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Keywords:

  • Regulator of G protein signaling 7;
  • Gαq/11;
  • Brain localization;
  • Immunohistochemistry;
  • Rat;
  • Confocal microscopy

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RNA isolation
  5. Quantitative real-time RT-PCR
  6. RESULTS
  7. RGS7 and Gαq/11 immunohistochemical distribution
  8. Regulation of RGS7 and RGS4 by PdBu
  9. DISCUSSION

Abstract : Regulators of G protein signaling (RGS) proteins serve as potent GTPase-activating proteins for the heterotrimeric G proteins αi/o and αq/11. This study describes the immunohistochemical distribution of RGS7 throughout the adult rat brain and its cellular colocalization with Gαq/11, an important G protein-coupled receptor signal transducer for phospholipase Cβ-mediated activity. In general, both RGS7 and Gαq/11 displayed a heterogeneous and overlapping regional distribution. RGS7 immunoreactivity was observed in cortical layers I-VI, being most intense in the neuropil of layer I. In the hippocampal formation, RGS7 immunoreactivity was concentrated in the strata oriens, strata radiatum, mossy fibers, and polymorphic cells, with faint to nondetectable immunolabeling within the dentate gyrus granule cells and CA1-CA3 subfield pyramidal cells. Numerous diencephalic and brainstem nuclei also displayed dense RGS7 immunostaining. Dual immunofluorescence labeling studies with the two protein-specific antibodies indicated a cellular selectivity in the colocalization between RGS7 and Gαq/11 within many discrete brain regions, such as the superficial cortical layer I, hilus area of the hippocampal formation, and cerebellar Golgi cells. To assess the ability of Gαq/11-mediated signaling pathways to modulate dynamically RGS expression, primary cortical neuronal cultures were incubated with phorbol 12,13-dibutyrate, a selective protein kinase C activator. A time-dependent increase in levels of mRNA for RGS7, but not RGS4, was observed. Our results provide novel information on the region- and cell-specific pattern of distribution of RGS7 with the transmembrane signal transducer, Gαq/11. We also describe a possible RGS7-selective neuronal feedback adaptation on Gαq/11-mediated pathway function, which may play an important role in signaling specificity in the brain.

Regulators of G protein signaling (RGS) proteins are GTPase-activating proteins for several G protein α-subunit members (Gαi and Gαq) and serve to attenuate G protein signaling (Koelle, 1997 ; Yan et al., 1997 ; Berman and Gilman, 1998 ; Shuey et al., 1998). Activation of G protein-coupled receptors catalyzes the exchange of Gα-bound GDP for GTP to cause the dissociation of Gα from Gβγ dimer and initiate downstream signal propagation ; RGS proteins terminate signaling by accelerating the relatively slow intrinsic Gα-GTPase activity and recycling the G protein complex back to its inactive GDP-bound heterotrimeric configuration (Dohlman and Thorner, 1997 ; Koelle, 1997 ; Neer, 1997).

Of 19 mammalian genes that express mRNAs with a conserved RGS core domain, many of these demonstrate regional specificity and heterogeneous expression within the rat CNS (Gold et al., 1997 ; Shuey et al., 1998). Both RGS4 and RGS7 mRNAs are prevalent in brain (Druey et al., 1996 ; Koelle and Horvitz, 1996 ; Gold et al., 1997 ; Shuey et al., 1998) and have become the focus of growing interest as to their potential physiological as well as pathological roles in vivo. A neuroadaptive function for the RGS family has been suggested in recent studies. In the first study, the expression of mRNA for RGS7, RGS8, and RGS10 was temporally modulated within specific rat brain regions after acute electroconvulsive shock treatment (Gold et al., 1997) ; in the second study, the expression levels of RGS2, RGS3, RGS5, and RGS8 were increased, whereas that of RGS9 was decreased, and those of RGS4 and RGS16 remained unaltered in the rat striatal caudate-putamen following repeated psychostimulant administration (Burchett et al., 1998).

Genetic evidence from Saccharomyces cerevisiae, Caenorhabditis elegans, and Aspergillus nidulans suggested that members of the RGS family could attenuate G protein-mediated signaling in vivo (Dohlman et al., 1996 ; Koelle and Horvitz, 1996 ; Yu et al., 1996. Several other studies have indicated that RGS proteins, such as RGS4, potently stimulate the rate of GTP hydrolysis for both Gαi- and Gαq-mediated cellular signaling (Berman et al., 1996 ; Hunt et al., 1996 ; Watson et al., 1996 ; Hepler et al., 1997). RGS7 is also a potent GTPase-activating protein for Gαq/11, inhibiting serotonin (5-hydroxytryptamine) 5-HT2c-mediated calcium mobilization and inositol phosphate generation, while exerting no obvious effect on either Gαs- or Gαi-mediated signaling in a Chinese hamster ovary-K1 cell assay system (DiBello et al., 1998 ; Shuey et al., 1998). Emerging evidence also indicates that RGS proteins may be essential regulatory components involved in neuronal excitability (Doupnik et al., 1997 ; Saitoh et al., 1997).

In situ hybridization studies have revealed strong similarities in the regional expression of RGS7, Gα11, and Gαq mRNAs in the adult rat brain (Shuey et al., 1998). We report on the immunohistochemical identification of RGS7 and its regional distribution and cellular selectivity in colocalizing with the G protein αq/11 in adult rat brain. We also report on the selective up-regulation of RGS7 mRNA levels in primary cortical neurons by a protein kinase C pathway activator. The findings provide new information on the specificity of G protein-coupled receptor signaling components in the CNS and illustrate a possible dynamic feedback regulatory mechanism of G protein function.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RNA isolation
  5. Quantitative real-time RT-PCR
  6. RESULTS
  7. RGS7 and Gαq/11 immunohistochemical distribution
  8. Regulation of RGS7 and RGS4 by PdBu
  9. DISCUSSION

Preparation of antibodies and recombinant proteins

A 123-amino acid recombinant core domain of RGS7 [amino acids 267-389 of the est U32439 (Shuey et al., 1998)] was used to generate polyclonal antisera (Genosys Biotechnologies, Woodlands, TX, U.S.A.). This was affinity-purified according to the method of Harlow and Lane (1988). In brief, 1 mg of protein was immobilized on NHS-Sepharose (Pharmacia Biotech, Piscataway, NJ, U.S.A.) and washed with 100 mM glycine (pH 2.5) and 100 mM triethylamine (pH 11.5). The serum was diluted 1 : 1 in phosphate-buffered saline (PBS) and passed through the column twice. Antibodies bound by acid-sensitive interactions were eluted with 100 mM glycine (pH 2.5), and alkali-sensitive antibodies were eluted with 100 mM triethylamine (pH 11.5). The fractions were pooled and dialyzed against PBS containing 0.02% sodium azide. Polyclonal affinity-purified antibody against the Gαq/11 subunit (lot no. LO87) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.). Recombinant RGS7 was prepared as previously described (Shuey et al., 1998), and recombinant rat G protein αq was purchased from Santa Cruz Biotechnology.

Tissue preparation

Adult male Wistar rats (weighing 250-350 g) were killed by CO2 asphyxiation. Various brain regions were dissected out and homogenized in 5 volumes of buffer medium (10 mM Tris-HCl, 1 mM EDTA, and 0.25 M sucrose, pH 7.4), containing a protease inhibitor cocktail (Complete ; Boehringer Mannheim, Indianapolis, IN, U.S.A.). An aliquot of the resultant homogenates was stored at —80°C and represented the homogenate fraction. The remainder of the homogenates was centrifuged at 1,240 g for 10 min at 4°C to pellet the nuclear fraction. The supernatant was centrifuged at 40,000 g for 60 min at 4°C to obtain a membrane pellet fraction, which was resuspended in 0.5 ml of ice-cold homogenization buffer, and the resulting supernatant was retrieved as a cytosolic fraction. Membrane, cytosolic, and nuclear fractions were stored at —80°C until required. The protein content of each preparation was determined by the method of Bradford (1976).

Western immunoblots

Crude homogenate, nuclear, cytosolic, and membrane preparations were diluted in 2 × Laemmli sample buffer (Bio-Rad, Hercules, CA, U.S.A.) containing 350 mM dithiothreitol and heated for 5 min at 90°C. Twenty micrograms of protein per well was resolved on 10% Tris-glycine-sodium dodecyl sulfate polyacrylamide gels, and molecular mass markers (4-250 kDa ; Novex, San Diego, CA, U.S.A.) were coelectrophoresed for size estimation. Protein was transferred electrophoretically to a nitrocellulose membrane in cold transfer buffer consisting of 48 mM Tris, 39 mM glycine, 0.0375% sodium dodecyl sulfate, and 20% methanol. The nitrocellulose sheets were incubated in PBS containing 0.1% Tween 20 (PBS-T), 5% nonfat dry milk, and 1.5% normal goat serum for 1 h at room temperature and then overnight at 4°C with rabbit affinity-purified polyclonal RGS7 antiserum at a dilution of 1:2,000-1:20,000 or with preimmune serum as control in PBS-T blocking buffer plus 1.5% normal goat serum. The nitrocellulose blots were washed in PBS-T (4 × 5 min) at room temperature followed by incubation for 1 h at room temperature with horseradish peroxidase-conjugated anti-rabbit IgG (1:1,000 dilution in PBS-T ; Amersham, Arlington Heights, IL, U.S.A.). The blots were washed in PBS-T (4 × 10 min) at room temperature, and immunoreactive proteins were visualized using the enhanced chemiluminescence (ECL) technique (Amersham).

Light microscope immunohistochemistry

Under sodium pentobarbital (60 mg/kg, i.p.) anesthesia, adult male Wistar rats (weighing 250-350 g) were perfused through the ascending aorta with 50 ml of 0.9% NaCl, followed by 400 ml of freshly prepared 4% paraformaldehyde in PBS (pH 7.4). Whole brains were removed, postfixed in 4% paraformaldehyde in PBS overnight, and immersed in 30% sucrose in PBS for a 48-h period at 4°C. The brains were frozen in powdered dry ice and stored at —80°C. Individual brains were serially sectioned (30 μm thickness) using a sliding microtome (Microm, Germany). Free-floating coronal sections were washed for 30 min in 0.3% H2O2 in PBS to neutralize endogenous peroxidase activity and rinsed in PBS (2 × 10 min). Tissue sections were blocked with 1.5% normal goat serum in PBS for 20 min and incubated on a shaker platform at 4°C overnight with varying dilutions of RGS7 antibody (1:2,000-1:20,000). Immunohistochemical controls of adjacent sections included omitting the primary antibody or replacing the primary antibody with preimmune serum. Sections were rinsed in PBS (2 × 10 min) and incubated with biotinylated goat antirabbit antibody for 30 min at room temperature. After rinsing in PBS (2 × 10 min), sections were incubated with the Elite avidin-biotin complex (Vector Laboratories, Burlingame, CA, U.S.A.) for 30 min and subsequently rinsed in PBS (3 × 10 min). Colorimetric development was with 0.05% 3,3′-diaminobenzidine tetrahydrochloride/0.05% H2O2 in 60 mM Tris buffer for 4 min. Immunostained sections were rinsed in PBS and distilled water, mounted onto glass slides, dehydrated in graded ethanol solutions, cleared in toluene, and coverslipped in DPX mountant (Fluka, Germany). Sections were examined and photographed using a Zeiss Axiophot photomicroscope. The nomenclature of Paxinos and Watson (1986) was used in the identification of anatomical structures within individual rat brain sections. The relative intensities of immunostaining for the individual antibodies were classified into five major categories (from nondetectable to very dense staining) and relate to each antibody's intensity of staining of the individual sections throughout the whole brain.

Immunofluorescence dual staining and confocal microscopy

Free-floating coronal sections (30 μm thickness) were processed using tyramide signal amplification kits (NEN Life Science, Boston, MA, U.S.A.). Tissue sections were washed in PBS (3 × 5 min) and preincubated with 0.3% H2O2 in PBS. After rinsing in PBS (2 × 10 min), the tissue sections were blocked in 0.5% blocking reagent in TNT buffer [0.1 M Tris-HCl (pH 7.5), 0.15 M NaCl, and 0.05% Tween 20] for 30 min and incubated overnight with polyclonal RGS7 antibody (1:2,000) on a shaker platform at 4°C. Sections were rinsed in TNT wash buffer for 3 × 5 min, followed by incubation with biotinylated goat anti-rabbit IgG (Vector) for 30 min at room temperature. After rinsing in TNT buffer (3 × 5 min), sections were incubated with streptavidin-horseradish peroxidase (1:100) for 30 min and further washed (3 × 5 min) in TNT buffer. Fluorescent tagging was with fluorescein tyramide (1:50) in fluorescein tyramide dilutant buffer for 5 min. Sections were washed (3 × 5 min) in TNT buffer followed by overnight application of the polyclonal antibody to Gαq/11 (1:2,000) at 4°C. The immunostaining procedure for Gαq/11 was identical to that for RGS7 except for the use of tetramethylrhodamine tyramide for fluorescence tagging. Finally, sections were rinsed in TNT buffer (3 × 10 min) and briefly in distilled water, then mounted onto glass slides, and coverslipped using Prolong Antifade solution (Molecular Probes, Eugene, OR, U.S.A.). Confocal microscopy was performed with a Leica TCS 4D system attached to a Leitz DMIRB microscope (Leica Lasertechnik, Heidelberg, Germany). The fluorescein and rhodamine markers were excited by a 488 nm line and a 568 nm line of an air-cooled ArKr laser, respectively.

Primary cell culture

Cortical neuronal cultures were prepared from prenatal Sprague-Dawley rat embryo cortices. Pregnant female rats were killed by asphyxiation with CO2 gas, and 16-18-day embryos were removed. The brains were dissected, and the cortex was removed and placed in cold Ca2+ - and Mg2+ -free Hanks' buffered saline solution. The cortex was dissociated by trituration through a 1,000-μl pipette tip, and the cells were centrifuged and resuspended in Neurobasal medium supplemented with 5% fetal bovine serum, 5% horse serum, l-glutamine (2 mM), and penicillin/streptomycin (5 units/ml and 5 μg/ml) and plated in poly-d-lysine-coated multiwell plates. The plating medium was replaced after 5 days with Neurobasal medium plus B27 supplement (GibcoBRL, Gaithersburg, MD, U.S.A.) and the antimitotic 5-fluoro-2′ -deoxyuridine-ribose (Sigma). Cell cultures were maintained at 37°C in 95% air/5% CO2, and the medium changed every 3 days.

RNA isolation

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RNA isolation
  5. Quantitative real-time RT-PCR
  6. RESULTS
  7. RGS7 and Gαq/11 immunohistochemical distribution
  8. Regulation of RGS7 and RGS4 by PdBu
  9. DISCUSSION

For isolation of total RNA from cultured cortical neurons, the medium was replaced with Neurobasal medium free of glutamine and glutamate for 24 h before the experiment. Neurobasal medium containing 500 nM phorbol 12,13-dibutyrate (PdBu) was then added for 1-24 h, and the total RNA was isolated at various time points using a single-step method with the isolation reagent RNA-Stat 60 (Tel-Test, Friendswood, TX, U.S.A.). In brief, the medium was removed, and the cells were lysed by addition of RNA-Stat 60. The cell lysate was passed through a pipette tip several times, chloroform was added, and the phases were separated by centrifugation. The aqueous phase containing the RNA was precipitated with isopropanol, washed with ethanol, and solubilized in water.

Quantitative real-time RT-PCR

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RNA isolation
  5. Quantitative real-time RT-PCR
  6. RESULTS
  7. RGS7 and Gαq/11 immunohistochemical distribution
  8. Regulation of RGS7 and RGS4 by PdBu
  9. DISCUSSION

Target amplification primers were synthesized for RGS4 and RGS7, with cyclophilin serving as the internal reference amplification. The RGS4 primers used were forward 5′ -TCTGCAGACTGCACTTCCCTAGT-3′ and reverse 5′-GAGTCTTGGCATTTCGGCATTTCGGTTCTC-3′, RGS7 primers were forward 5′ -TGGCAGTGGAGGACCTGAA-3′, and reverse 5′ -CAGAAATTCTTGCCATATTTCCTGTACT-3′, and cyclophilin primers were forward 5′-TCCCAGTTTTTTATCTGCACTGC-3′ and reverse 5′ -GCCTTCTTTCACCTTCCCAAA-3′. The RGS4 probe was 6FAM-CCTCAGTGTGCCTAATTCTCACACAGAGGC-TAMARA, the RGS7 probe was 6FAM-AAGGCCTATCCGAGAGGTCCCCTCTC-TAMARA, and the cyclophilin probe was JOE-AAGACTGAGTGGCTG-GATGGCAAGCATGTGGTC-TAMARA. The dyes were conjugated through a linker arm nucleotide linkage (Livak et al., 1995). FAM and JOE served as the reporter fluorochrome, and TAMARA served as the quencher. Probes were synthesized by Applied Biosystems (Foster City, CA, U.S.A.), and the PCR primers by GibcoBRL. The ABI 7700 detection system incorporated a 96-well thermocycler heat block, coupled to a laser detector. RT-PCR amplifications were performed using a 96-well optical tray and optical caps (MicroAmp ; Perkin-Elmer, Norwalk, CT, U.S.A.). Real-time quantitative RT-PCR required PCR amplification of the target, annealing of a fluorescently labeled probe, use of a 5′ nuclease assay, and detection of the cleaved reporter dye (Gibson et al., 1996). The probe was a fluorescently labeled oligonucleotide with a 5′ reporter dye and a 3′ quencher dye. The reporter dye was released from the probe during PCR by the 5′[RIGHTWARDS ARROW] 3′ exonuclease activity of Taq DNA polymerase, and the accumulation of signal was calculated over time. An internal reference, cyclophilin, was coamplified with the target for normalization of accumulated signal. The 50-μl master mix contained the following : each primer (GibcoBRL) at 100 nM, both probes (Perkin-Elmer) at 100 nM, dATP, dGTP, and dCTP at 200 μM each and 400 μM dUTP, 3 units of Amplitaq Gold, 1 × Taqman A buffer, 5.5 mM MgCl2 (Perkin-Elmer), 6 units of Moloney murine leukemia virus reverse transcriptase, and 10 units of RNAse inhibitor (GibcoBRL). The RT-PCR cycle was performed at 50°C for 30 min (first-strand cDNA synthesis) and 95°C for 10 min (activation of Taq Gold), followed by 35 cycles at 95°C for 15 s and 60°C for 1 min. Multiplexing in a single well required that both target and reference amplify with equal efficiencies. After this determination, relative quantitation could be calculated by the comparative Ct method (ABI Prism 7700 Sequence Detection System, user buletin no. 2, 1997).

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RNA isolation
  5. Quantitative real-time RT-PCR
  6. RESULTS
  7. RGS7 and Gαq/11 immunohistochemical distribution
  8. Regulation of RGS7 and RGS4 by PdBu
  9. DISCUSSION

Western immunoblot analysis

The protein specificities of anti-RGS7 and of anti-Gαq/11 rabbit polyclonal affinity-purified antibodies were confirmed by western blot analysis (Fig. 1A). Using rat thalamic tissue homogenates, as well as hippocampal membrane, nuclear, and cytosolic preparations, a single antigenic band corresponding to the RGS7 protein was revealed with an apparent molecular size of ~62 kDa (Fig. 1A). This was observed at RGS7 antibody dilutions from 1:2,000 to 1:20,000. Of all the deduced full-length RGS protein sequences reported to date, only RGS7 would be expected to migrate at this molecular size. Moreover, the RGS7 antibody recognized purified recombinant RGS7 protein, and immunoblots performed using preimmune serum exhibited no RGS7-like immunoreactivity (ir) (data not shown). RGS7 protein was generally more abundant in membrane fractions compared with cytosolic fractions ; the nuclear fraction displayed very faint antigenicity, which probably reflected a small amount of membrane contamination during the preparative centrifugation steps, because no positive nuclear staining was exhibited in subsequent immunohistochemical brain sections. Antigenicity of the Gαq/11 antibody, purchased from Santa Cruz Biotechnology, revealed a single protein band of correct apparent molecular size (42-43 kDa) using crude homogenate and membrane fractions from rat frontal cortex ; the cytosolic and nuclear fractions did not display any antigenic properties (Fig. 1A). Figure 1B illustrates the widespread distribution of RGS7 within all brain regions examined ; it was predominant in the membrane fractions but was also detectable within most cytosolic fractions, except for the olfactory bulb, pontine nucleus, and cerebellum (Fig. 1B), which may indicate regional subcellular compartmentalization. Our findings indicate that both RGS7 and Gαq/11 antibodies are specific for their respective antigenic proteins in brain extracts and thereby provide useful tools for the immunohistochemical identification of RGS7 and Gαq/11 in rat CNS. The RGS7 antibody was also shown to recognize an antigenic protein of similar molecular size in human cerebellar tissue fractions (J.-J.L., unpublished data).

image

Figure 1. Western immunoblots. A : Characterization of RGS7 and G protein αq/11 polyclonal affinity-purified antibodies. Rat whole homogenate (T), membrane (M), cytosolic (C), and nuclear (N) fractions (20 μg of protein per lane) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10% acrylamide) and immunoblotted overnight with either RGS7 antibody (1:2,000) or Gαq/11 antibody (1:2,000). The nitrocellulose membrane was stained and developed as described in Materials and Methods. αq, recombinant rat Gαq protein. B : Distribution pattern of RGS7 immunolabeling in various regional membrane and cytosolic preparations of the adult rat brain. OB, olfactory bulb ; FC, frontal cortex ; STR, striatum ; Hip and HIP, hippocampus ; Thl and THL, thalamus ; PN, pontine nucleus ; CB, cerebellum.

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RGS7 and Gαq/11 immunohistochemical distribution

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RNA isolation
  5. Quantitative real-time RT-PCR
  6. RESULTS
  7. RGS7 and Gαq/11 immunohistochemical distribution
  8. Regulation of RGS7 and RGS4 by PdBu
  9. DISCUSSION

Optimized antibody dilutions were 1:2,000 for both anti-RGS7 and anti-Gαq/11 as determined by western immunoblots and were subsequently used for the immunohistochemical studies. The limit of detection was 1:20,000 for the antibody to RGS7 and 1:10,000 for Gαq/11. Each immunohistochemical study was performed a minimum of three times using different brain preparations, and the information presented in Table 1 summarizes the distribution of RGS7 and of Gαq/11 within the various brain regions of the adult rat. Controls performed with preimmune serum or omitting primary antibody were negative, and no positive staining was detected in white matter.

Table 1. Regional labeling densities for RGS7 and Gαq/11 antibodies within adult rat brain Relative densities were determined by visual inspection of immunolabeled brain sections derived from three separate experiments. Two individuals independently assessed the same immunohistochemical sections. Evaluation of the immunohistochemical staining was divided into five major ir categories : -, nondetectable, i.e., equal to background ; ±, faint (visible above background) ; +, moderate ; ++, strong ; and +++, very dense. This classification relates to the range of immunostaining observed for that particular antibody throughout the various brain regions but is not directly comparable with the intensity range of the other antibody.
 RGS7 Gαq/11
Neocortex  
Cortical layer I+++++
Cortical layer II-III++
Cortical layer IV±±
Cortical layer V-VI++
Piriform cortex--
Hippocampus  
Dentate gyrus granule cells--
CA1-3 pyramidal cells--
Polymorphic cell++++
Molecular cell++
Stratum radiatum±+
Stratum lucidum+±
Stratum lucidum-moleculare++
Stratum oriens++++
Striatum  
Caudate-putamen++
Nucleus accumbens++
Globus pallidus+±
Amygdala  
Central amygdala±±
Lateral amygdala±±
Basolateral amygdala++++
Medial amygdala±±
Medial habenula+++
Lateral habenula--
Hypothalamus  
Anterior hypothalamic central±+
Paraventricular nucleus±++
Dorsomedial hypothalamic nuclei±+
Ventromedial nucleus±±
Arcuate nucleus±+
Medial mammillary nucleus+++
Lateral mammillary nucleus+±
Thalamus  
Anterodorsal thalamic nucleus+++
Anteroventral thalamic nucleus, ventrolateral++++
Anteroventral thalamic nucleus, dorsomedial++++
Reticular thalamic nuclei++++
Mediodorsal thalamic nuclei+++
Ventral posteromedial thalamic nuclei+++
Ventral posterolateral thalamic nuclei++++
Ventral lateral thalamic nuclei+++
Dorsal lateral geniculate nuclei++++
Ventral lateral geniculate nuclei+±
Lateral postthalamic nuclei, mediorostral±±
Medial geniculate nucleus+++++
Posterior thalamic nuclear group+++
Gustatory thalamic nuclei+++
Parafascicular thalamic nuclei+++
Entopeduncular nuclei+±
Subthalamic nucleus++
Substantia nigra  
Pars compacta++
Pars reticulata++
Superior colliculus+++
Interpeduncular nuclei+++++
Pontine nuclei+++++
Dorsal raphe nuclei±+
Cerebellum  
Molecular layer+++++
Granule cell layer++±
Purkinje cells-±

A laminar distribution of RGS7-ir was observed in cortical layers I-VI, being most dense in the deep cortical layer I neuropil (Fig. 2A). Immunolabeling was located in the neuropil of cortical layer I and in the arising processes of cortical layers II-VI, whereas the neuronal cell bodies were immunonegative. In the hippocampal formation, RGS7 immunolabeling was concentrated in the dendritic subfields of CA1 pyramidal cells, namely, strata oriens and strata radiatum, the mossy fibers, and polymorphic cells, with faint or nondetectable ir within the granule cells of the dentate gyrus and pyramidal cell body layer of the CA1-CA3 region (Fig. 2B). The medial habenula displayed strong RGS7-ir in the cell bodies and fibers that was not observed in the lateral portion of the habenula (Fig. 2C). RGS7-ir in the striatum was moderate throughout the caudate-putamen, nucleus accumbens, and globus pallidus (Table 1). Numerous cells within various thalamic nuclei exhibited positive immunostaining for RGS7 (Table 1). Highest levels of ir were found in the dorsal (Fig. 2D) and medial lateral geniculate nuclei, as well as in the thalamic anteroventral ventrolateral (Fig. 2E), anteroventral dorsomedial, and ventral posterolateral nuclei. The paraventricular nucleus of the hypothalamus displayed faint RGS7-ir, which was primarily confined to the neurosomata of the lateral magnocellular subdivision, with no visible staining observed in the medial parvocellular subdivision (Fig. 2F). The amygdala exhibited faint to moderate RGS7-ir, with strongest staining occurring in the basolateral portion. Within the brainstem, the interpeduncular and pontine nuclei displayed very dense immunostaining (Fig. 2G). In the cerebellum, strong ir for RGS7 was observed in the molecular layer, whereas moderate staining occurred in the Golgi cells of the granule layer ; staining was absent in the Purkinje cells (Fig. 2H). Although the cDNA sequences of RGS6 and the protein have a very high degree of similarity to that of RGS7 (74% identity in the RGS domain), the regional distribution of RGS6 mRNA in the brain has been found to be very different compared with that of RGS7 mRNA by in situ hybridization studies (Gold et al., 1997). This makes it highly improbable that the RGS7-specific antibody recognized RGS6 protein in our immunohistochemical studies.

image

Figure 2. Immunohistochemical distribution pattern of RGS7-ir in coronal sections of adult rat brain : (A) retrosplenial granular cortex, (B) hippocampus, (C) habenula, (D) dorsal lateral geniculate nucleus, (E) thalamic anteroventral ventrolateral nucleus, (F) paraventricular nucleus, (G) pontine nucleus, and (H) cerebellum. Bar = 50 μm for A, 100 μm for F and H, and 400 μm for B-E and G. Arrows point to specific areas of interest. G, granular layer ; M, molecular layer.

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The regional pattern of Gαq/11-ir staining was located to the neuronal dendrites and cell body peripheries and overlapped to a large degree with the observed RGS7 distribution (Table 1). However, Fig. 3 highlights some notable variations in the distribution of Gαq/11-ir relative to RGS7-ir. Gαq/11-ir was more intense in cortical layer I (Fig. 3A), the subdivisions of the paraventricular hypothalamic nucleus (magnocellular and parvocellular) (Fig. 3B), and the molecular layer of the cerebellum (Fig. 3C) but was less intense in the medial habenula and most thalamic nuclei (Table 1). In the Purkinje cell layer, which displayed no apparent RGS7-ir, faint labeling of Gαq/11-ir was observed, and the granular layer revealed scattered, faintly reactive cells (Fig. 3C). Table 1 further highlights areas in which both RGS7 and Gαq/11 are codistributed but display differences in relative staining intensities, e.g., hypothalamus and thalamus, denoting a possible lack of protein equivalence in these regions.

image

Figure 3. Immunohistochemical distribution pattern of Gαq/11-ir in coronal sections of adult rat brain : (A) frontal cortex layers I-III, (B) hypothalamic paraventricular nucleus (arrow), and (C) cerebellum (arrows point to Purkinje cell layer). Bar = 50 μm for A and 100 μm for B and C. G, granular layer ; M, molecular layer.

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Dual immunofluorescence studies were performed to determine the extent of cellular colocalization occurring between the two components in the same immunohisto-chemical sections. Control sections in which RGS7 or Gαq/11 primary antibodies were omitted or replaced with preimmune serum were negative. Furthermore, to ensure that no nonspecific sequential labeling was occurring in the multistep dual-labeling procedure, one of the primary antibodies (RGS7 or Gαq/11) was omitted but the secondary and tertiary antibody steps were performed to completion. Under these conditions, no cross-reactivity or dual labeling was observed. The merged fluorescent images depicting RGS7 and Gαq/11 immunostaining confirmed a widely parallel pattern of cellular distribution for both protein components that was not entirely uniform throughout the CNS. The most pronounced areas of colocalization were exhibited in the neuropil region of cortical layer I (Fig. 4A), which consists of a dense subpopulation of intrinsic cholinergic, serotonergic, and multipolar and horizontal bipolar GABAergic neuronal fibers. Within the hilus area of the hippocampal formation, the mossy fibers and membrane lining of the interneurons displayed discrete cellular colocalization, whereas the neighboring granule cells were predominantly Gαq/11-enriched (Fig. 4B. Somatodendritic areas of the subthalamic neurons exhibited highest colocalization (Fig. 4C), and similarly throughout most nuclei of the thalamus, RGS7 was closely associated with Gαq/11. In the cerebellum, RGS7 and Gαq/11 were predominantly colocalized in the interneuronal Golgi cells of the granule cell layer (Fig. 4D), as well as the molecular layer. The cell bodies and fibers of the medial habenula, which exhibits a profuse cholinergic innervation, displayed a diffuse colocalization of both protein components (Fig. 4E). It is interesting that despite the presence of both RGS7 and Gαq/11 immunolabeling in the hypothalamic magnocellular subdivision of the paraventricular nucleus, poor, if any, colocalization was found (Fig. 4F).

image

Figure 4. Double fluorescence immunolabeling of RGS7 (green signal) and Gαq/11 (red signal) in rat brain coronal sections determined by confocal microscopy : (A) cortical layers I-V (low magnification), (B) hippocampal formation, (C) subthalamic nucleus, (D) cerebellar granule cell layer, (E) medial habenula, and (F) paraventricular nucleus. Bar = 200 μm for A and 30 μm for B-F. Colocalization of RGS7 and Gαq/11 signals are represented as yellow.

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Regulation of RGS7 and RGS4 by PdBu

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RNA isolation
  5. Quantitative real-time RT-PCR
  6. RESULTS
  7. RGS7 and Gαq/11 immunohistochemical distribution
  8. Regulation of RGS7 and RGS4 by PdBu
  9. DISCUSSION

The data presented thus far suggest that RGS7 is colocalized at a cellular level with Gαq/11. Taken together with previous studies suggesting that RGS7 can regulate Gαq/11, these data suggest that this RGS protein may play an important role in regulating Gαq/11-mediated signaling in discrete brain regions. We next wanted to determine whether signaling pathways regulated by Gαq/11 could modulate expression of RGS7. Thus, primary cortical neurons were used to assess the temporal effect of PdBu-mediated protein kinase C pathway activation on RGS7 and RGS4 mRNA levels. The results shown in Fig. 5 indicate that the RGS7 mRNA content was significantly increased by 2.5-fold (p < 0.03) after a 12-h exposure to PdBu and was still elevated at the 24-h time point. This effect was consistently observed in three independent experiments performed using separate batches of primary cortical neurons. In contrast, RGS4 mRNA levels from similar sample preparations were not altered over the same time course (Fig. 5).

image

Figure 5. RGS7 up-regulation after PdBu treatment of primary cortical neurons. Following PdBu application (500 nM to primary rat cortical neuron cultures, total RNA was isolated at the time points indicated. RGS4 and RGS7 mRNA levels were determined and compared with cyclophilin levels using a dual-detection 5′ nuclease assay system. Values are reported as change in relative expression of RGS compared with cyclophilin using the untreated (0) time point as 100%. RGS7 data are mean ± SEM (bars) values from the three independent batches of cortical neurons. *p < 0.03 by unpaired t test comparing indicated time points with values obtained at 1 h. RGS4 data are mean values from two independent batches of cortical neurons.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RNA isolation
  5. Quantitative real-time RT-PCR
  6. RESULTS
  7. RGS7 and Gαq/11 immunohistochemical distribution
  8. Regulation of RGS7 and RGS4 by PdBu
  9. DISCUSSION

The present study describes the cellular distribution of immunoreactive RGS7 and of Gαq/11 proteins in the adult rat brain by immunohistochemistry using specific affinity-purified polyclonal antibodies. Western blot analysis with the anti-RGS7 antibody demonstrated the presence of RGS7 throughout most brain regions, and the immunospecific labeling of a single antigenic band of ~62 kDa was observed, corresponding to that predicted for the native form of RGS7 protein. The regional distribution of RGS7 in both membrane and cytosolic fractions suggests its presence within various neuronal subcellular compartments, either tightly associated within membrane-bound components or as a soluble cytosolic protein weakly associated with components involved in the transmembrane signal transduction machinery. Although the RGS core domains are relatively short, each consisting of ~120 amino acids, RGS7 has extended N- and C-terminal residues that could provide additional motifs involved in subcellular compartmentalization, transmembrane insertion, and/or posttranslational modifications, as has been reported for RGS1 and GAIP (De Vries et al., 1996 ; Faurobert and Hurley, 1997). In contrast, the Gαq/11 subunits were predominantly associated with brain membrane fractions. This transmembrane insertion could be accounted for by N-terminal posttranslational thioester-linked palmitoylation of endogenous Gαq/11 as has been reported previously for NG108-15 cell populations (Parenti et al., 1993).

Our immunohistochemical findings indicate a wide-spread and overlapping distribution of both RGS7 and Gαq/11 throughout the cortex, hippocampus, hypothalamus, striatum, thalamus, and cerebellum and together with the immunofluorescence colocalization studies support their close association throughout many of these regions within cell body and/or neuronal dendritic networks. RGS7 protein was not detected in white matter or in astrocytes, but this does not exclude its presence, as we have shown rat C6 glioma cell lines to express this protein in western immunoblots (J.-J.L., unpublished data). Although the Gαq/11 antibody was unable to distinguish between the separate Gαq and Gα11 subunits, current evidence tends to suggest little functional difference between the two G protein members either in their G protein-coupled receptor interactions or in phospholipase Cβ regulation (Blank et al., 1991 ; Berstein et al., 1992). Moreover, Milligan (1993) has reported that both Gα subunits are found in the same brain regions, with the levels of Gαq being approximately two- to threefold higher than those of Gα11. Without more detailed information at present, it may be assumed that RGS7 interacts nondiscriminately with both Gα subtypes.

The results confirm earlier in situ hybridization data on the morphological distribution of RGS7 and Gαq/11 mRNA (Gold et al., 1997 ; Shuey et al., 1998), as well as expand on previous immunohistochemical observations for Gαq/11 protein distribution in rat brain (Mailleux et al., 1992). However, despite a strong correlation in the regional distribution of protein and previously reported mRNA expression (Shuey et al., 1998) for both RGS7 and Gαq/11, their regional labeling densities differed substantially. For example, some of the highest levels of RGS7 and Gαq/11 mRNA expression were found in the medial mammillary nucleus, cerebellar granule cell layer, and subthalamic nucleus (Gold et al., 1997 ; Shuey et al., 1998), whereas their respective protein immunolabeling densities for the same regions were low. More striking is that specific regions such as the piriform cortex, dentate gyrus granule cells, pyramidal cells of the CA1-CA3 subfields, and lateral habenula displayed no detectable protein labeling (Table 1), whereas their mRNA was reported to be strongly expressed in similar regions (Gold et al., 1997 ; Shuey et al., 1998). We may conclude that, in many instances, the distribution of mRNA expressed in specific brain regions also represents sites of protein synthesis and colocalization. However, as mentioned above, obvious regional mismatches are apparent between protein and mRNA distribution, possibly owing to differences in half-life and/or translocation rates of protein from neuronal cell bodies toward distal terminal dendritic layers located at different sites in the brain.

The colocalization of RGS7 and Gαq/11 within discrete cellular structures is one relevant factor in determining the ability of a neuronal cell to channel information through specific second messenger pathways, and the role of RGS proteins would be to regulate negatively specific signaling events (Berman and Gilman, 1998 ; Shuey et al., 1998). The most prevalent areas of RGS7 and Gαq/11 colocalization were demonstrated in the superficial laminar layers of the cerebral cortex, hippocampal subfields, thalamus, and cerebellum, all of which represent areas enriched with a diverse array of serotonergic-, GABAergic-, cholinergic-, and glutaminergic-mediated pathways, many of which are known to promote signaling via the Gαq/11 component. A cellular selectivity in the colocalization between RGS7 and Gαq/11 within many discrete brain regions, such as the superficial cortical layer I, hilus area of the hippocampal formation, and cerebellar Golgi cells, was further noted and may suggest an even greater complexity in RGS7 and Gαq/11 interactions. It is currently unclear as to how RGS proteins regulate the kinetics of G protein-dependent biological responses, but they have been reported to play a crucial role in the timing of G protein-coupled receptor-mediated information transfer and in the rapid regulation of neuronal excitability (Doupnik et al., 1997 ; Saitoh et al., 1997).

In vitro transfection studies using Chinese hamster ovary-K1 cell populations have shown that recombinant RGS7 can rapidly attenuate 5-HT2c/Gαq/11-mediated activation of Ca2+ mobilization and inositol trisphosphate production (DiBello et al., 1998 ; Shuey et al., 1998), an effect that may be selective for Gαq/11-mediated events, because further studies failed to demonstrate an effect of recombinant RGS7 on Gαs- or Gαi-mediated adenylyl cyclase signaling events (DiBello et al., 1998 ; Shuey et al., 1998). Phorbol esters, such as PdBu, are direct activators of protein kinase C and thereby act as potent agonists of this pathway (Horsburgh et al., 1991). In primary cortical neurons, we observed the ability of PdBu to increase selectively RGS7 mRNA content but not that of RGS4. This suggests that a corresponding increase in protein levels would result in a neuroadaptive feedback regulation of neurotransmitter-Gαq/11-coupled pathways and provides scope for a possible functional association between Gαq/11 and RGS7, to regulate negatively excessive pathway activity. The precise physiological role played by RGS7 remains uncertain, but a dynamic fluctuation in the level of RGS7 mRNA expression has been reported after acute seizures in rat hippocampus (Gold et al., 1997). At present, it remains unclear as to which receptor(s) may be specifically associated with the Gαq/RGS7 pathway and the reason for the prolonged lag time involved before an increase in RGS7 expression occurred. Further investigations to elucidate these questions are currently underway.

At present, we cannot exclude in situ functional interactions between RGS7 and other Gα subunits or between Gαq/11 and other RGS subtypes, because the distribution of RGS7 could not be completely overlaid with that of Gαq/11, and the dual immunofluorescence portion of our study highlighted instances of identical cellular presence for both protein components with minimal colocalization. To this effect, RGS7 is reported to be an efficient GTPase-activating protein for Gαil and Gαo subunits in cell-free systems, and in mammalian cell populations, several alternative RGS subtypes have been shown to attenuate Gαq-mediated signaling (Huang et al., 1997 ; Neill et al., 1997 ; Yan et al., 1997 ; Saugstad et al., 1998). The hypothalamic paraventricular nucleus and cerebellum represent two strong regional candidates for Gαq/11 interaction with alternative RGS subtypes. The hypothalamic paraventricular nucleus is involved in serotonergic 5-HT2-mediated stimulation of oxytocin release (Van de Kar et al., 1995) and is a primary site of biosynthesis of several neuroendocrine hormone precursors and of nor-adrenergic afferent innervation of each type of neurosecretory somata in the magnocellular subdivision. Both the magno- and parvocellular subdivisions of the paraventricular nucleus demonstrated positive labeling for the Gαq/11 subunits (Fig. 3B), whereas RGS7 was clearly confined to the magnocellular subdivision (Fig. 2F) and exhibited no obvious cellular colocalization with Gαq/11 in this area (Fig. 3F). In the cerebellar Purkinje cell layer, RGS7 protein was not visibly colocalized with the Gαq/11 subunits but was clearly observed in the inhibitory Golgi cell interneurons of the granule layer (Fig. 3D). The cerebellar granule cell dendrites receive terminal GABAergic input from the Golgi cells, which are a principal target of glutamate axons. Overlapping regional mRNA expression of other RGS subtypes has been documented (Gold et al., 1997 ; Shuey et al., 1998), and it is probable that one or more of these RGS proteins constitute the preferred GTPase-activating protein for Gαq/11 in these subregions. This awaits further immunohistochemical investigation.

In conclusion, these studies provide the first immunohistochemical analysis of the relative distribution of RGS7 in brain, together with its cellular colocalization with the phosphoinositidase C-linked G protein Gαq/11. The general patterns of RGS7 and Gαq/11 labeling suggested a heterogeneous and overlapping distribution for both of these proteins and correlated well with previously published in situ hybridization studies within the CNS. Some interesting selectivities in cellular RGS7 and Gαq/11 colocalization were observed, and further studies are currently underway to determine the colocalization of RGS7 with the Gαi subfamily, another important G protein member. We also demonstrated a selective increase in RGS7 mRNA expression after prolonged protein kinase C-mediated stimulation in primary rat cortical neurons. In general, the findings improve our understanding of the relationship between RGS proteins and Gα subunits in discrete brain regions and provide important information on the functional significance of RGS protein subtype in the regulation of receptor-mediated G protein signal transduction pathways.

  • 1
    Berman D.M. & Gilman A.G. (1998) Mammalian RGS proteins : barbarians at the gate.J. Biol. Chem. 273,12691278.
  • 2
    Berman D.M., Wilkie T.M., Gilman A.G. (1996) GAIP and RGS4 are GTPase-activating proteins for the Gi subfamily of G protein alpha subunits.Cell 86,445452.
  • 3
    Berstein G., Blank J.L., Smrcka A.V., Higashijima T., Sternweis P.C., Exton J.H., Ross E.M. (1992) Reconstitution of agonist stimulated phosphatidylinositol 4,5-bisphosphate hydrolysis using purified m1 muscarinic receptor, Gq/11 and phospholipase C-β1. J. Biol. Chem. 267,80818088.
  • 4
    Blank J.L., Ross A.H., Exton J.H. (1991) Purification and characterization of two G-proteins that activate the β1 isozyme of phosphoinositidase-specific phospholipase C. J. Biol. Chem. 266,1820618216.
  • 5
    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. 72,248254.
  • 6
    Burchett S.A., Volk M.L., Bannon M.J., Granneman J.G. (1998) Regulators of G protein signaling : rapid changes in mRNA abundance in response to amphetamine.J. Neurochem. 70,22162219.
  • 7
    De Vries L., Elenko E., Hubler L., Jones T.L.Z., Farquhar M.G. (1996) GAIP is membrane-anchored by palmitoylation and interacts with the activated (GTP-bound) form of G alpha i subunits.Proc. Natl. Acad. Sci. USA 93,1520315208.
  • 8
    DiBello P.R., Garrison T.R., Apanovitch D.M., Hoffman G., Shuey D.J., Mason K., Cockett M.I., Dohlman H.G. (1998) Selective uncoupling of RGS action by a single point mutation in the G protein α-subunit. J. Biol. Chem. 273,57805784.
  • 9
    Dohlman H.G. & Thorner J. (1997) RGS proteins and signaling by heterotrimeric G proteins.J. Biol. Chem. 272,38713874.
  • 10
    Dohlman H.G., Song J., Ma D., Courchesne W.E., Thorner J. (1996) Sst2, a negative regulator of pheromone signaling in the yeast Saccharomyces cerevisiae : expression, localization, and genetic interaction and physical association with Gpal (the G-protein alpha subunit).Mol. Cell. Biol. 16,51945209.
  • 11
    Doupnik C.A., Davidson N., Lester H.A., Kofuji P. (1997) RGS proteins reconstitute the rapid gating kinetics of G-beta-gamma-activated inwardly rectifying K+ channels.Proc. Natl. Acad. Sci. USA 94,1046110466.
  • 12
    Druey K.M., Blumer K.J., Kang V.H., Kehrl J.H. (1996) Inhibition of G-protein-mediated MAP kinase activation by a new mammalian gene family.Nature 379,742746.
  • 13
    Faurobert E. & Hurley J.B. (1997) The core domain of a new retina specific RGS protein stimulates the GTPase activity of transducin in vitro.Proc. Natl. Acad. Sci. USA 94,29452950.
  • 14
    Gibson U.E.M., Heid C.A., Williams P.M. (1996) A novel method for real time quantitative competitive RT-PCR.Genome Res. 6,9951001.
  • 15
    Gold S.J., Ni Y.G., Dohlman H.G., Nestler E.J. (1997) Regulators of G-protein signaling (RGS) proteins : region-specific expression of nine subtypes in rat brain.J. Neurosci. 17,80248037.
  • 16
    Harlow E. & Lane D. (1988) Antibodies, a Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.
  • 17
    Hepler J.R., Berman D.M., Gilman A.G., Kozasa T. (1997) RGS 4 and GAIP are GTPase-activating proteins for Gqα and block activation of phospholipase Cβ by γ-thio-GTP-Gqα. Proc. Natl. Acad. Sci. USA 94,428432.
  • 18
    Horsburgh K., Dewar D., Graham D.I., McCulloch J. (1991) Autoradiographic imaging of [3H]phorbol 12,13-dibutyrate binding to protein kinase C in Alzheimer's disease. J. Neurochem. 56,11211129.
  • 19
    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. USA 94,61596163.
  • 20
    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 383,175177.
  • 21
    Koelle M.R. (1997) A new family of G-protein regulators—the RGS proteins. Curr. Opin. Cell Biol. 9,143147.
  • 22
    Koelle M.R. & Horvitz H.R. (1996) EGL-10 regulates G-protein signaling in the C. elegans nervous system and shares a conserved domain with many mammalian proteins.Cell 84,115125.
  • 23
    Livak K.J., Flood S.J., Marmarao J., Giusti W., Deetz K. (1995) Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization.PCR Methods Appl. 4,357362.
  • 24
    Mailleux P., Mitchell F., Vanderhaeghen J., Milligan G., Erneux C. (1992) Immunohistochemical distribution of neurons containing the G-proteins Gqα/G11α in the adult rat brain.Neuroscience 51,311316.
  • 25
    Milligan G. (1993) Regional distribution and quantitative measurement of the phosphoinositidase C-linked guanine nucleotide binding proteins G11α and Gqα in rat brain.J. Neurochem. 61,845851.
  • 26
    Neer E.J. (1997) Intracellular signalling : turning down G-protein signals.Curr. Biol. 7,R31R33.
  • 27
    Neill J.D., Duck L.W., Sellers J.C., Musgrove L.C., Scheschonka A., Druey K.M., Kehrl J.H. (1997) Potential role for a regulator of G protein signaling (RGS3) in gonadotropin-releasing hormone (GnRH) stimulated desensitization.Endocrinology 138,843846.
  • 28
    Parenti M., Vigano M.A., Newman C.M.H., Milligan G., Magee A.I. (1993) A novel N-terminal motif for palmitoylation of G-protein α subunits.Biochem. J. 291,349353.
  • 29
    Paxinos G. & Watson C. (1986) The Rat Brain in Stereotaxic Coordinates, 2nd edit. Academic Press, Sydney.
  • 30
    Saitoh O., Kubo Y., Miyatani Y., Asano T., Nakata H. (1997) RGS8 accelerates G-protein-mediated modulation of K+ currents.Nature 390,525529.DOI: 10.1038/37385
  • 31
    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. 18,905913.
  • 32
    Shuey D.J., Betty M., Jones P.G., Khawaja X.Z., Cockett M.I. (1998) RGS7 attenuates signal transduction through the Gαq family of heterotrimeric G proteins in mammalian cells.J. Neurochem. 70,19641972.
  • 33
    Van de Kar L.D., Rittenhouse P.A., Li Q., Levy A.D., Brownfield M.S. (1995) Hypothalamic paraventricular, but not supraoptic neurons, mediate the serotonergic stimulation of oxytocin secretion.Brain Res. Bull. 36,4550.
  • 34
    Watson N., Linder M.E., Druey K.M., Kehrl J.H., Blumer K.J. (1996) RGS family members : GTPase-activating proteins for heterotrimeric G-protein alpha-subunits.Nature 383,172175.
  • 35
    Yan Y., Chi P.P., Bourne H.R. (1997) RGS 4 inhibits Gq-mediated activation of mitogen-activated protein kinase and phosphoinositide synthesis.J. Biol. Chem. 272,1192411927.
  • 36
    Yu J., Wieser J., Adams T.H. (1996) The Aspergillus FlbA RGS domain protein antagonizes G-protein signaling to block proliferation and allow development.EMBO J. 15,51845190.