterations of S100 function have This work was supported by United States Public Health Service grant MH045372 (to K.A.N.), the Veterans Affairs Merit Review and Career Scientist programs (to K.A.N.), and the N. L. Tartar Trust (to Y.L.). Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.108.044925. 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. 0026-895X/08/7402-371–378 MOLECULAR PHARMACOLOGY Vol. 74, No. 2 U.S. Government work not protected by U.S. copyright 44925/3358914 Mol Pharmacol 74:371–378, 2008 Printed in U.S.A. 371 at A PE T Jornals on Jne 1, 2017 m oharm .aspeurnals.org D ow nladed from 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 Ca -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 histidinetagged 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 signalregulated kinases (ERKs) and inhibition of adenylate cyclase in HEK293 cells. Materials and Methods Materials. The B2H System and BacterioMatch II Rat Brain Library were purchase from Stratagene (La Jolla, CA). Quinpirole, 7-OH DPAT, ( )-butaclamol, 3-isobutyl-1-methylxanthine, adenine HCl, n-dodecylmaltoside, ethylene-bis(oxyethylenenitrilo)tetraacetic acid, 3-amino-1,2,4-triazole (3-AT), and culture media were purchased from Sigma-Aldrich (St. Louis, MO). Histidine dropout supplement (mixtures of amino acids and other nutrients) was purchased from BD Biosciences Clontech (Palo Alto, CA). [H]Spiperone (95 Ci/mmol) was purchased from GE Healthcare Bio-Sciences (Piscataway, NJ). Fetal and calf bovine sera for cell culture were purchased from HyClone (Logan, UT). The Lipofectamine 2000 cell transfection kit was purchased from Invitrogen (Carlsbad, CA). Precast gels and rat neostriatal neurons were purchased from Lonza Walkersville (Walkersville, MD). Protein G Plus agarose, normal rabbit IgG, and normal mouse IgG were from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies used include: rabbit anti-dopamine D2L/S (1/500 dilution; Millipore, Billerica, MA), mouse antiS100B (1/1000 dilution; GeneTex, San Antonio, TX), rabbit anti-His (1/500 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-FLAG M2 (1/1000 dilution; Sigma), rabbit anti-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 SuperSignal 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-D2IC3 and the BacterioMatch II Rat Brain Library (Stratagene) according to the manufacture’s protocol (Stratagene). Detection of proteinprotein 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 g/ml; puromycin 2 g/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 (mycD2/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-D2HEK cells using Lipofectamine 2000 transfection reagent and selection with puromycin (2 g/ml) and G418 (600 g/ml). Cell lines expressing the myc-tagged D2 receptor and FLAG-tagged S100B were isolated by screening via radioligand binding using [H]spiperone and via immunoblot analysis using a mouse anti-myc and a mouse anti-FLAG antibody. The binding of [H]spiperone was assessed as described previously (Liu et al., 2006), and the c-myctagged D2 receptor with coexpression of FLAG-tagged S100B had similar affinity for [H]spiperone as reported previously for the cmyc-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 372 Liu et al. at A PE T Jornals on Jne 1, 2017 m oharm .aspeurnals.org D ow nladed from 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/FLAGS100B-HEK293 cells from confluent 10-cm plates were washed and incubated twice for 3 min each time with calciumand 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 g 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 l 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 PullDown 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 BamHISalI 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 -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 g/ml) at 37°C to A600 0.5 and induced with 0.5 mM isopropyl -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 SDSPAGE, 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-568tagged 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 antiGAPDH 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 l) 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 M forskolin-stimulated cAMP accumulation was measured in intact myc-D2-HEK293 cells and myc-D2/FLAGS100B-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 M 3-isobutyl-1methylxanthine (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 M 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 l 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).