Characterization of regulators of G-protein signaling RGS4 and RGS10 proteins in the postmortem human brain

Neurochemistry International 57 (2010) 722–729

Contents lists available at ScienceDirect

Neurochemistry International journal homepage: www.elsevier.com/locate/neuint

Characterization of regulators of G-protein signaling RGS4 and RGS10 proteins in the postmortem human brain Guadalupe Rivero a,b,1, Ane M. Gabilondo a,b,*, Jesu´s A. Garcı´a-Sevilla c,d, Romano La Harpe e, Benito Morentı´n f, J. Javier Meana a,b a

Department of Pharmacology, University of the Basque Country, E-48940 Leioa, Bizkaia, Spain Centro de Investigacio´n Biome´dica en Red de Salud Mental (CIBERSAM), Spain Laboratory of Neuropharmacology, IUNICS, University of the Balearic Islands, E-07122 Palma de Mallorca, Spain d Red Tema´tica de Investigacio´n Cooperativa en Salud (RETICS-Trastornos Adictivos), Spain e Centre Universitaire Romand de Me´decine Le´gale-Site Gene`ve, Faculte´ de Me´decine, University of Geneva, CH-1211 Gene`ve 4, Switzerland f Basque Institute of Legal Medicine, E-48001 Bilbao, Bizkaia, Spain b c

A R T I C L E I N F O

A B S T R A C T

Article history: Received 21 January 2010 Received in revised form 29 July 2010 Accepted 11 August 2010 Available online 9 September 2010

Regulator of G-protein signaling (RGS) proteins are a large family of proteins that accelerate GTPase rate of the Ga subunits and therefore, negatively regulate G-protein signaling. Expression of RGS4 and RGS10 proteins was characterized in human prefrontal cortex attending to methodological (subcellular localization, antibody specificity and sensitivity, postmortem delay (PMD) and storage conditions of the samples) and demographic issues (age and gender of the subjects). Anti-RGS4 (N-16) antibody revealed a unique and specific band of 38 kDa that was highly enriched in the plasma membrane. Anti-RGS10 (C20) antibody revealed two specific bands of 24 and 27 kDa, corresponding to two possible isoforms of this protein, which were predominantly localized in the cytosol. Antibody dilution and protein linearity studies confirmed the sensitivity of the signal. A large number of samples from 58 individuals presenting well spread PMD, storage time, age of the subjects at the time of death, and male and female distribution were studied. A positive linear relationship between the age and RGS4 immunoreactivity was observed. There was a negative influence of PMD on the RGS10 27 kDa band immunoreactivity but a positive relationship emerged between the PMD and RGS4 immunoreactivity. Storage time of the samples did not have any influence on RGS4 nor on RGS10 immunoreactivity, showing their stability at 70 8C. When studying the RGS4 and RGS10 protein expression density in males and females, no significant difference was found between groups. This study demonstrates that RGS4 and RGS10 proteins can be detected by immunoreactive techniques in postmortem human brain cortex. The study provides important matching conditions that should be taken into account in postmortem brain studies of neuropsychiatric diseases. ß 2010 Elsevier Ltd. All rights reserved.

Keywords: Regulators of G-protein signaling RGS4 RGS10 Human brain Postmortem changes Aging Western blot

1. Introduction Heterotrimeric G-proteins mediate cellular responses of Gprotein coupled receptors (GPCRs) by serving to activate or inhibit cellular signaling pathways. They represent a widely used mechanism for signal transduction from membrane receptors to intracellular effectors. The amplitude and lifetime of this signaling are dictated by the lifetime of GTP, which suffers hydrolysis to GDP + Pi on the Ga subunit.

* Corresponding author at: Department of Pharmacology, University of the Basque Country, E-48940 Leioa, Bizkaia, Spain. Tel.: +34 946 015 572; fax: +34 946 013 220. E-mail address: [email protected] (A.M. Gabilondo). 1 Current address: Department of Physiology and Pharmacology, University of Bristol, BS81TD Bristol, United Kingdom. 0197-0186/$ – see front matter ß 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2010.08.008

Regulator of G-protein signaling (RGS) proteins function as GTPase activating proteins (GAPs), accelerating the GTPase rate of the G-protein and promoting its deactivation. Although early evidence suggested that RGS proteins acted primarily as negative regulators of G-protein signaling, later findings indicate that these proteins act as tightly regulated modulators and/or multifunctional integrators of G-protein signaling (see review in Hollinger and Hepler, 2002). RGS proteins appear to serve scaffolding functions for receptor signaling complexes (Zhong et al., 2003) and to directly modulate effectors (Sinnarajah et al., 2001; Anger et al., 2004; Ghavami et al., 2004; see review in Abramow-Newerly et al., 2006). All these regulatory mechanisms are of great physiological interest (Ishii and Kurachi, 2003) and probably also of pathophysiological relevance in various human diseases (Tekumalla et al., 2001; Nishiguchi et al., 2004; Emilsson et al., 2006; Talkowski et al., 2006).

G. Rivero et al. / Neurochemistry International 57 (2010) 722–729

RGS proteins modulate signaling from several mammalian GPCRs, including serotonin (Leone et al., 2000; Ghavami et al., 2004; Shi et al., 2006; Gu et al., 2007), dopamine (Yan et al., 1997), muscarinic acetylcholine (Ding et al., 2006), metabotropic glutamate (Saugstad et al., 1998), GABAB (Schiff et al., 2000), a2adrenoceptor (Cavalli et al., 2000; Hoffmann et al., 2001), m- and dopioid receptors (Garzo´n et al., 2005; Georgoussi et al., 2006). Twenty-one RGS genes have been identified in human and nineteen of them have shown to exert GAP activity (Sierra et al., 2002). RGS proteins have shown preference for certain Ga subunits over others. For instance, although they belong to different families (see review in Ross and Wilkie, 2000), both RGS4 and RGS10 proteins regulate Gi proteins activity (Berman et al., 1996; Hunt et al., 1996; Huang et al., 1997; Cavalli et al., 2000). Recent studies have involved the RGS4 protein in several CNS disorders, such as schizophrenia (Mirnics et al., 2001; Morris et al., 2004; Williams et al., 2004; Prasad et al., 2005; Talkowski et al., 2006; Ding and Hegde, 2009), anxiety (Leygraf et al., 2006), Alzheimer’s disease (Muma et al., 2003; Emilsson et al., 2006), regulation of pain and sensitivity to morphine (Garnier et al., 2003), dependence (Gold et al., 2003) and tolerance to morphine (Garzo´n et al., 2005) and other drugs of abuse (Bishop et al., 2002; Schwendt et al., 2006). On the other hand, association of RGS10 gene and schizophrenia (Hishimoto et al., 2004) and the modulation of both RGS4 and RGS10 by acute and chronic electroconvulsive seizures in rat brain (Gold et al., 2002) have been studied. The only approach that permits an analysis of the pathophysiological conditions of human brain at subcellullar level or protein level is represented by the use of autopsy tissue. However, postmortem human brain studies present several methodological issues as the unavoidable delay between death and tissue dissection, the age of subjects and the length and temperature of sample storage prior to assay (Perry and Perry, 1983). Due to several difficulties such as high rates of proteolysis (Krumins et al., 2004) and the scarceness of commercially available antibodies, most of the studies have measured mRNA expression of RGS proteins instead of direct protein density. Tissue distribution and levels of mRNA expression for the known human RGS members have been characterized (Larminie et al., 2004). However, mRNA expression is not always representative of protein density and thus, not much is known about the protein expression of RGS4 and RGS10 in the human brain. In this context, the aim of the present study was, first, to characterize the immunodetection of RGS4 and RGS10 proteins in postmortem human brain by commercially available antibodies, attending to subcellullar localization, specificity and sensitivity of the signal, and second, to investigate the effect of normal aging, gender and limiting parameters such as the postmortem delay (PMD) and the storage time of the samples at 70 8C on the immunodetection of RGS4 and RGS10 proteins in postmortem human brain samples. 2. Experimental procedures 2.1. Subject selection Human brains were obtained at autopsy from the Basque Institute of Legal Medicine, Bilbao, Spain and the Romand University Center of Legal Medicine, Geneva, Switzerland. The study was developed in compliance with policies of research and ethical review boards for postmortem brain studies (Basque Institute of Legal Medicine, Bilbao and Department of Psychiatry, University of Geneva, Geneva). In typical conditions, the corpse is stored at refrigeration temperature (4 8C) until autopsy. Samples from the right prefrontal cortex (Brodmann’s area 9) were dissected at the time of autopsy and immediately stored at 70 8C until assay. Toxicological screening (quantitative assays for antidepressants, antipsychotics, other psychotropic drugs, opiates and ethanol) was performed on blood samples at the National Institute of Toxicology, Madrid, Spain, or at the Toxicology Unit, University Center of Legal Medicine, Geneva, using a variety of standard procedures including radioimmunoassay, enzymatic immunoassay, high-performance liquid chromatography and gas chromatography–mass spectrometry.

723

A total of 66 brain specimens, that provided a wide range for the variables to be evaluated, were used. Samples of prefrontal cortex from eight subjects not included in the rest of the study were used in the characterization of RGS4 and RGS10 protein inmunodetection. Six brain specimens of the eight used in the characterization were pooled and used for the standard sample preparation. This preparation was used in routine experiments as a value of control (referring each value as a percentage of this standard). A group of samples from 58 different subjects was used to evaluate the effect of age, gender, PMD and storage time in RGS4 and RGS10 immunoreactivity. The samples were obtained from 35 men and 23 women. The age of the subjects at the time of death ranged from 15 to 88 years (mean: 41  2 years). The PMD ranged from 3 to 76 h (mean: 27  2 h) and the storage time at 70 8C ranged from 26 to 151 months (mean: 74  4 months). The pH of the human samples was 6.44  0.05. The group of 58 brain specimens was chosen on the basis of the following criteria: (a) medical information on the absence of neuropsychiatric disorders or drug abuse; (b) cause of death: accidental (n = 37), natural (n = 12), burned (n = 2), and homicide (n = 8); (c) toxicological screening of psychotropic drugs with negative results except for ethanol. The past medical records of the selected subjects revealed the presence of two subjects with cardiomyopathy. Toxicological screening in blood samples was performed in 55 from the 58 subjects and any of them revealed the presence of psychotropic drugs, except ethanol that was detected in 13 subjects (blood concentration: range 0.11–3.09 g/l; mean: 1.10  0.26 g/l). A possible influence of acute ethanol ingestion was discarded as its levels did not correlate with RGS4 and RGS10 protein immunoreactivity (data not shown). 2.2. Brain sample preparations Prefrontal cortex tissue samples of each subject (100 mg) were homogenized in 15 volumes of homogenization buffer (1 mM EDTA, 10 mM MG132 (Calbiochem, San Diego, CA, USA), 50 ml/g tissue Protease inhibitor cocktail (P-8340 Sigma, St Louis, MO, USA) and 50 mM Tris–HCl, pH 7.5 using an Ultra-Turrax T8 (IKA Labortechnik, Staufen, Germany) at maximum speed for 10 s (4 8C). Crude homogenates were centrifuged for 10 min at 600  g (4 8C). The pellet (P1 fraction) was discarded and the supernatants were then recentrifuged for 10 min at 40,000  g (4 8C). The resultant supernatant and the pellet (P2) corresponded to the cytosolic fraction and the plasma membrane fraction, respectively. Protein content was measured according to the method of Bradford (1976) using bovine serum albumine (BSA) as standard, and it was similar in the different brain samples. Samples of total homogenates and nuclear fractions proteins from human brain frontal cortex were prepared as described in Ramos-Miguel et al. (2009). All samples were aliquoted and stored at 70 8C until use. In routine experiments, different preparations of the samples from human brain or full-length recombinant GST-tagged human RGS4 and RGS10 proteins (Abnova Corporation, Taipei, Taiwan) were diluted in Laemmli sample buffer (final buffer concentrations: 50 mM Tris–HCl, pH 6.8, 2% SDS, 10% glycerol, 2.5% bmercaptoethanol and 0.01% bromophenol blue, Bio-Rad Laboratories, Hercules, CA, USA) to reach the final protein concentration (plasma membrane fractions: 0.48  0.05 mg/ml, cytosolic fractions: 0.45  0.01 mg/ml; total homogenates: 1.5 mg/ml; nuclear fractions: 2.5 mg/ml; recombinant RGS4 and RGS10 proteins: 0.1 mg/ml). The samples were then denatured at 95 8C for 5 min.

2.3. Immunoblot analysis and quantitation of immunoreactivity of the target proteins 1–46 mg of protein of each brain sample and 1 mg of full-length recombinant GST-tagged human RGS4 and RGS10 proteins were loaded and separated by electrophoresis at 120 V for about 90 min on 15-well (6 cm  8 cm gels, 0.75– 1.5 mm-thick, Bio-Rad Laboratories, Hercules, CA, USA) minigels of sodium dodecyl sulphate-polyacrilamide (12%). Proteins were electrophoretically transferred (90 V for 1 h) to nitrocellulose membranes (Schleicher & Schuell GmbH, Dassel, Germany). The nitrocellulose membranes were incubated 1 h at room temperature in blocking solution (phosphate-buffered saline solution with 5% nonfat dry milk, 0.2% Tween-20, and 0.5% BSA, pH 7.4) and then, they were incubated overnight at 4 8C in blocking solution containing the appropriate primary antibody. For the different proteins, the following primary antibodies were used: (a) goat polyclonal anti-RGS4 antibody (raised against a peptide mapping at the amino terminus of RGS4 of human origin, N-16, sc-6204, lots C221 and K1802, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA); (b) goat polyclonal anti-RGS10 antibody (raised against a peptide mapping at the carboxy terminus of RGS10 of human origin, C-20, sc-6206, lot F018, Santa Cruz Biotechnology Inc.). Various dilutions of anti-RGS4 and anti-RGS10 antibodies were checked and 1:5000 was selected as the optimal dilution for the immunodetection of both proteins by Western blot. The secondary antibodies, either horseradish peroxidase-linked rabbit anti-goat IgG (Sigma) (1:25,000 dilution for the detection of both proteins) or Alexa Fluor 680 conjugated donkey anti-goat IgG (Molecular Probes, Eugene, OR, USA) (1:25,000 dilution for the detection of RGS4 and 1:10,000 dilution for the detection of RGS10) were incubated in blocking solution at room temperature for 1 h. In the first case, bound antibody (immunoreactivity) was detected using the enhanced chemiluminescence (ECL) Western blot detection system (Amersham, Buckinghamshire, UK) and visualized by exposure to autoradiographic film (AGFA CURIX RP2 from AgfaGevaert, Mortsel, Belgium or Hyperfilm, Amersham, Buckinghamshire, UK) for 5 s to

724

[(Fig._2)TD$IG]

G. Rivero et al. / Neurochemistry International 57 (2010) 722–729

2 h (autoradiograms). The autoradiograms were scanned with the EPSON Perfection 1240U scanner (Seiko Epson Corporation, Nagano, Japan) and analyzed by quantitative densitometry (measurements of optical density), using the NIH Image or Scion Image (based on NIH Image for Macintosh) from the National Institute of Health created by Wayne Rasband, Bethesda, USA (available from http://rsb.info.nih.gov/nih-image (accessed December 2009) and http://www.scioncorp.com/pages/ scion_image_windows.htm (accessed December 2009), respectively). On the other hand, those membranes incubated with fluorescent conjugated antibodies were detected and quantified using the Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA) and integrated intensity values (II) were obtained. Each gel contained a prestained broad-range protein ladder to measure the molecular masses of individual bands. Equal loading of the total protein content in the gel was verified by the immunodetection of b-actin. For that purpose, the nitrocellulose membranes detected by chemiluminescence were stripped and b-actin was detected using mouse monoclonal anti-b-actin antibody (clone AC-15, lot 104K4787, Sigma) at 1:5000 dilution as primary antibody and horseradish peroxidase-linked sheep antimouse IgG antiserum (Amersham) (1:10,000 dilution) as secondary antibody. On the other hand, for the fluorescence detection method, the nitrocellulose membranes were not stripped and b-actin was detected at the same time as target proteins using mouse monoclonal anti-b-actin antibody at 1:5000 dilution as primary antibody and IR Dye 800 conjugated donkey anti-mouse IgG (Rockland Immunochemicals, Philadelphia, PA, USA) as secondary antibody (1:10,000 dilution). To assess the effect of PMD, storage time, age and gender of the subjects on RGS protein expression, duplicate problem samples were evaluated using a standard sample loaded on the same gel and this quantification procedure was repeated at least twice in different gels, for a total number of four measurements for each subject sample. Immunoreactivity values of target proteins were corrected for the amount of b-actin and given as a percentage of b-actin immunoreactivity. In order to prevent interexperimental variability, the relative amount of the target protein was also calculated as a percentage of the value obtained for the standard sample loaded on the same gel. 2.4. Statistics Generally, results are expressed as means  S.E.M. (standard error of the mean). The multiple regression analyses were carried out using the SPSS 14.0 programme (SPSS Inc., Chicago, IL, USA) to assess the contribution of gender and continuous predictor variables (age, PMD, storage time) to the immunoreactivity of both RGS4 and RGS10 proteins. Pearson’s correlation coefficients were calculated by the method of least squares to test for possible association between continuous descriptive variables (age, PMD and storage time) and RGS4 and RGS10 protein immunoreactivities by using GraphPad Prism 4.01 software (GraphPad Software Inc., La Jolla, CA, USA). The level of significance was chosen as p = 0.05.

3. Results 3.1. Subcellular distribution of RGS4 and RGS10 proteins in human prefrontal cortex: cytosolic and plasma membrane fractions Immunoblot analysis of samples of prefrontal cortex with antiRGS4 antibody demonstrated the presence of a single immunoreactive band with a molecular mass of 38 kDa (Figs. 1 and 2). The subcellular localization of RGS4 protein was shown to be almost

[(Fig._1)TD$IG]

Fig. 1. Representative immunoblots depicting labelling of immunodetectable regulator of G-protein signaling (RGS) RGS4 and RGS10 proteins in postmortem human brain (prefrontal cortex, Brodmann’s area 9). Subcellular distribution of RGS4 and RGS10 proteins is shown for the corresponding total homogenates (TH) and plasma membrane (P2), cytosolic (S), and nuclear (N) fractions. The amount of protein from each cellular preparation loaded on the gel (8 mg or 16 mg) is indicated at the bottom of the figure. The immunoblots show the predominant expression of RGS4 in plasma membrane and RGS10 in cytosolic fraction.

Fig. 2. Immunoblots of RGS4 and RGS10 proteins in plasma membrane and cytosolic fractions, respectively. The apparent molecular masses of RGS4 (38 kDa) and RGS10 (24–27 kDa) proteins were determined by calibrating the blots with prestained molecular weight markers as shown on the left side. The specificity of the antibodies anti-RGS4 (top) and anti-RGS10 (bottom) was assessed by preincubating the corresponding antibody with its antigenic peptide, RGS4 peptide (p4) and RGS10 peptide (p10) (preadsorbed antibody), which resulted in the blockade of the immunoreaction for the specific protein. Afterwards, the antibodies were preincubated with the opposing antigenic peptide and with RGS5 N-terminal peptide (p5), which resulted in no blockade of the immunoreaction for the specific protein.

exclusive of the plasma membrane (Fig. 1). Anti-RGS10 antibody demonstrated the presence of two immunoreactive bands with molecular masses of 27 and 24 kDa (Figs. 1 and 2). The analysis of the subcellular localization showed the predominance of RGS10 protein in the cytosolic fraction (Fig. 1). Taking into account these observations, plasma membrane fractions were used to perform the routine immunoblot analyses for the RGS4 protein and cytosolic fractions for the RGS10 protein. 3.2. Characterization of antibodies for the immunodetection of RGS4 and RGS10 proteins in human prefrontal cortex The antibodies used were tested for their specificity on Western blots of human brain tissue. Previous incubation of the anti-RGS4 antibody with the antigenic peptide for RGS4 protein (p4) as a preadsorbed antibody test, resulted in the blockade of the immunoreaction for the specific protein, which confirmed the specificity of the antibody (Fig. 2). When preadsorbing the antiRGS4 antibody with the antigenic RGS10 peptide (p10) and also with the RGS5 N-terminal peptide (p5) the RGS4 band was still detected, revealing no cross-reaction of the antibody anti-RGS4 with RGS10 and with RGS5 proteins (Fig. 2). The two bands of 27 and 24 kDa resulting of the incubation of the blotting membrane with anti-RGS10 antibody were blocked when the antibody was preincubated with the corresponding antigenic RGS10 peptide (p10) (Fig. 2). However, preincubation of the anti-RGS10 antibody with the antigenic RGS4 peptide (p4) or with the RGS5 N-terminal peptide (p5), resulted in no blockade of the RGS10 signal, which confirmed the specificity of the antibody (Fig. 2). Anti-RGS4 and anti-RGS10 antibodies were secondly tested for specificity on Western blots of 1 mg of full-length recombinant GST-tagged human RGS4 (r4) and RGS10 (r10) proteins (Fig. 3). The recombinant RGS4 protein was detected by the commercial anti-

[(Fig._3)TD$IG]

[(Fig._4)TD$IG]

G. Rivero et al. / Neurochemistry International 57 (2010) 722–729

725

Fig. 3. Identification of RGS4 (top) and RGS10 (bottom) proteins by Western blot in human brain plasma membranes (P2) and cytosolic fraction (S) and in samples of full-length recombinant GST-tagged human RGS4 protein (r4) and full-length recombinant GST-tagged human RGS10 protein (r10) using the N-terminal (N-16) anti-RGS4 and the C-terminal (C-20) anti-RGS10 antibodies from Santa Cruz Biotechnology Inc. The specificity of the signal was confirmed by the lack of immunoreaction of RGS4 antibody with the full-length recombinant GST-tagged human RGS10 protein (r10) and viceversa.

RGS4 antibody (N-16) with a molecular mass of approximately 49 kDa, that would correspond to the sum of the 23 kDa of the 205 amino acidic sequence of the RGS4-1 and/or RGS4-2 isoforms (Ensembl Transcript ID: ENST00000367909) (Ding et al., 2007) plus the 26 kDa due to the GST fraction. Another band of 31 kDa was also detected in the lane loaded with recombinant RGS4 protein (r4) but it was attributed to degraded fragments of this recombinant RGS4 protein (Fig. 3). The recombinant RGS10 protein was detected by the commercial anti-RGS10 antibody (C-20) with a molecular mass of approximately 46 kDa, corresponding to the sum of the 20 kDa of the 173 amino acidic sequence of the RGS10 protein plus the 26 kDa due to the GST fraction (Fig. 3). The recombinant RGS10 protein could not be detected by the antiRGS4 antibody and the recombinant RGS4 protein was not detected by the anti-RGS10 antibody, further confirming the specificity of both antibodies (Fig. 3). When incubating the Western blots of prefrontal cortex cytosolic fraction with anti-RGS10 antibody, sometimes other unknown bands were also visualized. These bands were ascribed to the incubation with the secondary antibody because they still appeared in the absence of the primary antibody whereas no signal of the RGS10 protein was detected (data not shown). 3.3. Protein linearity for quantitation of immunoreactivity of RGS4 and RGS10 proteins in human prefrontal cortex In the 1–8 mg range, a linear relationship was established between the optical density of RGS4 and the total protein content

Fig. 4. Saturation of the immunoreactivity signal of target proteins (expressed by optical density units, OD) by increasing total protein content loaded in the gel. (A) For RGS4 protein, 1–46 mg of total protein content (plasma membrane fraction) were loaded in the same gel resulting in increasing immunoreactivity, which became saturated at 10 mg. Inset of the first graph shows the linear relationship between total protein content 1–8 mg (plasma membrane fraction) and the immunoreactivity of RGS4 protein in a representative experiment. This observation led to fix 4 mg as the appropriate protein content to perform the routine experiments. (B) For RGS10 protein, 1–26 mg of total protein content (cytosolic fraction) were loaded in the same gel resulting in saturation of the signal obtained for the 24 and 27 kDa isoforms at 20 mg. Insets of these graphs show the linear relationship between total protein content 4–18 mg (cytosolic fraction) and the immunoreactivity of both 24 and 27 kDa isoforms of the RGS10 protein in a representative experiment. These observations led to fix 8 mg as the appropriate protein content to perform the routine experiments.

of the plasma membrane sample (inset of Fig. 4A). In the superior range (10–40 mg), the optical density became saturated (Fig. 4A). Thus, 4 mg of plasma membrane proteins was selected as the total protein content most suitable for the immunodetection of RGS4 in the human brain. For RGS10 protein, increasing concentrations of total cytosolic proteins were loaded on the gel and the 27 and 24 kDa band immunoreactivities were measured. The optical signal of both 27 and 24 kDa bands linearly augmented in the 4–18 mg range (insets of Fig. 4B) and became saturated with total protein contents superior to 18 mg (Fig. 4B). These observations led to use a total

[(Fig._5)TD$IG]

726

G. Rivero et al. / Neurochemistry International 57 (2010) 722–729

Fig. 5. Effect of the variables age and postmortem delay (PMD) on the immunoreactivity of RGS4 (n = 58) and RGS10 (n = 57) proteins in the prefrontal cortex of human brain. Duplicate problem samples were evaluated using a standard sample loaded on the same gel and this quantification procedure was repeated at least twice in different gels, for a total number of four measurements for each subject sample. The problem sample values were corrected for the amount of b-actin and given as a percentage of immunoreactivity. The corrected problem sample values were referred to the standard sample’s value (control value) as a percentage. (A) Effect of the age (15–88 years) of the subjects included in the study on the immunoreactivity of RGS4 and RGS10 proteins. (B) Effect of PMD (3–76 h) on the immunoreactivity of RGS4 and RGS10 proteins. The influence of the age of the subjects and the PMD on the target proteins was studied by linear regression analyses, where r values are the Pearson’s correlation coefficients and the lines represent the regressions of the correlations (for age: RGS4 38 kDa: y = 61.45 + 0.967x, n = 58; RGS10 27 kDa: y = 103.1 + 0.019x, n = 57; RGS10 24 kDa: y = 117 0.456x, n = 57; and for PMD: RGS4: y = 84.32 + 0.618x, n = 58; RGS10 27 kDa: y = 123.3 0.740x, n = 57; RGS10 24 kDa: y = 109.9 0.439x, n = 57). (*) RGS10 27 kDa, solid line; (*) RGS10 24 kDa, dashed line. For numeric data see Section 3.

cytosolic protein content of 8 mg to perform the routine immunodetection of the 27 and 24 kDa bands. 3.4. Methodological and demographic variables contributing to RGS4 and RGS10 immunoreactive protein expression in the human brain Multiple regression analyses in the entire group of human brain samples (n = 58) revealed that the expression of RSG4 and RGS10 proteins was affected by methodological and demographic variables. The contribution to the variance of the predictor variables entered in the multiple regression models varied from 32% to 47% (Pearson’s correlation r = 0.32–0.47, p values <0.05), depending on the measured protein and the predictor variable. For the RGS4 protein, a significant model emerged (F[3,54] = 10.709, adjusted R2 = 0.338, p < 0.05). According to this model the best predictors of individual protein immunoreactivity in the plasma membrane were the age at death of the subject (b = 0.371, p = 0.001) and the PMD (b = 0.277, p < 0.05), regardless of the gender of the subjects (b = 0.078, p = 0.513) and the storage time of the samples (b = 0.127, p = 0.293). The contribution of the age at death of the subject and the PMD to the RGS4 protein expression were further assessed by a linear regression model. RGS4 immunoreactivity significantly augmented with the age (slope: 0.97  0.24, r = 0.467, p = 0.0002, range 15–88 years) (Fig. 5A). For the PMD, linear regression analysis between PMD and RGS4 immunoreactivity revealed a positive linear relationship (slope: 0.62  0.24, r = 0.320, p = 0.0144, range 3–76 h) (Fig. 5B). Multiple regression analysis was also carried out in order to investigate the contribution of the same methodological and demographic variables to the immunoreactivity of both RGS10 27 kDa and RGS10 24 kDa bands in the cytosolic fraction. The immunoreactivity of RGS10 27 kDa band was only predicted by the PMD, which accounted for the 9% of the variance (F[1,55] = 6.650, adjusted R2 = 0.092, p < 0.05, b = 0.328), resulting in a negative linear relationship between PMD and RGS10 27 kDa immunoreac-

tivity levels (slope: 0.74  0.29, r = 0.328, p = 0.0126, range 3– 76 h) (Fig. 5B). The variables age and gender of the subject and storage time of the sample did not account for modifications on RGS10 27 kDa immunoreactivity (age: b = 0.076, p = 0.566; gender: b = 0.089, p = 0.498; storage time: b = 0.063, p = 0.637). RGS10 24 kDa immunoreactivity was the least sensitive and its expression was not significantly affected by any of the variables analyzed. These results are in concordance with those obtained in total homogenates of human prefrontal cortex from 11 random control subjects. As in plasma membranes, RGS4 protein immunoreactivity in total homogenates also showed a positive linear relationship with the age at death of the subjects (slope: 1.93  0.60, r = 0.585, p = 0.004, range 23–52 years) and with the PMD (slope: 0.67  0.19, r = 0.611, p = 0.002, range 10–102 h), without a significant effect of the gender of the subjects (p = 0.264, 7 male and 4 female) and the storage time of the samples (p = 0.545, range 30–72 months). Moreover, as in the cytosolic fraction, RGS10 immunoreactivity (both 27 and 24 kDa bands) in total homogenates showed a negative linear relationship with the PMD (slope: 0.67  0.17, r = 0.718, p = 0.001) without a significant effect of the age at death (p = 0.146), gender of the subjects (p = 0.100), and the storage time of the samples (p = 0.563). 4. Discussion This study provides a complete characterization of RGS4 and RGS10 proteins in the postmortem human brain by means of an appropriate and reliable method for the quantification of the expression of these proteins. In the human brain different patterns of subcellular localization were observed for the target proteins RGS4 and RGS10. In agreement with current knowledge, RGS4 protein exhibited majoritary plasma membrane localization. A recent immunogold electron microscopic analysis in prefrontal cortex of rhesus macaques found RGS4 protein to be highly localized in plasma membranes being strategically positioned to regulate synaptic transmission (Paspalas et al., 2009).

G. Rivero et al. / Neurochemistry International 57 (2010) 722–729

This predominant localization of RGS4 protein in the plasma membrane is probably due to the amphipathic a-helix formed by its N-terminal 33 amino acids (Bernstein et al., 2000) and it is tightly coupled with the ability of RGS4 to inhibit signaling (Srinivasa et al., 1998; Tu et al., 2001). The immunoreactive band obtained for the RGS4 protein resulted in a molecular mass of 38 kDa. Recently, five splice variants of RGS4 protein have been described in the human brain: RGS4-1 and RGS4-2 of 205 amino acids (Ensembl Transcript ID: ENST00000367909; available from: http://www.ensembl.org/ Homo_sapiens/Gene/Summary?db=core;g=ENSG00000117152;r= 1:163038396-163046592;t=ENST00000367909, accessed December 2009), RGS4-3 of 302 amino acids (Ensembl Transcript ID: ENST00000421743) and the truncated forms RGS4-4 and RGS4-5 of 93 amino acids and 187 amino acids, respectively (Ensembl Transcript IDs: ENST00000367908 and ENST00000367906) (Ding et al., 2007). Therefore, the 38 kDa band from the present work may well correspond to the RGS4-3 denominated variant, which is detected with a 35 kDa molecular weight (Ding et al., 2007). Other 25 kDa and 15 kDa bands, probably corresponding to the other isoforms have been previously described (Schwendt et al., 2006; Ding et al., 2007) but they were not detected by the anti-RGS4 (N-16) antibody probably due to the predominancy of the 38 kDa RGS4-3 isoform in the human brain. The RGS4-3 isoform (302 amino acids) differs from the RGS4-1 and RGS4-2 isoforms (205 amino acids) in that it possesses a 97 amino acid N-terminal extension. This Nterminal extension could confer different receptor selectivity and/or particularly higher plasma membrane localization. Specific analysis of different RGS4 isoforms can be of great importance. Thus, in dorsolateral prefrontal cortex of schizophrenic subjects the expression of the splice variant RGS4-3 has been described to be decreased compared with a normal healthy group, whereas overall RGS4 expression and expression of other RGS4 isoforms did not differ between groups (Ding and Hegde, 2009). In contrast, RGS10 protein appeared to be primarily a cytosolic protein and could be detected as a double band of 24–27 kDa. These two bands may probably correspond to two of the three suggested isoforms of the human RGS10 protein (Haller et al., 2002) of 181, 173 and 167 amino acids (Ensembl Transcript IDs: ENST00000369103, ENST00000369101 and ENST00000392865, respectively; available from: http://www.ensembl.org/Homo_sapiens/Gene/Summary? db=core;g=ENSG00000148908;r=10:121259340-121302220;t= ENST00000369101, accessed December 2009). Although in the present study the RGS10 protein could not be detected in the nuclear fraction from human brain samples, numerous laboratories have described nuclear localization (Chatterjee and Fisher, 2000; Burgon et al., 2001; Waugh et al., 2005) as well as cytoplasmic localization (Rimler et al., 2006) of this protein in other systems. Phosphorylation via cAMP-dependent protein kinase A (PKA) has been proposed as a mechanism for translocation of RGS10 from the plasma membrane and cytosol into the nucleus (Burgon et al., 2001). Thus, RGS10 protein may localize in the plasma membrane, consistent with a role in modulating synaptic G-protein coupled receptor signaling but it also may be located in the nucleus, the cytoplasm or shuttle between the nucleus and the cytoplasm as nucleo-cytoplasmic shuttle protein (Chatterjee and Fisher, 2000; Rimler et al., 2006). Functional role of nuclear RGS10 remains unknown being one of the possible hypothesis the regulation of gene expression that has already been suggested for RGS12 and RGS9 proteins (Chatterjee and Fisher, 2002; Bouhamdan et al., 2004). In the absence of specific antibodies against endogenous proteins many researchers have relied on in situ hybridization to semiquantitatively predict the level of expressed protein (Gold et al., 2002; Geurts et al., 2003; Erdely et al., 2004). However, expression of RGS mRNA and protein densities do not necessarily change in unison (Gold et al., 2003) and monitoring protein

727

becomes a point of great importance in evaluating the impact of modulators on the physiological expression and function of RGS proteins. Some other investigators have questioned the ability of the antibody herein used to detect bona fide endogenous RGS4 (Krumins et al., 2004; Schwendt et al., 2006). A set of controls for the commercial antibodies from Santa Cruz Biotechnology, Inc. anti-RGS4 (N-16) and anti-RGS10 (C-20) were performed. In order to authenticate the staining pattern of the antibodies, both antiRGS4 and anti-RGS10 antibodies were subjected to preadsorption control tests (blockade of the specific signal by previous incubation with the antigenic peptide) resulting in antiserums of great specificity. It was also possible that the antiserums could exhibit cross reactivity with other RGS family members. Therefore, we incubated each antiserum with the blocking peptide corresponding to the other target protein and also with the RGS5 protein Nterminal peptide (N-14), which has around 70% sequence homology with the N-terminus of RGS4 protein and observed no cross-reaction of anti-RGS4 antibody nor of anti-RGS10 antibody. Furthermore, the incubation of the antibody with Western blots from full-length recombinant GST-tagged human RGS4 protein revealed an immunoreactive band of the expected size (49 kDa). The same procedure was carried out with Western blots from full-length recombinant GST-tagged human RGS10 protein but, in favour of the specificity of anti-RGS4 (N-16) antibody, anti-RGS4 failed to recognize RGS10 protein. In the same way, the anti-RGS10 antibody recognized the full-length recombinant GST-tagged human RGS10 protein, but not the full-length recombinant GST-tagged human RGS4 protein. The decline in cognitive and motor capabilities that often accompanies aging might represent the functional consequence of a decrease in the activities of signal transduction processes at neuronal level. Basal G-protein activity (i.e., [35S]GTPgS binding in the absence of agonist) was found to progressively decrease with the age of the subject at death (Gonza´lez-Maeso et al., 2002), even though the immunoreactivity of the majority of the G-protein a subunits were not significantly changed with aging. In the present work, RGS4 protein was found to significantly increase with age, which could lead to a decrease in G-protein activity. These present results extend previous findings indicating that the basal activity of G-protein/adenylyl cyclase signal transduction pathway declines with aging (Cowburn et al., 1992; Ozawa et al., 1999). On the other hand, RGS10 protein immunoreactivity was not altered during normal aging process, suggesting no implication of this protein in basal G-protein activity declining with age. Gender of the subjects was other demographic variable to study and, as expected, no significant differences in RGS4 and RGS10 immunoreactivity values were found between males and females. By contrast, Lipska et al. (2006) found RGS4 mRNA expression to differ between men and women. However, this mRNA differences may not be a general fact, as they did not replicate in all the brain collections included in their study and as they do not translate into differences in protein expression. Neuropsychiatric disease studies in postmortem human brain tissue are performed usually by matching cases and controls by gender and age of the subjects but inherent variables such as the PMD or the freezing storage time may produce changes that could alter the result of the analysis (Perry and Perry, 1983; Whitehouse et al., 1984; Burke and Greenbaum, 1987; Paul et al., 1997). In this context, the present data showed that unexpectedly, immunoreactivity values of RGS4 augmented with the length of PMD (range 3–76 h), suggesting strong postmortem stability of this protein. In disagreement with present results, RGS4 protein had been reported to be susceptible to degradation by the proteasome pathway (Davydov and Varshavsky, 2000; Kim et al., 1999) and to increase its optical signal significantly in PC12 and AtT20 cells

728

G. Rivero et al. / Neurochemistry International 57 (2010) 722–729

when exposed to proteasomal inhibitor MG132 or lactacystin, correlating with an increase in GAP activity (Krumins et al., 2004; Bodenstein et al., 2007). Whether in vitro pattern of degradation of RGS4 protein in cellular systems reproduces the postmortem degradation pattern of a human brain stored at 4 8C remains to be elucidated. Indeed, the lack of influence of the PMD on RGS4 mRNA expression had also been described by Lipska et al. (2006). On the other hand, immunoreactivity levels of the RGS10 27 kDa protein significantly decreased with the PMD suggesting a strong and rapid degradation of this protein that did not occur for the RGS10 24 kDa band. Finally, the effect of storage time of the human brain samples at 70 8C was analyzed and no alteration of the immunoreactivity of RGS4 and RGS10 proteins was found. In general, it is assumed that most biochemical activities are stable for several years when tissue is stored at very low temperatures ( 70 8C or less) (Perry and Perry, 1983) whereas decreases of the receptor density have been demonstrated when postmortem human brain samples are stored at 25 8C (Rodrı´guez-Puertas et al., 1996). Therefore, the present findings confirm the importance of examining potential artifacts that may alter RGS proteins immunoreactivity and so, impair a clear interpretation of the specific role of transduction mechanisms when control and cases for neuropsychiatric diseases are compared. Acknowledgements This study was supported by grants from the MICINN (SAF 04/ 02784 and SAF 09/08460 to J.J.M. and SAF 08/01311 to J.A.G.-S.), FIS 01/0358 and the Basque Government (IT 199/07) to J.J.M., and RETICS (RD06/0001/0003) to J.A.G.-S.; G.R. was recipient of a predoctoral fellowship from the Basque Government, Spain. The authors wish to thank the staff members of the Basque Institute of Legal Medicine, Bilbao, and the Romand University Center of Legal Medicine, Geneva, for their cooperation in the study. Dr. Javier Ballesteros, Department of Neurosciences, University of the Basque Country is thanked for the useful statistical assistance and Aintzane Garcı´a-Bea, Department of Pharmacology, University of the Basque Country for the technical assistance. References Abramow-Newerly, M., Roy, A.-A., Nunn, C., Chidiac, P., 2006. RGS proteins have a signaling complex, interactions between RGS proteins and GPCRs, effectors, and auxiliary proteins. Cell. Signal. 18, 579–591. Anger, T., Zhang, W., Mende, U., 2004. Differential contribution of GTPase activation and effector antagonism to the inhibitory effect of RGS proteins on Gq-mediated signaling in vivo. J. Biol. Chem. 279, 3906–3915. Berman, D.-M., Wilkie, T.-M., Gilman, A.-G., 1996. GAIP and RGS4 are GTPaseactivating proteins for the Gi subfamily of G protein alpha subunits. Cell 86, 445–452. Bernstein, L.-S., Grillo, A.-A., Loranger, S.-S., Linder, M.-E., 2000. RGS4 binds to membranes through an amphipathic alpha-helix. J. Biol. Chem. 275, 18520– 18526. Bishop, G.-B., Cullinan, W.-E., Curran, E., Gutstein, H.-B., 2002. Abused drugs modulate RGS4 mRNA levels in rat brain, comparison between acute drug treatment and a drug challenge after chronic treatment. Neurobiol. Dis. 10, 334–343. Bodenstein, J., Sunahara, R.-K., Neubig, R.-N., 2007. N-Terminal residues control proteasomal degradation of RGS2, RGS4, and RGS5 in human embryonic kidney 293 cells. Mol. Pharmacol. 71, 1040–1050. Bouhamdan, M., Michelhaugh, S.-K., Calin-Jageman, I., Ahern-Djamali, S., Bannon, M.-J., 2004. Brain-specific RGS9-2 is localized to the nucleus via its unique proline-rich domain. Biochim. Biophys. Acta 1691, 141–150. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Burgon, P.-G., Lee, W.-L., Nixon, A.-B., Peralta, E.-G., Casey, P.-J., 2001. Phosphorylation and nuclear translocation of a regulator of G protein signaling (RGS10). J. Biol. Chem. 276, 32828–32834. Burke, R.-E., Greenbaum, D., 1987. Effect of postmortem factors on muscarinic receptor subtypes in rat brain. J. Neurochem. 49, 592–596. Cavalli, A., Druey, K.-M., Milligan, G., 2000. The regulator of G protein signaling RGS4 selectively enhances alpha 2A-adrenoreceptor stimulation of the GTPase activity of Go1alpha and Gi2alpha. J. Biol. Chem. 275, 23693–23699.

Chatterjee, T.-K., Fisher, R.-A., 2000. Cytoplasmic, nuclear, and golgi localization of RGS proteins. Evidence for N-terminal and RGS domain sequences as intracellular targeting motifs. J. Biol. Chem. 275, 24013–24021. Chatterjee, T.-K., Fisher, R.-A., 2002. RGS12TS-S localizes at nuclear matrix-associated subnuclear structures and represses transcription, structural requirements for subnuclear targeting and transcriptional repression. Mol. Cell. Biol. 22, 4334–4345. Cowburn, R.-F., O’Neill, C., Ravid, R., Alafuzoff, I., Winblad, B., Fowler, C.-J., 1992. Adenylyl cyclase activity in postmortem human brain, evidence of altered G protein mediation in Alzheimer’s disease. J. Neurochem. 58, 1409–1419. Davydov, I.-V, Varshavsky, A., 2000. RGS4 is arginylated and degraded by the N-end rule pathway in vitro. J. Biol. Chem. 275, 22931–22941. Ding, J., Guzman, J.-N., Tkatch, T., Chen, S., Goldberg, J.-A., Ebert, P.-J., Levitt, P., Wilson, C.-J., Hamm, H.-E., Surmeier, D.-J., 2006. RGS4-dependent attenuation of M4 autoreceptor function in striatal cholinergic interneurons following dopamine depletion. Nat. Neurosci. 9, 832–842. Ding, L., Mychaleckyj, J.C., Hegde, A.-N., 2007. Full length cloning and expression analysis of splice variants of regulator of G-protein signaling RGS4 in human and murine brain. Gene 401, 46–60. Ding, L., Hegde, A.-N., 2009. Expression of RGS4 splice variants in dorsolateral prefrontal cortex of schizophrenic and bipolar disorder patients. Biol. Psychiatry 65, 541–545. Emilsson, L., Saetre, P., Jazin, E., 2006. Low mRNA levels of RGS4 splice variants in Alzheimer’s disease, association between a rare haplotype and decreased mRNA expression. Synapse 59, 173–176. Erdely, H.-A., Lahti, R.-A., Lopez, M.-B., Myers, C.-S., Roberts, R.-C., Tamminga, C.-A., Vogel, M.-W., 2004. Regional expression of RGS4 mRNA in human brain. Eur. J. Neurosci. 19, 3125–3128. Garnier, M., Zaratin, P.-F., Ficalora, G., Valente, M., Fontanella, L., Rhee, M.-H., Blumer, K.-J., Scheideler, M.-A., 2003. Up-regulation of regulator of G protein signaling 4 expression in a model of neuropathic pain and insensitivity to morphine. J. Pharmacol. Exp. Therap. 304, 1299–1306. ˜ oz, M., de la Torre-Madrid, E., Sa´nchez-Bla´zquez, P., 2005. Garzo´n, J., Rodrı´guez-Mun Effector antagonism by the regulators of G protein signalling (RGS) proteins causes desensitization of mu-opioid receptors in the CNS. Psychopharmacology 180, 1–11. Georgoussi, Z., Leontiadis, L., Mazarakou, G., Merkouris, M., Hyde, K., Hamm, H., 2006. Selective interactions between G protein subunits and RGS4 with the Cterminal domains of the mu- and delta-opioid receptors regulate opioid receptor signaling. Cell. Signal. 18, 771–782. Geurts, M., Maloteaux, J.-M., Hermans, E., 2003. Altered expression of regulators of G-protein signaling (RGS) mRNAs in the striatum of rats undergoing dopamine depletion. Biochem. Pharmacol. 66, 1163–1170. Ghavami, A., Hunt, R.-A., Olsen, M.-A., Zhang, J., Smith, D.-L., Kalgaonkar, S., Rahman, Z., Young, K.-H., 2004. Differential effects of regulator of G protein signaling (RGS) proteins on serotonin 5-HT1A, 5-HT2A, and dopamine D2 receptormediated signaling and adenylyl cyclase activity. Cell. Signal. 16, 711–721. Gold, S.-J., Heifets, B.-D., Pudiak, C.-M., Potts, B.-W., Nestler, E.-J., 2002. Regulation of regulators of G protein signaling mRNA expression in rat brain by acute and chronic electroconvulsive seizures. J. Neurochem. 82, 828–838. Gold, S.-J., Han, M.-H., Herman, A.-E., Ni, Y.-G., Pudiak, C.-M., Aghajanian, G.-K., Liu, R.-J., Potts, B.-W., Mumby, S.-M., Nestler, E.-J., 2003. Regulation of RGS proteins by chronic morphine in rat locus coeruleus. Eur. J. Neurosci. 17, 971–980. Gonza´lez-Maeso, J., Torre, I., Rodrı´guez-Puertas, R., Garcı´a-Sevilla, J.A., Guimo´n, J., Meana, J.-J., 2002. Effects of age, postmortem delay and storage time on receptor-mediated activation of G-proteins in human brain. Neuropsychopharmacology 26, 468–478. Gu, Z., Jiang, Q., Yan, Z., 2007. RGS4 modulates serotonin signaling in prefrontal cortex and links to serotonin dysfunction in a rat model of schizophrenia. Mol. Pharmacol. 71, 1030–1039. Haller, C., Fillatreau, S., Hoffmann, R., Agenes, F., 2002. Structure, chromosomal localization and expression of the mouse regulator of G-protein signaling10 gene (mRGS10). Gene 297, 39–49. Hishimoto, A., Shirakawa, O., Nishiguchi, N., Aoyama, S., Ono, H., Hashimoto, T., Maeda, K., 2004. Novel missense polymorphism in the regulator of G-protein signaling 10 gene, analysis of association with schizophrenia. Psychiatry Clin. Neurosci. 58, 579–581. Hoffmann, M., Ward, R.-J., Cavalli, A., Carr, I.-C., Milligan, G., 2001. Differential capacities of the RGS1, RGS16 and RGS-GAIP regulators of G protein signaling to enhance alpha2A-adrenoreceptor agonist-stimulated GTPase activity of G(o1)alpha. J. Neurochem. 78, 797–806. Hollinger, S., Hepler, J.-R., 2002. Cellular regulation of RGS proteins, modulators and integrators of G protein signaling. Pharmacol. Rev. 54, 527–559. Huang, C., Hepler, J.-R., Gilman, A.-G., Mumby, S.-M., 1997. Attenuation of Gi- and Gq-mediated signaling by expression of RGS4 or GAIP in mammalian cells. Proc. Natl. Acad. Sci. U.S.A. 94, 6159–6163. Hunt, T.-W., Fields, T.-A., Casey, P.-J., Peralta, E.-G., 1996. RGS10 is a selective activator of G alpha i GTPase activity. Nature 383, 175–177. Ishii, M., Kurachi, Y., 2003. Physiological actions of regulators of G-protein signaling (RGS) proteins. Life Sci. 74, 163–171. Kim, E., Arnould, T., Sellin, L., Benzing, T., Comella, N., Kocher, O., Tsiokas, L., Sukhatme, V.-P., Walz, G., 1999. Interaction between RGS7 and polycystin. Proc. Natl. Acad. Sci. U.S.A. 96, 6371–6376. Krumins, A.-M., Barker, S.-A., Huang, C., Sunahara, R.-K., Yu, K., Wilkie, T.-M., Gold, S.-J., Mumby, S.-M., 2004. Differentially regulated expression of endogenous RGS4 and RGS7. J. Biol. Chem. 279, 2593–2599.

G. Rivero et al. / Neurochemistry International 57 (2010) 722–729 Larminie, C., Murdock, P., Walhin, J.-P., Duckworth, M., Blumer, K.-J., Scheideler, M.A., Garnier, M., 2004. Selective expression of regulators of G-protein signaling (RGS) in the human central nervous system. Mol. Brain Res. 122, 24–34. Leone, A.-M., Errico, M., Lin, S.-L., Cowen, D.-S., 2000. Activation of extracellular signal-regulated kinase (ERK) and Akt by human serotonin 5-HT1B receptors in transfected BE(2)-C neuroblastoma cells is inhibited by RGS4. J. Neurochem. 75, 934–938. Leygraf, A., Hohoff, C., Freitag, C., Willis-Owen, S.-A., Krakowitzky, P., Fritze, J., Franke, P., Bandelow, B., Fimmers, R., Flint, J., Deckert, J., 2006. Rgs2 gene polymorphisms as modulators of anxiety in humans? J. Neural Transm. 113, 1921–1925. Lipska, B.-K., Mitkus, S., Caruso, M., Hyde, T.-M., Chen, J., Vakkalanka, R., Straub, R.-E., Weinberger, D.-R., Kleinman, J.-E., 2006. RGS4 mRNA expression in postmortem human cortex is associated with COMT Val158Met genotype and COMT enzyme activity. Hum. Mol. Genet. 15, 2804–2812. Mirnics, K., Middleton, F.-A., Stanwood, G.-D., Lewis, D.-A., Levitt, P., 2001. Diseasespecific changes in regulator of G-protein signaling 4 (RGS4) expression in schizophrenia. Mol. Psychiatry 6, 293–301. Morris, D.-W., Rodgers, A., McGhee, K.-A., Schwaiger, S., Scully, P., Quinn, J., Meagher, D., Waddington, J.-L., Gill, M., Corvin, A.-P., 2004. Confirming RGS4 as a susceptibility gene for schizophrenia. Am. J. Med. Genet. B: Neuropsychiatr. Genet. 125, 50–53. Muma, N.-A., Mariyappa, R., Williams, K., Lee, J.-M., 2003. Differences in regional and subcellular localization of Gq/11 and RGS4 protein levels in Alzheimer’s disease, correlation with muscarinic M1 receptor binding parameters. Synapse 47, 58–65. Nishiguchi, K.-M., Sandberg, M.-A., Kooijman, A.-C., Martemyanov, K.-A., Pott, J.-W., Hagstrom, S.-A., Arshavsky, V.-Y., Berson, E.-L., Dryja, T.-P., 2004. Defects in RGS9 or its anchor protein R9AP in patients with slow photoreceptor deactivation. Nature 427, 75–78. Ozawa, H., Ukai, W., Kornhuber, J., Yamaguchi, T., Froelich, L., Ikeda, H., Saito, T., Riederer, P., 1999. Postnatal ontogeny of GTP binding protein in the human frontal cortex. Life Sci. 65, 2315–2323. Paspalas, C.-D., Selemon, L.-D., Arnsten, A.-F., 2009. Mapping the regulator of G protein signaling 4 (RGS4): presynaptic and postsynaptic substrates for neuroregulation in prefrontal cortex. Cereb. Cortex 19, 2145–2155. Paul, D., Gauthier, C.-A., Minor, L.-D., Gonzales, G.-R., 1997. The effects of postmortem delay on mu, delta and kappa opioid receptor subtypes in rat brain and guinea pig cerebellum evaluated by radioligand binding. Life Sci. 61, 1993– 1998. Perry, E.-K., Perry, R.-H., 1983. Human brain neurochemistry, some postmortem problems. Life Sci. 33, 1733–1743. Prasad, K.-M., Chowdari, K.-V., Nimgaonkar, V.-L., Talkowski, M.-E., Lewis, D.-A., Keshavan, M.-S., 2005. Genetic polymorphisms of the RGS4 and dorsolateral prefrontal cortex morphometry among first episode schizophrenia patients. Mol. Psychiatry 10, 213–219. Ramos-Miguel, A., Garcı´a-Fuster, M.-J., Callado, L.-F., La Harpe, R., Meana, J.-J., Garcı´a-Sevilla, J.-A., 2009. Phosphorylation of FADD (Fas-associated death domain protein) at derine 194 is increased in the prefrontal cortex of opiate abusers: relation to mitogen activated protein kinase, phosphoprotein enriched in astrocytes of 15 kDa, and Akt signalling pathways involved in neuroplasticity. Neuroscience 161, 23–38. Rimler, A., Jockers, R., Lupowitz, Z., Sampson, S.-R., Zisapel, N., 2006. Differential effects of melatonin and its downstream effector PKCalpha on subcellular localization of RGS proteins. J. Pineal Res. 40, 144–152. Rodrı´guez-Puertas, R., Pascual, J., Pazos, A., 1996. Effects of freezing storage time on the density of muscarinic receptors in the human postmortem brain, an autoradiographic study in control and Alzheimer’s disease brain tissues. Brain Res. 728, 65–71.

729

Ross, E.-M., Wilkie, T.-M., 2000. GTPase-activating proteins for heterotrimeric G proteins, regulators of G protein signaling (RGS) and RGS-like proteins. Annu. Rev. Biochem. 69, 795–827. Saugstad, J.-A., Marino, M.-J., Folk, J.-A., Hepler, J.-R., Conn, P.-J., 1998. RGS4 inhibits signaling by group I metabotropic glutamate receptors. J. Neurosci. 18, 905– 913. Schiff, M.-L., Siderovski, D.-P., Jordan, J.-D., Brothers, G., Snow, B., De Vries, L., Ortiz, D.-F., Diverse-Pierluissi, M., 2000. Tyrosine-kinase-dependent recruitment of RGS12 to the N-type calcium channel. Nature 408, 723–727. Schwendt, M., Gold, S.-J., McGinty, J.-F., 2006. Acute amphetamine down-regulates RGS4 mRNA and protein expression in rat forebrain, distinct roles of D1 and D2 dopamine receptors. J. Neurochem. 96, 1606–1615. Shi, J., Damjanoska, K.-J., Zemaitaitis, B.-W., Garcia, F., Carrasco, G., Sullivan, N.-R., She, Y., Young, K.-H., Battaglia, G., Van de Kar, L.-D., Howland, D.-S., Muma, N.-A., 2006. Alterations in 5-HT (2A) receptor signaling in male and female transgenic rats over-expressing either Gq or RGS-insensitive Gq protein. Neuropharmacology 51, 524–535. Sierra, D.-A., Gilbert, D.-J., Householder, D., Grishin, N.-V., Yu, K., Ukidwe, P., Barker, S.-A., He, W., Wensel, T.-G., Otero, G., Brown, G., Copeland, N.-G., Jenkins, N.-A., Wilkie, T.-M., 2002. Evolution of the regulators of G-protein signaling multigene family in mouse and human. Genomics 79, 177–185. Sinnarajah, S., Dessauer, C.-W., Srikumar, D., Chen, J., Yuen, J., Yilma, S., Dennis, J.-C., Morrison, E.-E., Vodyanoy, V., Kehrl, J.-H., 2001. RGS2 regulates signal transduction in olfactory neurons by attenuating activation of adenylyl cyclase III. Nature 409, 1051–1055. Srinivasa, S.-P., Bernstein, L.-S., Blumer, K.-J., Linder, M.-E., 1998. Plasma membrane localization is required for RGS4 function in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. U.S.A. 95, 5584–5589. Talkowski, M.-E., Seltman, H., Bassett, A.-S., Brzustowicz, L.-M., Chen, X., Chowdari, K.-V., Collier, D.-A., Cordeiro, Q., Corvin, A.-P., Deshpande, S.-N., Egan, M.-F., Gill, M., Kendler, K.-S., Kirov, G., Heston, L.-L., Levitt, P., Lewis, D.-A., Li, T., Mirnics, K., Morris, D.-W., Norton, N., O’Donovan, M.-C., Owen, M.-J., Richard, C., Semwal, P., Sobell, J.-L., St Clair, D., Straub, R.-E., Thelma, B.-K., Vallada, H., Weinberger, D.R., Williams, N.-M., Wood, J., Zhang, F., Devlin, B., Nimgaonkar, V.-L., 2006. Evaluation of a susceptibility gene for schizophrenia, genotype based metaanalysis of RGS4 polymorphisms from thirteen independent samples. Biol. Psychiatry 60, 152–162. Tekumalla, P.-K., Calon, F., Rahman, Z., Birdi, S., Rajput, A.-H., Hornykiewicz, O., Di Paolo, T., Bedard, P.-J., Nestler, E.-J., 2001. Elevated levels of DeltaFosB and RGS9 in striatum in Parkinson’s disease. Biol. Psychiatry 50, 813–816. Tu, Y., Woodson, J., Ross, E.-M., 2001. Binding of regulator of G protein signaling (RGS) proteins to phospholipid bilayers—contribution of location and/or orientation to GTPase-activating protein activity. J. Biol. Chem. 276, 20160–20166. Waugh, J.-L., Lou, A.-C., Eisch, A.-J., Monteggia, L.-M., Muly, E.-C., Gold, S.-J., 2005. Regional, cellular, and subcellular localization of RGS10 in rodent brain. J. Comp. Neurol. 481, 299–313. Whitehouse, P.-J., Lynch, D., Kuhar, M.-J., 1984. Effects of postmortem delay and temperature on neurotransmitter receptor binding in a rat model of the human autopsy process. J. Neurochem. 43, 553–559. Williams, N.-M., Preece, A., Spurlock, G., Norton, N., Williams, H.-J., McCreadie, R.-G., Buckland, P., Sharkey, V., Chowdari, K.-V., Zammit, S., Nimgaonkar, V., Kirov, G., Owen, M.-J., O’Donovan, M.-C., 2004. Support for RGS4 as a susceptibility gene for schizophrenia. Biol. Psychiatry 55, 192–195. Yan, Y., Chi, P.-P., Bourne, H.-R., 1997. RGS4 inhibits Gq-mediated activation of mitogen-activated protein kinase and phosphoinositide synthesis. J. Biol. Chem. 272, 11924–11927. Zhong, H., Wade, S.-M., Woolf, P.-J., Linderman, J.-J., Traynor, J.-R., Neubig, R.-R., 2003. A spatial focusing model for G protein signals. Regulator of G protein signaling (RGS) protein-mediated kinetic scaffolding. J. Biol. Chem. 278, 7278–7284.