Localisation of the GPRC5B receptor in the rat brain and spinal cord

Molecular Brain Research 106 (2002) 136–144 www.elsevier.com / locate / bres

Research report

Localisation of the GPRC5B receptor in the rat brain and spinal cord Melanie J. Robbins a , *, Kelly J. Charles b , David C. Harrison b , Menelas N. Pangalos b a

Department of Schizophrenia and BPD, Psychiatry CEDD, GlaxoSmithKline Pharmaceuticals, New Frontiers Science Park ( North), Third Avenue, Harlow, Essex CM19 5 AW, UK b Neurology CEDD, GlaxoSmithKline Pharmaceuticals, New Frontiers Science Park ( North), Third Avenue, Harlow, Essex CM19 5 AW, UK Accepted 5 August 2002

Abstract Recently a novel subfamily of closely related orphan G protein-coupled receptors (GPCRs) was identified, called GPRC5A, GPRC5B, GPRC5C and GPRC5D. Based on sequence homology, these receptors were classified as family C GPCRs, which include metabotropic GABA B receptors, metabotropic glutamate receptors, the calcium sensing receptor and a number of pheromone receptors. GPRC5 receptors share approximately 30–40% sequence homology to each other and 25% homology to the other family C members. It has been shown human GPRC5B mRNA is predominantly expressed in the central nervous system. In order to further characterise this receptor, we investigated both the mRNA and protein expression profiles in rodent tissues. Western blot analysis, using affinity-purified antisera specific to GPRC5B, identified a protein migrating at approximately 68 kDa, close to the predicted molecular weight for GPRC5B. Immunocytochemical analysis of GPRC5B-transfected cells revealed a cell surface localisation. In addition, immunohistochemical analysis of GPRC5B in rat brain and spinal cord demonstrated receptor expression in many areas, with highest levels of immunoreactivity in the neocortex, all subfields of the hippocampus, the granule cell layer of the cerebellum and throughout the spinal cord.  2002 Elsevier Science B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters, and receptors Topic: Regional localization of receptors and transmitters Keywords: GPRC5B; CNS; Family C GPCR

1. Introduction G-protein coupled receptors (GPCRs) are divided into three main families dependent upon their sequence homology and function. Family A form the rhodopsin-like receptor class, family B are secretin-like receptors and family C are the metabotropic-like receptors. The principle differentiating characteristic of the family C GPCRs is their large extracellular amino terminal domain which is thought to be essential for ligand binding and initiation of signal transduction. In addition, family C GPCRs are further subdivided into four subfamilies based on their sequences and ligand selectivity. These include the metabotropic glutamate receptors (mGluR), the GABA B receptor, the calcium sensing receptor (CaSR) and the *Corresponding author. Tel.: 144-1279-622-968; fax: 144-1279-622230. E-mail address: melanie j [email protected] (M.J. Robbins). ]]

pheromone receptors [5,9,14,17]. More recently a new sub-class of family C has been identified which includes four orphan receptors, GPRC5A, GPRC5B, GPRC5C and GPRC5D [2,4,6,15]. Unlike other family C members, these receptors contain only a short amino terminus of 30–50 amino acids, suggesting that domains other than the Nterminus may be important for ligand binding and signal transduction, a feature more typical of family A and family B receptors [3,13,15] GPRC5A was the first of this family to be identified by differential display analysis of a small lung carcinoma cell line. Subsequently, GPRC5B, GPRC5C and GPRC5D were identified by homology searching of EST and genomic databases [6,15]. A common biological characteristic between GPRC5A, GPRC5B and GPRC5C is the ability of all trans-retinoic acid (ATRA) to stimulate mRNA expression in a number of cell lines of different origin [2,15]. This is not the case for GPRC5D whose mRNA levels are not affected by ATRA [2]. As a result of these observa-

0169-328X / 02 / $ – see front matter  2002 Elsevier Science B.V. All rights reserved. PII: S0169-328X( 02 )00420-5

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tions, these receptors have also been termed retinoic acidinducible receptors (RAIG1, RAIG2 and RAIG3). The mRNA expression profile of GPRC5A, GPRC5B, GPRC5C and GPRC5D has previously been reported in human adult central nervous system (CNS) and peripheral tissues [2,4,15]. GPRC5A is expressed almost exclusively in human lung. In contrast, GPRC5B is expressed predominantly in tissues of the CNS, whereas GPRC5C and GPRC5D are expressed predominantly in peripheral tissues. Despite extensive efforts by us and other groups, a ligand has yet to be identified for this family of orphan receptors. Therefore, to further our understanding of the potential role that this group of receptors may play within the CNS, we generated antisera and mRNA primers to GPRC5B. We have analysed the expression of GPRC5B in both adult rat brain and spinal cord at the mRNA and protein levels. It is hoped that this information will facilitate the generation of mRNA expression libraries and / or protein extract libraries suitable for identifying the ligand for this CNS specific receptor.

2. Materials and methods

2.1. Taqman quantitative polymerase chain reaction Total RNA from rat was prepared using Trizol reagent (Life Technologies) following the manufacturer’s protocols. Tissues were dissected from three rats and pooled before RNA preparation, as previously described [12]. Total RNA from primary rat neuronal cells was prepared using the RNeasy kit (Qiagen) following the manufacturer’s protocol. cDNA synthesis was performed in triplicate as previously described [12]. Parallel reactions for each mRNA sample were performed in the absence of reverse transcriptase to determine the level of contaminating genomic DNA. Primer sequences for rat were— ratGPRC5B-F 59-AGGGATGCTGGAGATGCCTAG-39, ratGPRC5B-R 59-ACCAGTTGTCATTTGTCATGGCT-39 and ratGPR5B-probe 59-CGCAAAGGATTGACTTGCTCAGGACA-39. Data were captured as previously described and analysed using the relative standard curve method [10]. Each sample was normalised to cyclophilin to correct for differences in RNA quality and quantity [12].

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mg / ml L-cysteine, 40 mg / ml DNAse type IV and incubated at 37 8C for 60–75 min. The supernatant was removed and replaced by 1 ml ovomucoid / trypsin inhibitor solution [1 mg / ml trypsin inhibitor, 50 mg / ml bovine serum albumin (BSA) fraction V and 40 mg / ml DNAse I type IV in L-15 media]. The media was replaced twice before generating a single cell suspension by trituration with a flame-polished Pasteur pipette. The cell suspension was transferred to a plastic centrifuge tube and diluted to 10 ml with Dulbecco’s modified Eagle’s medium (DMEM) / 10% fetal calf serum (FCS) containing 4 mM L-glutamine. The cells were harvested by centrifugation at 1000 rpm for 5 min, resuspended in DMEM / 10% FCS and plated onto poly-L-lysine coated T75 flasks. The culture media was changed 4 days after seeding and then every 3–4 days. After 12 days in culture, O2A progenitor cells were removed from the underlying type 1 astrocytes by mechanical shaking as described by McCarthy and de Vellis [11]. The astrocytes were cultured separately and the medium changed every 3–4 days. The resultant O2A cell suspension was incubated in a bacterial culture dish for 15 min to differentially remove microglia and the supernatant then placed in a 50-ml tube for 2 min. Cell aggregates sediment at the bottom of the tube allowing the single cell suspension to be removed and centrifuged at 1000 rpm for 5 min. The cell pellet was resuspended in Sato’s medium and plated at the required cell density on poly-L-lysine coated wells. After 24 h in culture this preparation contained predominantly O2A progenitor cells and between 2 and 5% type 1 astrocytes. Cells were used for the experiments described here after 24 h, 3 days or 6 days in culture. During this period the O2A cells differentiate to O4 1 pro-oligodendrocytes.

2.3. Culture and transfection of HEK-293 cells All cell culture reagents were obtained from Life Technologies, Paisley, UK. HEK-293 cells were maintained in (DMEM) supplemented with 10% fetal calf serum and 1% non-essential amino acids. Exponentially growing cells were transfected using Lipofectamine Plus (Life Technologies) according to the manufacturer’s instructions and then incubated for 24 h to allow for protein expression before analysis.

2.2. Culturing of rat oligodendrocytes and rat atsrocytes 2.4. Antibodies and immunofluorescence O2A progenitor cells were prepared as described in Ref. [1] with modifications. Primary mixed brain cell cultures were prepared from brains of 1–2-day-old Sprague–Dawley rats. The rats were decapitated, the brains removed and the cerebral cortices dissected and stripped of the meninges. The cortices were minced in ice-cold MEM-Hepes and allowed to settle before the media was replaced by MEMHepes containing 30 U / ml papain (Worthington), 0.25

A rabbit antiserum specific for the intracellular region of the human GPRC5B C-terminus was raised against the 15 amino acid peptide—DTSQPRMRETAFEED (amino acids 416–439). This antiserum also matches closely to two rat EST sequences (BF562991 and BF567929) found by similarity searching of public databases. The antiserum was generated and affinity purified by Research Genetics.

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Two rabbits were injected with 0.1 mg of each peptideKLH conjugate in Freund’s complete adjuvant, and boosted four times with the same amount of antigen in incomplete adjuvant. Terminal bleeds were clotted overnight at 4 8C and the serum separated from blood cells by centrifugation at 8000 g. Antigens were immobilised on activated supports to allow for the affinity purification of antisera. Elution from columns was via a pH gradient and fractions were collected and stored in 0.125 M borate buffer. For immunocytochemistry, cells on glass coverslips were fixed with 4% paraformaldehyde for 5 min then permeabilised with 0.1% Triton X-100 for 10 min. Cells were incubated in primary antibody [anti-GPRC5B, 1:2000 in phosphate-buffered saline (PBS), 60 min], washed in PBS, and then incubated in goat anti-rabbit IgG, fluorescein isothiocyanate (FITC) obtained from Sigma, UK and used at 1:100 in PBS, 45 min. Cells were then washed in PBS, mounted in Citifluor (UKC, Canterbury, UK) and viewed using a Leica laser scanning confocal microscope. Pre-absorption of the sera was carried out overnight at 4 8C using 20 mg peptide / ml blocking solution including 1:2000 anti-GPRC5B primary antibody.

2.5. Western immunoblotting Transiently transfected HEK293 cells, untransfected HEK293 cells, whole rat brain, rat cortex and rat astrocytes were lysed with ice-cold RIPA buffer [1% Nonidet P-40, 1% DOC(Na), 0.1% sodium dodecyl sulfate (SDS), 0.15 M NaCl, 0.01 M sodium phosphate, pH 7.2] including protease inhibitors (protease inhibitor cocktail tablets, Roche, UK). Samples were diluted in sample buffer containing 2% (v / v) 2-mercaptoethanol and boiled for 5 min. Eluted proteins were resolved by discontinuous SDS– polyacrylamide gel electrophoresis (PAGE), and transferred to a polyvinylidene difluoride membrane using a semi-dry transfer system (Bio-Rad). The membranes were blocked with 5% non-fat milk in PBS / 0.05% Tween-20 and then incubated overnight with either anti-GPRC5B antibody at 0.1–1 mg / ml, or anti-c-myc (rabbit polyclonal) at a dilution of 1:200 in blocking solution. Immunoreactive bands were detected with a goat anti-rabbit antibody (Santa Cruz Biotechnology, CA, USA) conjugated to horseradish peroxidase (1:15,000) followed by chemiluminescence detection (Supersignal chemiluminescent substrate, Pierce, UK). Pre-absorption of the sera was carried out overnight at 4 8C using 20 mg peptide / ml blocking solution including 0.1 mg / ml anti-GPRC5B primary antibody.

2.6. Tissue preparation Male Sprague–Dawley rats were anaesthetized with sodium pentobarbital prior to being transcardially perfused with 4% paraformaldehyde (PFA) in PBS (pH 7.4). Brains and spinal cords were removed and post-fixed in 4% PFA

for 24 h before being transferred to 30% sucrose for 48 h at 4 8C. Tissues were frozen in isopentane at 240 8C and 40 mm brain or 35 mm spinal cord sections were cut at 220 8C using a cryostat. Brain and spinal cord sections were stored in cryoprotectant containing glycerol and ethylene glycol at 220 8C prior to use. All procedures involving experimental animals were conducted in accordance with the United Kingdom Animals (Scientific Procedures) Act, 1986 and conformed to GlaxoSmithKline ethical standards.

2.7. Immunohistochemistry Immunostaining was revealed using the ABC detection system as described elsewhere [8]. Briefly, all sections were washed thoroughly in PBS, permeabilised in 0.1% Triton X-100 and endogenous peroxidase activity removed by 1% H 2 O 2 in PBS. Non-specific antibody binding was reduced by pre-incubation in a PBS blocking solution containing 1% bovine serum albumin (Boehringer Mannheim, Germany) and 10% normal goat serum (Vector Labs., Burlingame, CA, USA). Sections were incubated with primary antibody (1.8 mg / ml) in blocking solution for 48 h at 4 8C. In control experiments, primary antibodies were pre-absorbed with an excess amount of the immunogenic peptide (10 mg / ml) at 4 8C for 72 h. Sections were then incubated with a biotinylated anti-rabbit secondary antibody (1:200, Vector Labs.) followed by a peroxidaseconjugated avidin–biotin complex (Vector Labs. Peroxidase Rabbit Kit) and 3,39-diaminobenzidine tetrachloride as the colour substrate (Vector Labs.). Sections were dehydrated in 100% ethanol, immersed in Histolene (CellPath, Leeds, UK) for 10 min and coverslipped with DPX (BDH, Lutterworth, UK). Sections were analysed using a Leica DMR microscope equipped with a Leica DC200 digital camera. Images were prepared using PaintShop Pro Version 7.0 (JASC, Eden Prairie, MN, USA) for contrast and brightness adjustments and cropping.

3. Results

3.1. Analysis of GPRC5 B mRNA expression in rat tissues We designed polymerase chain reaction (PCR) primers and a fluorogenic probe to specifically amplify the rat GPRC5B receptor. Taqman analysis of cDNA reverse transcribed from total RNA was carried out in both rat brain and peripheral tissues for GPRC5B (Fig. 1A). GPRC5B was found to be highly and predominantly expressed in all tissues of the CNS and in the dorsal root ganglion (DRG). Highest levels of GPRC5B mRNA expression were observed in amygdala, cerebellum, areas of the neocortex, spinal cord, striatum, substantia nigra, thalamus and pituitary, with the lowest level of expression

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Fig. 1. Taqman RT-PCR analysis of GPRC5B in rat tissues. (A) Taqman distribution profiles of GPRC5B are shown in peripheral and brain rat tissues. Units are expressed as arbitrary fluorescence units and have been normalised to the housekeeping gene cyclophilin. Measurements are from three independent samples derived from total RNA and are mean6S.E.M. GPRC5B shows a widespread distribution throughout all the rat tissues investigated with the highest levels of expression seen in the CNS tissues. (B) Taqman distribution profiles of GPRC5B are shown in rat astrocytes that have been cultured for 21 days (Astro 21DIV) and rat oligodendrocytes that have been cultured for either 1 day (Oligod 1DIV), 3 days (Oligod 3DIV), or 6 days (Oligod 6DIV). Units are expressed as arbitrary fluorescence units and have been normalised to the housekeeping gene cyclophilin. Measurements are from three independent samples derived from total RNA and are mean6S.E.M.

detectable in the DRG. In peripheral tissues, expression was low or undetectable (heart, adipose, mammary gland, lung and testis) apart from a moderate level of mRNA expression observed in the placenta (Fig. 1A). Taqman analysis was also carried out in rat astrocytes and oligodendrocytes for GPRC5B (Fig. 1B). GPRC5B was found to be expressed predominantly in astrocytes and to a lesser extent in oligodendrocytes.

3.2. Characterisation of the GPRC5 B antibody by Western blotting and immunocytochemistry In order to investigate the distribution of GPRC5B at the protein level we generated polyclonal antisera against a peptide sequence in the intracellular region of the GPRC5B receptor. Initial characterisation of the antisera was performed by Western blotting of HEK293 cell

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extracts over-expressing epitope tagged c-myc GPRC5B receptor and lysates from whole rat brain, rat cortex lysates and rat astrocytes. A predominant band was detected in HEK293 extracts over-expressing the GPRC5B receptor, and in lysates from rat whole brain and rat cortex migrating at a molecular weight of approximately 68 kDa using the anti-GPRC5B antisera (Fig. 2A). A band of identical size was also observed with anti-myc antisera confirming the specificity of the anti-GPRC5B antisera. In addition, higher molecular weight protein bands were observed that could potentially represent GPRC5B dimers

(at approximately 130 kDa) or higher molecular weight protein aggregates. The higher molecular weight band is the predominant band observed in rat astrocytes. All molecular weight bands were abolished using antiGPRC5B pre-incubated in the presence of the respective immunizing peptide. We further characterised the GPRC5B antisera by examining the expression of GPRC5B in transiently-transfected HEK293 cells using immunocytochemistry (Fig. 2B). After permeabilising the cells with Triton X-100, GPRC5B immunopositive cells were detected. Staining

Fig. 2. Characterisation of the GPRC5B antibody. (A) Immunoblot showing expression of GPRC5B. Lysates prepared from untransfected HEK293 cells (lane 1), HEK293 cells transiently transfected with c-myc tagged GPRC5B (lanes 2, 3 and 6), whole rat brain (lane 4), rat cortex (lane 5) and rat astrocytes (lane 6) were analysed by SDS–PAGE (10%). Immunoblotting was performed with antibodies specific to GPRC5B (lanes 1–6) or the c-myc epitope (lane 7). GPRC5B migrates at approximately 68 kDa. The specificity of the GPRC5B antibody was determined by preabsorption of the serum with peptide (lane 3). (B) Subcellular localization of GPRC5B. HEK293 cells transiently transfected with GPRC5B were examined for expression, after permeabilisation with 0.1% Triton X-100, using the specific GPRC5B antibody. Staining of GPRC5B could be seen intracellularly and within the plasma membrane. The specificity of the GPRC5B staining was determined by preabsorption of the serum with peptide. Photomicrographs were taken using a confocal laser microscope.

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was observed within intracellular perinuclear membrane compartments, similar to Golgi or endoplasmic reticular staining. In addition, strong staining was observed around the circumference of the cells in a pattern typical of cell surface receptor localisation. All staining was abolished by pre-incubation of the primary antibody with the immunizing peptide (Fig. 2B).

3.3. Analysis of GPRC5 B protein expression in the rat brain and spinal cord GPRC5B showed a widespread localisation throughout the rat brain with most intense immunoreactivity (IR) observed in the cortex, caudate putamen, hippocampus and cerebellum and was comparable to the ubiquitous expression pattern observed at the mRNA level (data not shown). GPRC5B IR was also observed in the nucleus accumbens, all nuclei of the septum, thalamic and hypothalamic nuclei, and in particular in the ventral nuclei of the hypothalamus. Strong GPRC5B IR was also observed in the superficial gray layer of the superior colliculus and dorsal cortex of the inferior colliculus and was less intense in the deeper layers of both colliculi. Both nuclei of the substantia nigra expressed moderate degrees of GPRC5B IR as did the pontine nuclei. In the brain stem IR was wide spread in areas including the reticular and vestibular nuclei. Specificity controls of the antisera with the immunogenic peptide at 10 mg / ml abolished all positive immunoreactivity as seen in the CA1 subfield of the hippocampus (Fig. 4E). A more detailed immunohistochemical characterisation of GPRC5B IR in specific areas of the brain and spinal cord is described below. Cortex—Layers II–VI of the cortex were densely immunoreactive for GPRC5B, with the majority of IR observed in the cytoplasm, but not the nuclear space, as shown in the frontal cortex (Fig. 3A), some labelling of apical dendrites was also seen in the large pyramidal cells of layer V of the cortex (Fig. 3B). GPRC5B IR was also seen in the corpus callosum (Fig. 3A). Hippocampus—All subfields of the hippocampus demonstrated GPRC5B immunoreactivity, particularly in the CA1–CA3 sub-regions and the dentate gyrus (Fig. 4A); relatively little IR was seen in the stratum oriens or stratum radiatum, nor in the molecular layer of the dentate. In the CA1 subfield, GPRC5B IR was associated with the cell body of pyramidal cells and to a lesser extent with the proximal apical dendrites of these cells, a few scattered interneurons were observed in the stratum oriens (Fig. 4B). In the CA3 subfield fewer pyramidal cells were immunoreactive for GPRC5B, though again the immunoreactivity observed was strongest in the cell membrane with little labelling in the proximal apical dendrites of these cells (Fig. 4C). Very few stained interneurons were seen in the stratum oriens or the stratum radiatum of the CA3 subfield. Granule cells of the dentate gyrus were densely immuno-

Fig. 3. Distribution of GPRC5B in the prefrontal cortex and cerebellum. Photomicrographs showing GPRC5B IR in the frontal cortex (A, B), and cerebellum (C, D). GPRC5B IR was seen in cortical layers II–VI (A), cell bodies (arrows) and apical dendrites (arrowheads) in layer V of the cortex were GPRC5B IR (B). In the cerebellum GPRC5B IR was seen in all layers (C), and was most strong in the purkinje cells (D). Corpus callosum–cc; molecular cell layer—ml; granule cell layer—gl; basket cell—B; Purkinje cell—P; stellate cell—s; granule cell—g. Scale bars5 500 mm (A), 50 mm (B, D) and 100 mm (C).

reactive for GPRC5B (Fig. 4D), with labelling evenly distributed between both the upper and lower blades of the gyrus. Some interneurons in the hilus of the dentate gyrus were immunoreactive for GPRC5B. Cerebellum—Cerebellar Purkinje neurons demonstrated intense GPRC5B IR, though dendritic labelling was not very intense (Fig. 3C). In addition, granule cells demonstrated GPRC5B IR, as did Golgi cells in the Purkinje cell layer and small stellate cells in the molecular layer (Fig. 3D). Spinal cord—Anti-GPRC5B antisera labelled cells in most layers of the spinal cord (Fig. 5A). In the dorsal horn, neuronal somata in layers I, II and III were all densely immunoreactive for GPRC5B, and neuropil labelling was more intense than in other CNS areas (Fig. 5B). GPRC5B IR was also observed in the large motor neurons of the ventral horn (Fig. 5C). White matter IR was observed in both the dorsal and ventral regions of the spinal cord.

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Fig. 4. Distribution of GPRC5B in hippocampus. Photomicrographs showing GPRC5B IR in the hippocampus. GPRC5B IR was seen in all subfields of the hippocampus (A). Stratum pyramidale of the CA1 (B) and CA3 (C) subfields apical dendrites were lightly IR (arrows), and granule cells and interneurons (arrow heads) of the dentate hilus (D). Pre-absorption of the antisera with 10 mg / ml cognate peptide completely abolished all IR (E). Stratum oriens—so; stratum radiatum—sr; stratum pyramidale—p; molecular layer of dentate—m; upper blade of dentate—u; lower blade of dentate—l. Scale bars5200 mm (A); 50 mm (B–E).

4. Discussion Pharmacological modulation of family C GPCRs, such as GABA B and mGlu receptors, is thought to present potentially useful approaches for the treatment of a variety of neurological and neuropsychiatric disorders, ranging from epilepsy to addiction [7]. It is therefore of importance for us to increase our understanding of novel genes falling within this receptor family. In this report, we describe for the first time the mRNA and protein distribution of the orphan receptor GPRC5B in rat brain and spinal cord tissues. GPRC5B is a member of the recently identified

small sub-subfamily of (retinoic acid induced) family C GPCRs (GPRC5A, B, C and D) [2,4,6,15]. Although these receptors share closest sequence homology to family C GPCRs, their short N-terminal domain makes them more similar in structure to family A or family B GPCRs. Similarity searching of EST databases, using the human GPRC5B sequence, has identified partial sequence for the rat orthologue of GPRC5B. Using this information, we have extended the expression profiling of GPRC5B to rat central and peripheral tissues and produced antisera able to detect both the human and rat GPRC5B receptor. Previous studies have analysed the expression profile of

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Fig. 5. Distribution of GPRC5B in the spinal cord. Photomicrographs showing GPRC5B IR in the spinal cord (A), substantia gelatinosa (B) and ventral motor neurons (C). White matter—wm. Scale bars55 mm (A) 50 mm (B, C).

GPRC5A, GPRC5B and GPRC5C at the mRNA level in human tissues of the periphery and the CNS. GPRC5B has previously been found to be highly expressed in the CNS [4,15], in marked contrast to GPRC5A, GPRC5C and GPRC5D which were predominantly expressed in a variety of peripheral tissues [2,6,15]. In this study, rat GPRC5B mRNA distribution was similar to that previously observed in human tissues [4,15]. There was widespread expression across the majority of CNS tissues, with low but detectable levels of expression observed in some peripheral tissues, particularly placenta. These findings are in general agreement with other mRNA studies on human tissue, which detected low levels of GPRC5B mRNA expression in the periphery although somewhat higher mRNA levels have

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been reported in pancreas, kidney and testis [4,15]. Any differences between studies in peripheral mRNA expression levels of GPRC5B, are likely a result of species differences between rat and human, as well as the differing sensitivities of the techniques used [e.g., reverse transcriptase (RT) PCR, Taqman RT-PCR and Northern blot analysis]. At the protein level no data has previously been reported on the expression of GPRC5B. In this study, we have generated a GPRC5B specific antisera to an intracellular epitope within the C-terminal tail of the receptor. Western blot analysis demonstrated the human GPRC5B migrating as a protein of about 68 kDa. This is slightly higher than the predicted molecular weight of approximately 50 kDa, indicating possible post-translational modification (e.g., glycosylation and / or phosphorylation). The presence of higher molecular weight material in the Western blots, tempts us to speculate about the potential existence of dimeric forms of the receptor, migrating at approximately 130 kDa. This is not atypical of many family C GPCRs which are known to form both homodimers, as is seen with both mGluRs and CaSRs, or heterodimers, as seen with the GABA B receptor [9,16]. However, further work will be needed to confirm the existence of such GPRC5B dimers, as this may simply be an artefact of the over-expressing transfection based system. A weak immunoreactive band was also observed in extracts from untransfected cells. This band could be reflective of endogenous GPRC5B expression in HEK293 cells, since they have been shown to express low levels of GPRC5B mRNA [15]. Alternatively, we cannot discount the possibility that this immunoreactive band is non-specifically detected by the antisera but is effectively competed by the immunising peptide. Immunofluorescence studies of GPRC5B expressed in HEK293 demonstrated an intense staining around the cell surface with micro clusters of high level expression. This pattern of GPRC5B expression was indistinguishable from previous studies using an antibody to an epitope tag on GPRC5B or using a C-terminal GFP-tagged GPRC5B construct [2,15]. GPRC5B expression was generally widespread throughout the rat brain and spinal cord with strongest immunoreactivity detected in the cortex, cerebellum and hippocampus. This is in good agreement with the rat mRNA distribution data. Immunoreactivity was most pronounced in layers II–VI in all cortical regions examined. In the hippocampus, pyramidal and granule cells of CA1–CA3 and dentate sub regions were strongly positive, though little interneuronal IR was observed, possibly suggesting a lack of inhibitory control of this receptor in this structure. The known importance of the hippocampus in cognitive function suggests a potential but speculative role for this receptor in learning and memory. Astrocytes also expressed GPRC5B as determined by semi-quantitative PCR and Western immunoblotting and to a lesser extent in oligodendrocytes. The functional relevance of GPRC5B

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expression in glial cells is unclear, but may suggest a role in synaptic development and function. GPRC5B localisation was also high in the granule cell layer of the cerebellum and in the cell bodies but not the dendritic arbour of the Purkinje cells. Granule cells act as excitatory interneurons between mossy fibres and Purkinje cells and the fact that the cell bodies were IR in both cell types suggests that GPRC5B may exert a positive influence on the spino-cerebellar system and play a role in motor control. Expression of GPRC5B in the spinal cord also points to a possible role in nociceptive and / or mechanical processing, as both neuronal and neuropil labelling in the substantia gelatinosa were particularly high. Further work to see if receptor expression is in any way altered in the spinal cord or DRG, in animal models of nociception may help clarify the importance of this receptor in this area. Studies investigating GPRC5B expression in cell populations of the dorsal root ganglia may also yield clues as to the role of GPRC5B in thermal, mechanical and nociceptive processing. However, the fact that little mRNA expression was observed in the DRG suggests that this receptor may predominantly be post-synaptic and of spinal origin. GPRC5B immunoreactivity was also detected in white matter tracts of the brain and spinal cord in agreement with previous data in human tissue demonstrating high levels of mRNA expression in the corpus callosum [15]. In summary our data provides the first characterisation of the GPRC5B receptor in brain and spinal cord at the protein level. Future work to identify specific neuronal and / or glial populations expressing GPRC5B may go some way to increasing our understanding, however, identification of the endogenous ligand remains the most critical factor in determining function. Identification of the brain and spinal cord regions expressing this orphan receptor at a high level may help in the selection of suitable tissues for such ligand identification. Further screening work to identify the ligand, in addition to the development of knockout animals, will improve our understanding of this orphan receptor and facilitate the study of its potential role in the central nervous system.

Acknowledgements The authors would like to thank Mrs. Shabina Nasir and Mrs. Elli Enayat for technical assistance, and Dr. Peter R. Maycox for valuable comments on the manuscript.

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