Studies in Primate Brain and Transfected Mammalian Cells (Epitope-Tagging/Prefrontal Cortex/Di Receptor Family) CLARE BERGSON*T, LADISLAV Mrzljakt, MICHAEL S
Total Page：16
File Type：pdf, Size：1020Kb
Proc. Natl. Acad. Sci. USA Vol. 92, pp. 3468-3472, April 1995 Neurobiology Characterization of subtype-specific antibodies to the human D5 dopamine receptor: Studies in primate brain and transfected mammalian cells (epitope-tagging/prefrontal cortex/Di receptor family) CLARE BERGSON*t, LADISLAV MRZLJAKt, MICHAEL S. LIDOWt, PATRICIA S. GOLDMAN-RAKICt, AND ROBERT LEVENSON* *Department of Pharmacology, Pennsylvania State College of Medicine, Milton S. Hershey Medical Center, P.O. Box 850, Hershey, PA 17033; and tSection of Neuroanatomy, Yale University School of Medicine, New Haven, CT 06510 Contributed by Patricia S. Goldman-Rakic, December 30, 1994 ABSTRACT To achieve a better understanding of how D5 MATERIALS AND METHODS dopamine receptors mediate the actions of dopamine in brain, we have developed antibodies specific for the D5 receptor. D5 Fusion Protein Constructs. A cDNA fragment encoding aa antibodies reacted with recombinant baculovirus-infected Sf9 375-477 of the human D5 receptor (3, 4) was generated by cells expressing the D5 receptor but not with the D1 receptor PCR using human D5 cDNA (generously provided by D. K. or a variety of other catecholaminergic and muscarinic re- Grandy, Vollum Institute, Portland, OR) as template and the ceptors. Epitope-tagged D5 receptors expressed in mammalian following primers: D55' (5'-TTGGAATTCAGCCACTTCT- cells were reactive with both D5 antibodies and an epitope- GCTCCCGCACG-3'); and D53' (5'-GCGTCGACAGTTTA- specific probe. A mixture of N-linked glycosylated polypep- ATGGAATCCATTCGGG-3'). PCR was done with Pfu DNA tides and higher molecular-mass species was detected on polymerase and Pfu buffer 1 (Stratagene) for 35 cycles (1 min immunoblots of membrane fractions of D5-transfected cells at 95°C, 1 min at 50°C, 1 min at 72°C). The PCR products were and also of primate brain. D5 receptor antibodies intensely inserted into the EcoRI and Sal I sites of bacterial expression labeled pyramidal neurons in the prefrontal cortex, whereas vectors pMalc2 (New England Biolabs) and pGEX-4T-1 spiny medium-sized neurons and aspiny large interneurons of (Pharmacia) to yield plasmids encoding maltose-binding pro- the caudateinucleus were relatively lightly labeled. Antibodies tein (MBP)-D5 fusion protein MBP-D5, and glutathione to the D5 dopamine receptor should prove important in S-transferase (GST)-D5 fusion protein GST-D5. Both DNA D1 constructs were confirmed by dideoxynucleotide chain- experimentally determining specific roles for the D5 and termination sequencing. receptors in cortical processes and diseases. Fusion Proteins, Rabbit Immunizations, and Antibody Pu- rification. MBP-D5 and GST-D5 were induced in Escherichia The effects of dopamine in brain are mediated by two phar- coli strain XL-1 Blue in the presence of 1 mM isopropyl 13-D macologically distinguishable classes of receptors, D1 and D2 thiogalactoside and purified using amylose (New England (1). Previous studies suggest that activation of D1-like recep- Biolabs) and glutathione agarose (Pharmacia) resins, respec- tors is important in the working memory process mediated by tively. Three New Zealand White rabbits were immunized with the primate prefrontal cortex (PFC) (2). Recently, molecular MBP-D5 as described (10). Antibodies reactive with the D5 cloning has revealed the existence of two D1-like receptors, D1 portion of MBP-D5 were affinity-purified on nitrocellulose and Ds (3, 4), raising the possibility that both receptors are strips containing GST-D5 fusion protein (11). involved in this mnemonic process. Although receptor auto- Membrane Preparation, N-Glycosidase F Digestion, and radiography suggests that there is a high concentration of Immunoblotting. Membrane fractions from recombinant bac- D1-like binding sites in the PFC (5), analysis of the receptor ulovirus-infected Sf9 cells were generously provided by M. mechanisms involved in working memory has been limited by Dennis (Biosignal, Montreal). Crude microsomes from trans- the inability to distinguish D1 from D5 receptor sites. fected CV-1 cells and monkey brain were prepared as de- Antibodies specific for the D1 receptor protein have recently scribed (12), and protein concentrations were determined (13). been developed (6, 7) and used to localize D1 dopamine Solubilized proteins were fractionated by SDS/PAGE (14) and receptors in the primate brain (8). The D1 receptor is prefer- electroblotted to polyvinylidine difluoride (PVDF) or nitro- entially detected in spines of pyramidal neurons in the PFC, cellulose filters in transfer buffer/5% (vol/vol) methanol; suggesting that it is involved in the dopaminergic modulation filters were processed for immunoreactivity as described (15). of excitatory input. The distribution of the D5 receptor protein Filters were incubated with D5 antibodies (1 ,ug/ml), washed, has not yet been determined, although D5 mRNA has been and then incubated with horseradish peroxidase-conjugated localized in the human and monkey motor cortex and striatum goat anti-rabbit IgG (diluted 1:50,000) (Jackson ImmunoRe- (9). search). D5 antibody reactivity was detected by enhanced Here we report the development of D5 dopamine receptor- chemiluminescence (ECL) using an ECL kit (Amersham). specific antibodies. We have used these antibodies to analyze To remove N-linked sugars, 50 ,ug of crude microsomes were the expression and physical properties of D5 protein in brain heated at 65°C for 15 min in 0.5% SDS/1% 2-mercaptoethanol and in transfected mammalian cells. We find that, like the D1 and then digested at 37°C with 500 units of N-glycosidase F receptor, the D5 receptor protein preferentially localizes to (PNGase F; New England Biolabs), according to manufactur- pyramidal neurons in the PFC, raising the possibility that the er's instructions. Reactions were stopped by addition of an two D1-like receptors play complementary or synergistic roles equal volume of 2x Laemmli loading buffer (14). in cortical processes. Abbreviations: PFC, prefrontal cortex; MBP, maltose-binding protein; The publication costs of this article were defrayed in part by page charge HA, hemagglutinin; GST, glutathione S-transferase; mAb, monoclo- payment. This article must therefore be hereby marked "advertisement" in nal antibody. accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 3468 Downloaded by guest on September 26, 2021 Neurobiology: Bergson et aL Proc. Natl. Acadc ScL USA 92 (1995) 3469 Construction, Expression, and Immunofluorescent Detec- RESULTS tion of Epitope-Tagged D5 Receptors. A 9-aa peptide (Tyr- Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala) from the hemagglutinin D5 Antibody Production and Specificity. The D5 dopamine (HA) of influenza virus was inserted into the N terminus of the receptor shares 80% sequence similarity with the D1 dopamine D5 receptor between aa 1 and 2 (16). The epitope-tagged receptor in predicted transmembrane segments (3, 4). How- ever, the similarity between the D1 and D5 dopamine receptors construct was verified by dideoxynucleotide chain-termination is <10% in the predicted fourth cytoplasmic domain of these sequencing and subcloned into the expression vector receptors. No significant homology in this region of the D. D5 pTetsplice (generously provided by G. Schatz, Yale Uni- receptor was found with any other guanine nucleotide-binding versity) to yield plasmid pHA/D5. Transfections of African protein (G protein)-coupled receptor after search of the green monkey kidney CV-1 cells were done essentially as GenBank data base. To obtain subtype-specific antibodies, we described (17). Cells were grown in Dulbecco's modified immunized rabbits with MBP-D5, a fusion protein composed Eagle's medium (DMEM)/10% fetal calf serum/tetracycline of the bacterial MBP and the C-terminal 102 amino acids of the (0.5 ,ug/ml). For stable transfections, CV-1 cells were exposed human D5 dopamine receptor. D5-specific antibodies were to a calcium phosphate precipitate of the following plasmid then affinity-purified from rabbit serum using GST-D5 fusion DNAs: pHA/Ds (10 ,g); ptTA (10 ,g), encoding the tetra- protein. cycline-controlled transactivator (18); and pSV2a1 (4 ,ug), As shown in Fig. 1, the specificity of the affinity-purified encoding the at subunit ofthe rat Na,K-ATPase (19). Ouabain antibodies was tested on immunoblots of membrane prepara- selection was done as described (19); ouabain-resistant colo- tions of recombinant baculovirus-infected Sf9 insect cells nies were maintained in medium containing 0.5 ,uM ouabain. expressing a spectrum of G protein-coupled receptors, includ- Cells expressing epitope-tagged D5 receptors were identified ing the Dl, D2, D3, D4, and D5 dopamine receptors. The by immunofluorescence (17) using the HA epitope-specific antibodies bound to material exhibiting a broad molecular monoclonal antibody (mAb), 12CA5 (Babco, Emeryville, CA). mass distribution ('50-66 kDa) in the membrane fractions Tissue Preparation, Immunohistochemistry, and RNA in prepared from D5-expressing cells. In contrast, D5 antibodies Situ Hybridization. Perfusion and preparation of brain tissue did not react with any of the 14 other dopaminergic, seroton- from three adult male rhesus monkeys (Macaca mulatta) for ergic, adrenergic, and muscarinic receptors present on the immunohistochemistry was done as described (20). Sections immunoblot. To test the possibility that the reactive band from were processed using goat anti-rabbit biotinylated antibodies the D5 membrane fraction represented differentially glycosy-