Endosomal location of dopamine receptors in neuronal cell cytoplasm
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- Wolstencroft, E.C., Simic, G., Man, N.t. et al. J Mol Hist (2007) 38: 333. doi:10.1007/s10735-007-9106-5
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Five subtypes of dopamine receptor exist in two subfamilies: two D1-like (D1 and D5) and three D2-like (D2, D3 and D4). We produced novel monoclonal antibodies against all three D2-like receptors and used them to localize receptors in Ntera-2 (NT-2) cells, the human neuronal precursor cell line. Most of the immunostaining for all three receptors colocalized with mannose-6-phosphate receptor, a marker for late endosomes formed by internalization of the plasma membrane. This result was obtained with antibodies against three different epitopes on the D3 receptor, to rule out the possibility of cross-reaction with another protein, and controls without primary antibody or in the presence of competitor antigen were completely negative. In rat cerebral cortex and hippocampus, some of the dopamine receptor staining was found in similar structures in neuronal cell cytoplasm. Only some of the neurons were positive for dopamine receptors and the pattern was consistent with previously-reported patterns of innervation by dopamine-producing neurons. Endosomal dopamine receptors may provide a useful method for identifying cell bodies of dopamine-responsive neurons to complement methods that detect only active receptors in the neuronal cell membrane.
KeywordsG protein-coupled receptorEndosomeNT-2 cellsNtera-2Monoclonal antibodyBrainHippocampusCerebellumCerebral cortexMannose-6-phosphate receptor
Dopamine receptors are members of the G protein-coupled receptor (GPCR) family, which initiate their biological function by coupling to GTP regulatory proteins (G proteins). In humans, five dopamine receptor subtypes, DRD1 (Monsma et al. 1990), DRD2 (Bunzow et al. 1988), DRD3 (Sokoloff et al. 1990), DRD4 (van Tol et al. 1991) and DRD5 (Sunahara et al. 1991) have been identified. The D1-like receptors, DRD1 and DRD5, stimulate adenylyl cyclase (Monsma et al. 1990; Albert et al. 1990) whereas the D2-like receptors, DRD2, DRD3 and DRD4, are coupled to inhibitory G proteins and their stimulation leads to inhibition of adenylyl cyclase (Sokoloff et al. 1990). All five have an extracellular amino terminus, seven transmembrane domains and an intracellular carboxyl terminus. Abnormalities in dopamine neurotransmission are implicated in the aetiology of neurological and psychiatric disorders including schizophrenia (Williams et al. 1998) and Parkinson’s disease (Oliveri et al. 2000).
The distribution of D2-like dopamine receptor mRNAs in brain has been studied by in situ hybridization (Sokoloff et al. 1990; Mengod et al. 1989) and radioligand binding studies (Martres et al. 1985; Bouthenet et al. 1987; Camps et al. 1989) have revealed the distribution of biologically-active receptors, including the use of sub-type selective ligands (Levesque et al. 1992; Herroelen et al. 1994; Hall et al. 1996; Bancroft et al. 1998). Most immunohistochemical studies of D2-like receptors so far have used only a single antibody, with the notable exception of two studies of DRD4 that used antibodies against two epitopes (Defagot et al. 1997; Mauger et al. 1998). Use of antibodies against two or more epitopes is the best safeguard against cross-reaction, especially with monoclonal antibodies (Morris 1998). The reported sizes of D2-like dopamine receptors on western blots have varied widely in the literature (36–95 kDa) and cross-reaction with other proteins has not always been ruled out.
In the present study, we have used a new panel of antibodies against three different DRD3 epitopes to determine the cellular and subcellular localization of DRD3 in rat brain and Ntera-2 cells. Additional monoclonal antibodies against specific peptide sequences in DRD2 and DRD4 were used to support and confirm the subcellular localization of D2-like dopamine receptors. We suggest that the presence of D2-like dopamine receptors in cytoplasmic vesicles could provide a useful means to identify the cell bodies of dopamine-responsive neurons, since neuronal extensions with dopamine receptors often project far into other regions of the brain.
For monoclonal antibodies, Balb/c mice were immunized with peptides conjugated to bovine serum albumin (BSA) through cysteine residues using MBS (3-maleimidobenzoic acid N-succinimidyl ester). Suitable human peptide sequences were identified on the basis of specificity for each subtype and antigenicity. The peptides were synthesized by Alta Bioscience (University of Birmingham, UK). Hybridoma fusions with the Sp2/0 mouse myeloma cell line were performed as described previously (Nguyen and Morris 1996). Hybridoma culture supernatants were screened by enzyme linked immunosorbent assay (ELISA) using plates coated with the respective unconjugated peptide. ELISA positive wells were further screened by Western blotting on total Ntera-2 protein extract and total brain (rat and human) extract.
A rabbit polyclonal antiserum raised against the same N-terminal DRD3 peptide was obtained from Calbiochem. A polyclonal antiserum was produced against a DRD3 C-terminal recombinant protein (amino acids 205–400) by immunization of Balb/c mice. The corresponding cDNA was obtained from a full-length human DRD3 construct by PCR and was cloned into pET 32a expression vector (Novagen, Darmstadt, Germany). The expressed protein was purified for immunization by affinity chromatography using His.Bind resin (Novagen, Darmstadt, Germany) and used to immunize Balb/c mice. All animal procedures were performed under UK license.
Enzyme linked immunosorbent assay
A 96 well PVC microtitre plate (Dynatech, Billingshurst, UK) was coated with 50 μl per well of the peptide at a concentration of 5–10 μg/ml in PBS, and incubated at room temperature for 1 h. The antigen was then removed and the plate was washed 3 times with incubation buffer (IB, PBS + 1% Triton X-100). 300 μl of a 4% solution of skimmed milk powder in IB was added to each well for 30 min at room temperature. After washing 3 times with IB, 50 μl of culture supernatant was added at room temperature for 1 h. The primary antibody was removed and the plate washed 4 times with IB. Horseradish peroxidase-conjugated rabbit anti-mouse Ig (DAKOpatts, Denmark; 50 μl of 1/1,000 in dilution buffer: 0.1% BSA, 1% horse serum and 1% fetal calf serum in IB) was then added and incubated at room temperature for 1 h. The plate was washed 4 times with IB. One hundred and fifty microlitres of substrate solution (0.4 mg/ml o-phenylene-diamine in 25 mM phosphate-citrate buffer pH 5.0 with 0.1% H2O2) was added until the colour developed. The reaction was then stopped with 50 μl of 3 M H2SO4. Samples were read using a 492 nm filter on a Titertek Multiscan Plate reader.
Human brain samples were provided by the Medical Research Council Brain Bank, Kings College, London, UK. Total protein extracts of human or rat brain were made by Dounce homogenization in four volumes of SDS extraction buffer (10% SDS, 10% EDTA, 5% 2-mercaptoethanol, 10% glycerol, 50 mM Tris–HCl, pH 7.0) and clarified by centrifugation at 160,000 g for 30 min. After SDS-PAGE on 10% polyacrylamide gels, proteins were transferred to nitrocellulose electrophoretically overnight. After blocking for 1 h in 3% skimmed milk powder in incubation buffer (0.05% Triton X-100 in PBS), blots were incubated with mAb (1/100 dilution of culture supernatant in incubation buffer/1% horse serum/1% fetal calf serum/0.1% BSA) for 1 h. After washing 3 times in PBS, blots were incubated with biotinylated anti-mouse Ig and a peroxidase-avidin detection reagent (Vectastain ABC Elite, Vector Laboratories, Peterborough, UK) according to the manufacturer’s instructions. Blots were developed with diaminobenzidine in 25 mM phosphate-citrate buffer (pH 5.0), 0.012% H2O2.
The human embryonal carcinoma cell line, Ntera-2 (Andrews et al. 1984), also known as NT-2, was cultured in Dulbecco’s MEM with Glutamax-I (Invitrogen, Paisley, UK) and 10% fetal bovine serum (Invitrogen, UK). Cells were seeded onto coverslips at 5 × 105 cells and, after culture for 48 h, were fixed in 50:50 acetone–methanol for 5 min.
Preparation of rat brain sections
A whole brain was removed from an adult Sprague-Dawley rat immediately post-mortem and rinsed in ice-cold isotonic saline for 1 h. It was then mounted and frozen in isopentane cooled in liquid nitrogen and sectioned dorso-laterally into 5 μm frozen sections. The sections were mounted on microscope slides in the same orientation and allowed to air dry.
Cells and tissue sections were fixed using 50% acetone/50% methanol. The primary antibody was diluted in PBS/1% horse serum/1% fetal calf serum and incubated on the slide for 1 h at room temperature. After washing with PBS, FITC-conjugated rabbit anti-(mouse Ig) (1/50: Vector Labs, Burlingame, USA) in PBS/1% horse serum/1% fetal calf serum was applied for 30 min. After counterstaining nuclei with ethidium bromide, slides were mounted in 10% glycerol in PBS and photographed using a Leica DMRB epifluorescence photomicroscope with 10× or 63× objectives. For double labelling, fixed cells were incubated sequentially with rabbit antisera and mouse mAb for 1 h, followed by a mixture of FITC-conjugated anti-(mouse Ig) (1/50 dilution) (Vector Labs, Burlingame, CA, USA) and TRITC-conjugated anti-(rabbit Ig) (1/100 dilution) (DAKO, Glostrup, Denmark) for 30 min. Confocal images were captured using a Zeiss LSM510 Meta confocal system.
Production of antibodies that recognize 50 kDa dopamine receptors in human brain
A panel of monoclonal antibodies against different epitopes of each of the D2-like dopamine receptors
Antigen (amino acids)
3rd Cytoplasmic domain (210–218)
3rd Cytoplasmic domain (210–218)
3rd Cytoplasmic domain (210–218)
3rd Cytoplasmic domain (272–282)
3rd Cytoplasmic domain (272–282)
3rd Extracellular domain (176–185)
Most of the D2-like dopamine receptors in cultured Ntera-2 cells are found in endosomal vesicles in the cytoplasm
Some dopamine receptors were detected at the expected plasma membrane sites
One would normally expect to find D2-like dopamine receptors in the plasma membrane, close to synapses with dopamine-producing neurons, and Fig. 2d shows a section of rat basal ganglia with DRD3 staining of dendrite-like processes. Similar staining was observed in other areas of the brain, including cortical layer V of cerebral cortex, where dopaminergic innervation is also expected (not shown). Identification and proper characterization of different dendritic processes is a complex neuroscience study beyond the scope of this paper, but we show Fig. 2d to emphasize that our mAbs may detect dopamine receptors in their functional location in plasma membranes, as well as in endosomes. Indeed, DRD3 receptors are sometimes detectable in the plasma membrane of Ntera-2 cells (Fig. 2e), although cell culture appears to favour location in endosomes.
Endosome-like staining by dopamine receptor antibodies can be used to identify specific subsets of rat brain neurons
Figure 3a shows that all three receptors are also present as cytoplasmic vesicles in neurons of cortical layers V and VI of rat cerebral cortex. Antibody staining of the vesicles was prevented by pre-incubation with peptide antigen (see inset for DRD3 control). The DRD2 mAb shows additional staining in layer I, but, with only one mAb available, we cannot rule out cross-reaction with a non-DRD protein. Cross-reaction of MANDOP41 with DRD3 cannot be entirely ruled out. Figure 3b shows a section through the hippocampus and dentate gyrus. Cytoplasmic vesicles containing D2-like dopamine receptors were found in CA3 and other neurons, but were absent from CA1 neurons and from granule cells of the dentate gyrus. Similar endosome-like vesicles were observed in cerebellum (Purkinje neurons) and brain stem (large bipolar neurons) (data not shown). This shows that DRD-positive vesicle staining could be a useful means of identifying the cell bodies of neurons that are making different dopamine receptors.
Immunolocalization studies using monoclonal antibodies against the D2-like dopamine receptors have shown an endosomal distribution in the Ntera-2 cell line. This distribution pattern is not a staining artifact, since it is completely dependent on the presence of the primary DRD-specific mAb and is blocked by pre-incubation with antigen. We are confident of this localization for DRD3 since an identical pattern was produced by mAbs against two different epitopes in the N-terminal and third cytoplasmic domains, as well as by an antiserum against the C-terminal region and a commercial rabbit antiserum. This confidence was reinforced (a) by the fact that mAbs against DRD2 (different part of third cytoplasmic domain) and DRD4 (third extracellular domain) stained these vesicles, suggesting that all three D2-like dopamine receptors are co-distributed in these vesicles in Ntera-2 cells, and (b) by the observation of similar vesicles in rat brain sections. Because unique peptide sequences were chosen as immunogens for each subtype, cross-reaction between D2-like dopamine receptors is hardly possible, except for DRD4 where the possibility of cross-reaction with DRD3 cannot be entirely ruled out. Cross-reaction with some functionally-unrelated protein can be ruled out when antibodies against two or more epitopes are used (i.e. DRD3) but not when only one mAb is available (e.g. DRD2 in Fig. 3). According to current ideas about GPCR trafficking in general (Dohlman et al. 1991), and that of dopamine receptors in particular (Ng et al. 1994), receptors are internalized in “early endosomes” from which they may be recycled to the plasma membrane or targeted for turnover in “late endosomes”. It is interesting to note that the dopamine transporter protein, which regulates dopamine receptor activation, is known to be internalized into endosomes by a clathrin-dependent mechanism (Sorkina et al. 2005). Internalization and recycling of GPCRs involves arrestin and a phosphorylation/dephosphorylation cycle (Ng et al. 1994; Ferguson et al. 1996). A number of transfection studies of DRD1 and DRD2 have shown a predominantly cytoplasmic localization for DRD2, while DRD1 is localized in the plasma membranes after transfection (Ng et al. 1994; Vickery and von Zastrow 1999; Prou et al. 2001). Agonist stimulation, however, also results in internalization of DRD1 (Ng et al. 1994; Vickery and von Zastrow 1999). Internalization of the receptor following ligand binding is known to occur for a number of GPCRs, including the D2 type receptors, and this leads to a lowering of surface receptor density (Ferguson 2001; Sun et al. 2003). Agonist-independent internalization of D2 type receptors may be occurring in Ntera-2 cells under the culture conditions used in this study.
Previous immunohistological studies of dopamine receptors in the brain have shown the presence in neuronal cells of DRD1 and 2 (Levey et al. 1993) and DRD4 (Defagot et al. 1997; Wedzony et al. 2000; Rivera et al. 2002) but subcellular distribution of the receptors was not described. Our present antibody studies of rat brain sections have localized D2-like dopamine receptors to specific neuronal cells in the hippocampus and layers V and VI of the cerebral cortex. This distribution is consistent with known dopaminergic afferent systems to the hippocampus (Goldsmith and Joyce 1994) and to cortical pyramidal neurons of layers V and VI (Goldman-Rakic et al. 1989; Gulledge and Jaffe 1998). Unlike Ntera-2 cells in culture, post-mortem rat brain is exposed to very high levels of released dopamine within a few minutes of death (Vulto et al. 1988) and agonist-induced internalization of dopamine receptors can also be a very rapid process (Vickery and von Zastrow 1999; Macey et al. 2004). It is possible that receptors have been internalized as a result of this process and this may partly explain why their endosomal location has not been described previously. However, the presence of receptors in the cell body, rather than dendritic processes, may make it easier to identify dopamine-responsive neurons by immunohistochemistry. For example, radioligand binding studies detected D2-like receptors in layers V and VI of the cortex, but binding in layers I–III was also found (Martres et al. 1985; Bouthenet et al. 1987). This might be explained by projection of apical dendrites from pyramidal neurons in layers V and VI into layers I–III, since ligand binding detects active receptors on dendrites whereas endosomes will be most evident in the neuronal cell body. These two studies also found D2-like receptors in the hippocampus and this was confirmed by later studies with more specific ligands for DRD2 (Camps et al. 1989) and DRD3 (Levesque et al. 1992; Herroelen et al. 1994; Hall et al. 1996; Bancroft et al. 1998). The distribution of cytoplasmic vesicle labelling is therefore in broad agreement with earlier radioligand studies.
This study was supported by a studentship (Robert Hughes Jones Bequest for Research into Schizophrenia) from the Clwyd Institute of Health Studies for E.C.W. We thank Prof. P.W. Andrews (University of Sheffield, UK) for the Ntera-2 cell line. G.E.M. and G.S. thank the British Council in Croatia for support with travel grants.