mammalian cells - Europe PMC

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, ROBERT LEVENSON*

AND

*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

To achieve a better understanding of how D5 ABSTRACT dopamine receptors mediate the actions of dopamine in brain, we have developed antibodies specific for the D5 receptor. D5 antibodies reacted with recombinant baculovirus-infected Sf9 cells expressing the D5 receptor but not with the D1 receptor or a variety of other catecholaminergic and muscarinic receptors. Epitope-tagged D5 receptors expressed in mammalian cells were reactive with both D5 antibodies and an epitopespecific probe. A mixture of N-linked glycosylated polypeptides and higher molecular-mass species was detected on immunoblots of membrane fractions of D5-transfected cells and also of primate brain. D5 receptor antibodies intensely labeled pyramidal neurons in the prefrontal cortex, whereas spiny medium-sized neurons and aspiny large interneurons of the caudateinucleus were relatively lightly labeled. Antibodies to the D5 dopamine receptor should prove important in experimentally determining specific roles for the D5 and D1 receptors in cortical processes and diseases.

MATERIALS AND METHODS Fusion Protein Constructs. A cDNA fragment encoding aa 375-477 of the human D5 receptor (3, 4) was generated by PCR using human D5 cDNA (generously provided by D. K. Grandy, Vollum Institute, Portland, OR) as template and the following primers: D55' (5'-TTGGAATTCAGCCACTTCTGCTCCCGCACG-3'); and D53' (5'-GCGTCGACAGTTTAATGGAATCCATTCGGG-3'). PCR was done with Pfu DNA polymerase and Pfu buffer 1 (Stratagene) for 35 cycles (1 min at 95°C, 1 min at 50°C, 1 min at 72°C). The PCR products were inserted into the EcoRI and Sal I sites of bacterial expression vectors pMalc2 (New England Biolabs) and pGEX-4T-1 (Pharmacia) to yield plasmids encoding maltose-binding protein (MBP)-D5 fusion protein MBP-D5, and glutathione S-transferase (GST)-D5 fusion protein GST-D5. Both DNA constructs were confirmed by dideoxynucleotide chaintermination sequencing. Fusion Proteins, Rabbit Immunizations, and Antibody Purification. MBP-D5 and GST-D5 were induced in Escherichia coli strain XL-1 Blue in the presence of 1 mM isopropyl 13-D thiogalactoside and purified using amylose (New England Biolabs) and glutathione agarose (Pharmacia) resins, respectively. Three New Zealand White rabbits were immunized with MBP-D5 as described (10). Antibodies reactive with the D5 portion of MBP-D5 were affinity-purified on nitrocellulose strips containing GST-D5 fusion protein (11). Membrane Preparation, N-Glycosidase F Digestion, and Immunoblotting. Membrane fractions from recombinant baculovirus-infected Sf9 cells were generously provided by M. Dennis (Biosignal, Montreal). Crude microsomes from transfected CV-1 cells and monkey brain were prepared as described (12), and protein concentrations were determined (13). Solubilized proteins were fractionated by SDS/PAGE (14) and electroblotted to polyvinylidine difluoride (PVDF) or nitrocellulose filters in transfer buffer/5% (vol/vol) methanol; filters were processed for immunoreactivity as described (15). Filters were incubated with D5 antibodies (1 ,ug/ml), washed, and then incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (diluted 1:50,000) (Jackson ImmunoResearch). D5 antibody reactivity was detected by enhanced chemiluminescence (ECL) using an ECL kit (Amersham). To remove N-linked sugars, 50 ,ug of crude microsomes were heated at 65°C for 15 min in 0.5% SDS/1% 2-mercaptoethanol and then digested at 37°C with 500 units of N-glycosidase F (PNGase F; New England Biolabs), according to manufacturer's instructions. Reactions were stopped by addition of an equal volume of 2x Laemmli loading buffer (14).

The effects of dopamine in brain are mediated by two pharmacologically distinguishable classes of receptors, D1 and D2 (1). Previous studies suggest that activation of D1-like receptors is important in the working memory process mediated by the primate prefrontal cortex (PFC) (2). Recently, molecular cloning has revealed the existence of two D1-like receptors, D1 and Ds (3, 4), raising the possibility that both receptors are involved in this mnemonic process. Although receptor autoradiography suggests that there is a high concentration of D1-like binding sites in the PFC (5), analysis of the receptor mechanisms involved in working memory has been limited by the inability to distinguish D1 from D5 receptor sites. Antibodies specific for the D1 receptor protein have recently been developed (6, 7) and used to localize D1 dopamine receptors in the primate brain (8). The D1 receptor is preferentially detected in spines of pyramidal neurons in the PFC, suggesting that it is involved in the dopaminergic modulation of excitatory input. The distribution of the D5 receptor protein has not yet been determined, although D5 mRNA has been localized in the human and monkey motor cortex and striatum

(9).

Here we report the development of D5 dopamine receptorspecific antibodies. We have used these antibodies to analyze the expression and physical properties of D5 protein in brain and in transfected mammalian cells. We find that, like the D1 receptor, the D5 receptor protein preferentially localizes to pyramidal neurons in the PFC, raising the possibility that the two D1-like receptors play complementary or synergistic roles in cortical processes.

Abbreviations: PFC, prefrontal cortex; MBP, maltose-binding protein; HA, hemagglutinin; GST, glutathione S-transferase; mAb, monoclonal antibody. tTo whom reprint requests should be addressed.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

3468

Neurobiology: Bergson et aL

Proc. Natl. Acadc ScL USA 92 (1995)

Construction, Expression, and Immunofluorescent Detection of Epitope-Tagged D5 Receptors. A 9-aa peptide (TyrPro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala) from the hemagglutinin (HA) of influenza virus was inserted into the N terminus of the D5 receptor between aa 1 and 2 (16). The epitope-tagged construct was verified by dideoxynucleotide chain-termination sequencing and subcloned into the expression vector pTetsplice (generously provided by D. G. Schatz, Yale University) to yield plasmid pHA/D5. Transfections of African green monkey kidney CV-1 cells were done essentially as described (17). Cells were grown in Dulbecco's modified Eagle's medium (DMEM)/10% fetal calf serum/tetracycline (0.5 ,ug/ml). For stable transfections, CV-1 cells were exposed to a calcium phosphate precipitate of the following plasmid DNAs: pHA/Ds (10 ,g); ptTA (10 ,g), encoding the tetracycline-controlled transactivator (18); and pSV2a1 (4 ,ug), encoding the at subunit of the rat Na,K-ATPase (19). Ouabain selection was done as described (19); ouabain-resistant colonies were maintained in medium containing 0.5 ,uM ouabain. Cells expressing epitope-tagged D5 receptors were identified by immunofluorescence (17) using the HA epitope-specific monoclonal antibody (mAb), 12CA5 (Babco, Emeryville, CA). Tissue Preparation, Immunohistochemistry, and RNA in Situ Hybridization. Perfusion and preparation of brain tissue from three adult male rhesus monkeys (Macaca mulatta) for immunohistochemistry was done as described (20). Sections were processed using goat anti-rabbit biotinylated antibodies (Vector Laboratories) and an avidin-biotin-horseradish peroxidase complex for signal amplification (ABC Elite kit; Vector Laboratories). Peroxidase was visualized by using 0.05% diaminobenzidine (DAB) in the presence of 0.01% hydrogen peroxide in phosphate buffer. For in situ hybridization, monkeys were perfused with 4% (wt/vol) paraformaldehyde/phosphate-buffered saline. Brains were postfixed at 4°C in fixative/15% sucrose and then immersed in isopentane at -40°C for 5 min. Sections were cut on a cryostat, processed as described (21), then prehybridized with 75% (vol/vol) formamide, 10% (wt/vol) dextran sulfate, 3x standard saline/ citrate, 1 x Denhardt's solution, 10 mM dithiothreitol, 50 mM Na2PO4 (pH 7.4), and 0.3% Triton X-100. Sense and antisense cRNA probes were transcribed from a pGemBlue plasmid containing nt 779-1114 of the published human D5 receptor cDNA (3, 4) using T7 and SP6 RNA polymerases and digoxigenin-UTP as described (11). Probes (1.0 ng/,ul) in prehybridization solution were incubated with sections at 55°C for 16 hr. Sections were washed and treated with RNase as described (21), with a final wash at 55°C for 1 hr in 0.5x standard saline/citrate. Sections were incubated with alkaline phosphatase-conjugated anti-digoxigenin antibodies (Boehringer Mannheim) according to the manufacturer's instructions. Alkaline phosphatase was visualized using 5-bromo-4-chloro-3indolyl phosphate/nitroblue tetrazolium (Boehringer Mannheim).

116976645-

RESULTS D5 Antibody Production and Specificity. The D5 dopamine receptor shares 80% sequence similarity with the D1 dopamine receptor in predicted transmembrane segments (3, 4). However, the similarity between the D1 and D5 dopamine receptors is

>

fO

IC)

D

....... ......... ..... ..

..

....

.:...:.:.

19=

66-

';'.' 'u'.-,.'X,'.t

r...... ....

4529FIG. 4. Comigration of D5 receptor antibody immunoreactive proteins expressed in primate brain and in D5 receptor-transfected cells on SDS/PAGE. Immunoblot of membrane proteins prepared from monkey brain or peripheral tissue (20 ,ug) and untransfected (CV-1) or pHA/Ds-transfected CV-1 cells (2 ,ug) (D5 CV-1) as indicated. SN, substantia nigra. Proteins were separated on SDS/10% polyacrylamide gels and then electroblotted to poly(vinylidene difluoride) filters. Positions of molecular mass markers are indicated at

left.

Neurobiology: Bergson et aL

FIG. 5. Localization of D5 receptor protein and mRNA in the macaque PFC. Sections (30 gm thick) of comparable levels of Walker's area 9 of the PFC incubated with affinity-purified D5 antibodies (A) or D5 antisense RNA probes (B). Cells in layers III and V are the most

prominently labeled in both sections. (X90) (C) Higher magnification of a layer III pyramidal cell soma and apical dendrite shown in A. (x360) wm, White matter.

nucleus (data not shown). The concordance of labeling patterns obtained with both antibody and cRNA probes further supports the specificity of the antibodies for the D5 receptor.

DISCUSSION We have taken advantage of the sequence divergence in the C terminus of the D1 and D5 dopamine receptors to generate antibodies specific for the human D5 receptor. Antibody specificity was demonstrated by multiple criteria including (i) reactivity with the D5 receptor and not with a variety of other G protein-coupled receptors; (ii) reactivity and localization of epitope-tagged D5 receptors in transfected cells; and (iii) lack of reactivity with brain tissue and immunoblots in the presence of D5 fusion proteins and not in the presence of nonspecific fusion proteins. We observe antibody reactivity only with the D5 dopamine receptor and not with other dopaminergic, adrenergic, serotonergic, and muscarinic receptors on immunoblots, suggesting that the antibodies are selective for the D5 receptor and are unlikely to react with other members of the G protein-coupled receptor superfamily. We affinity-purified antibodies from crude rabbit serum using GST-D5 fusion protein to obtain antibodies specific for the D5 receptor portion of the MBP-D5 immunogen. These affinity-purified antibodies react with brain tissue and immunoblots containing membranes prepared from hippocampus, caudate, and substantia nigra in the presence of other GST or MBP fusion proteins. However, in the presence of GST-D5 or MBP-D5 fusion proteins, D5 immu-

Proc. Natl. Acad. Sci. USA 92 (1995)

3471

noreactivity is specifically eliminated. It therefore seems reasonable to conclude that the antibodies we have produced are specific for the D5 dopamine receptor. In general, affinity-purified D5 antibodies and the HAspecific mAb react with identical bands on immunoblots containing epitope-tagged D5 receptors. Most epitope-tagged D5 receptors expressed in transfected cells appear to result from N-linked glycosylation of the core D5 receptor protein. Both mAb 12CA5 and D5 antibodies also detect less abundant forms of the D5 receptor that appear resistant to N-glycosidase F digestion and which display apparent molecular masses of 66-200 kDa. Some of these higher-molecular-mass species are also detected in monkey brain along with protein species that co-migrate with the core and N-linked glycosylated forms of the D5 receptor expressed in CV-1 cells. It is possible that the higher molecular weight forms of the D5 receptor may result from post-translational mechanisms other than N-linked glycosylation or from aggregation. Treatment of the crude membrane preparations with a variety of reducing and denaturing agents failed to alter the mobility of these immunoreactive bands, consistent with the idea that the higher-molecular-weight forms represent aggregates. Other G protein-coupled receptors have also been found to behave as a mixture of presumably N-linked glycosylated forms and higher-molecular-mass aggregates when expressed in cell culture (22, 23). In addition, the major form of a splice variant of the D3 dopamine receptor, D3nf, expressed in brain migrates with a molecular mass on SDS/PAGE approximately twice that of D3nf receptors expressed in transfected mammalian cells, raising the possibility that other receptors in brain may also behave as aggregates (24). However, this does not appear to be a physical property common to all dopamine receptors expressed in brain because the most abundant forms of the D1 receptor in brain can be deglycosylated (C.B. and R.L., unpublished observations). Light microscopic analysis of D5 receptor protein expression indicates that the D5 receptor is relatively abundant in PFC where D5 receptor antibodies prominently label pyramidal neurons. The D1 receptor shows a similar distribution in the primate PFC at this level of analysis. At the electron microscopic level, the D1 receptor has been localized in numerous dendritic spines of pyramidal cells (8). The availability of subtype-specific antibodies for the D5 and D1 dopamine receptor provides a means of assessing the role of individual dopamine receptor subtypes in various behavioral paradigms, as well as in neuropathologies. For example, if the D5 receptor has a different distribution in pyramidal neurons of the PFC, it should be possible to determine the specific functional contributions of each receptor subtype to working memory (2, 25). It should also now be possible to investigate whether the D1 and/or D5 receptor subtypes exhibit alterations in abundance or subcellular localization in cortical regions related to diseases such as Parkinson disease or schizophrenia. We thank M. Pappy and Y. Cao for their excellent technical assistance as well as Dr. H. Komuro for help with confocal imaging. This research was supported by the National Alliance for Research on Schizophrenia and Depression (C.B.) and a National Institutes of Mental Health Center Grant P50-MH44866-05. 1. Stoof, J. C. & Kebabian, J. W. (1984) Life Sci. 35, 2281-2296. 2. Sawaguchi, T. & Goldman-Rakic, P. S. (1991) Science 251, 947-950. 3. Grandy, D. K., Zhang, Q.-Y., Bouvier, C., Zhou, Q.-Y., Johnson, R. A., Allen, L., Buck, K., Bunzow, J. R., Salon, J. & Civelli, 0. (1991) Proc. Natl. Acad. Sci. USA 88, 9175-9179. 4. Sunahara, R. K., Guan, H.-C., O'Dowd, B. F., Seeman, P., Laurier, L. G., George, S. R., Torchia, J., Van Tol, H. M. & Niznik, H. (1991) Nature (London) 350, 614-619. 5. Lidow, M. S., Goldman-Rakic, P. S., Gallager, D. W. & Rakic, P. (1991) Neuroscience (Oxford) 40, 667-671.

3472

Neurobiology: Bergson et al

6. Huang, Q., Zhou, D., Chase, K., Gusella, J. F., Aronin, N. & DiFiglia, M. (1992) Proc. Natl. Acad. Sci. USA 89, 11988-11992. 7. Levey, A. I., Hersch, S. M., Rye, D. B., Sunahara, R., Niznik, H. B., Kitt, C. A., Price, D. L., Maggio, R., Brann, M. R. & Ciliax, B. J. (1993) Proc. Natl. Acad. Sci. USA 90, 8861-8865. 8. Smiley, J. F., Levey, A. I., Ciliax, B. J. & Goldman-Rakic, P. S. (1994) Proc. Natl. Acad. Sci. USA 91, 5720-5724. 9. Huntley, G. W., Morrison, J. F., Prikhozhan, A. & Sealfon, S. C. (1992) Mol. Brain Res. 15, 181-188. 10. Shyjan, A. W. & Levenson, R. (1989) Biochemistry 28, 45314535. 11. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Lab. Press, Plainview, NY), 2nd Ed., Vol. 3. 12. Siddhu, A., Kassis, S., Kebabian, J. & Fishman, P. H. (1990) Biochemistry 25, 6695-6701. 13. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254. 14. Laemmli, U. K. (1970) Nature (London) 227, 680-685. 15. Towbin, H., Staehelin, T. & Gordon, J. (1979) Proc. Nati. Acad. Sci. USA 76, 4350-4354. 16. Wilson, M. S., Niman, H., Houghton, R., Cherenson, A., Connoly, M. & Lerner, R. (1984) Cell 37, 767-778.

Proc. Nati Acad Sci. USA 92 (1995) 17. Canfield, V. A. & Levenson, R. (1993) Biochemistry 32, 1378213786. 18. Gossen, M. & Bujard, H. (1992) Proc. Natl. Acad. Sci. USA 89, 5547-5551. 19. Kent, R. B., Emanuel, J. R., Neriah, Y. B., Levenson, R. & Housman, D. E. (1987) Science 237, 901-903. 20. Mrzljak, L., Levey, A. I. & Goldman-Rakic, P. S. (1993) Proc. Natl. Acad. Sci. USA 90, 5194-5198. 21. Whitfield, H. J., Jr., Brady, L. S., Smith, M. A., Mamalaki, E., Fox, R. J. & Herkenham, M. (1990) Cell. Mol. Neurobiol. 10, 145-157. 22. Mouillac, B., Caron, M., Bonin, H., Dennis, M. & Bouvier, M. (1992) J. Biol. Chem. 267, 21733-21737. 23. Ng, G. YK., Mouillac, B., George, S. R., Caron, M., Dennis, M., Bouvier, M. & O'Dowd, B. F. (1994) Eur. J. Pharmacol. 267, 7-19. 24. Liu, K., Bergson, C., Levenson, R. & Schmauss, C. (1994) J. Biol. Chem. 269, 29220-29226. 25. Goldman-Rakic, P. S., Funahashi, S. & Bruce, C. J. (1990) Cold Spring Harbor Symp. Quant. Biol. 55, 1025-1038.