Studies in Primate Brain and Transfected Mammalian Cells (Epitope-Tagging/Prefrontal Cortex/Di Receptor Family) CLARE BERGSON*T, LADISLAV Mrzljakt, MICHAEL S

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- (Vector Laboratories) and an avidin-biotin-horseradish per- lated forms of the D5 receptor, we treated these membranes oxidase complex for signal amplification (ABC Elite kit; with N-glycopeptidase F to remove N-linked sugars. As shown Vector Laboratories). Peroxidase was visualized by using in Fig. 1B, in membranes treated with N-glycosidase F, D5 0.05% diaminobenzidine (DAB) in the presence of 0.01% antibodies reacted with a single band of '51 kDa, which may hydrogen peroxide in phosphate buffer. For in situ hybridiza- represent a single protein species. The mobility of this band tion, monkeys were perfused with 4% (wt/vol) paraformalde- agrees well with the predicted size of the D5 receptor core hyde/phosphate-buffered saline. Brains were postfixed at 4°C protein (3, 4). in fixative/15% sucrose and then immersed in isopentane at To further test the specificity of affinity-purified D5 anti- -40°C for 5 min. Sections were cut on a cryostat, processed as bodies, we examined antibody reactivity with an HA epitope- tagged D5 receptor expressed in monkey CV-1 cells. The described (21), then prehybridized with 75% (vol/vol) form- reactivity of membrane fractions prepared from untransfected amide, 10% (wt/vol) dextran sulfate, 3x standard saline/ and HA-tagged D5 transfected cells with the HA epitope- citrate, 1 x Denhardt's solution, 10 mM dithiothreitol, 50 mM specific mAb 12CA5 and D5 antibodies is shown in Fig. 2 A Na2PO4 (pH 7.4), and 0.3% Triton X-100. Sense and antisense and B, respectively. The reaction of mAb 12CA5 with CV-1 cRNA a probes were transcribed from pGemBlue plasmid cells expressing an HA-tagged Na/K-ATPase al subunit containing nt 779-1114 of the published human D5 receptor ('110 kDa) (17) was included as a positive control (Fig. 2A). cDNA (3, 4) using T7 and SP6 RNA polymerases and digoxi- D5 antibodies did not react with any proteins in untransfected genin-UTP as described (11). Probes (1.0 ng/,ul) in prehy- CV-1 cells (Fig. 2B), whereas a nonspecific 90-kDa mAb- bridization solution were incubated with sections at 55°C for 16 reactive protein was present in both transfected and untrans- hr. Sections were washed and treated with RNase as described fected cells (Fig. 2A). In general, the D5 and 12CA5 antibodies (21), with a final wash at 55°C for 1 hr in 0.5x standard reacted with proteins of similar size in D5-transfected CV-1 saline/citrate. Sections were incubated with alkaline phos- cells (Fig. 2 A and B). The most abundant forms of the D5 phatase-conjugated anti-digoxigenin antibodies (Boehringer receptor migrated with a molecular mass of "50-66 kDa. In Mannheim) according to the manufacturer's instructions. Al- addition, a relatively abundant band of 135 kDa was also kaline phosphatase was visualized using 5-bromo-4-chloro-3- detected with both the D5 and 12CA5 antibodies. Each anti- indolyl phosphate/nitroblue tetrazolium (Boehringer Mann- body also reacted with a number of other bands -66-200 kDa heim). in size. To test whether these bands represent differentially A B + a0 0 00c 2 E LC LO) 116- 116- 0 0 97- 97- 66- 66- i 45- 45-

31- 31- FIG. 1. Specificity of D5 antibodies for the D5 dopamine receptor. D5 antibodies reacted with immunoblots containing crude membrane fractions (5 ,g) prepared from recombinant Sf9 cells expressing D1, D2, D3, D4, and D5 dopamingeric; Ml, M2, M3, M4, and M5 muscarinic; a2a, a2c, 1,3 and (32 adrenergic; and 5HT1A serotonergic receptors (A) and D5 membrane fractions (D5) and D5 membrane proteins treated with N-glycosidase F (D5 +) (B). Proteins were separated on SDS containing 10% (A) or 12% (B) polyacrylamide gels and transferred to poly(vinylidene difluoride) (A) or nitrocellulose (B) filters. Positions of molecular mass markers are indicated at left. Downloaded by guest on September 26, 2021 3470 Neurobiology: Bergson et aL Proc. Natl. Acad Sci. USA 92 (1995)

+ A a a A Z B 8 C a 200- 200- 200-1 116- _ * 16 -: 97_ 116=97= E 97R 66- 66- 66- 45- 45- 31- 31-

FIG. 2. Reactivity of D5 antibodies and HA epitope-specific mAb with epitope-tagged receptors in CV-1 cells. Immunoblots containing membranes (10 ,g) prepared from Na,K-ATPase al-transfected (Na,K-ATPase cil), -untransfected (CV-1), and pHA/Ds-transfected CV-1 (D5 CV-1) cells. The + designates membranes treated with N-glycosidase F. Filters were treated with HA epitope-specific mAb 12CA5 (A), or affinity-purified D5 receptor antibodies (B and C). Proteins were separated on SDS containing 12% (A and B) or 10% (C) FIG. 3. Subcellular localization of the epitope-tagged D5 receptors polyacrylamide gels and then electroblotted to poly(vinylidene di- visualized by confocal laser microscopy. Transfected CV-1 cells were fluoride) filters. Positions of molecular mass markers are indicated at grown in medium without tetracycline 24 hr before fixation. Cells were left. then fixed, permeabilized, and treated with affinity-purified D5 antibodies and fluorescein isothiocyanate-conjugated anti-rabbit IgG glycosylated forms of the D5 receptor, we treated membrane (Jackson ImmunoResearch) as described (17). (Bar = 15 ,um.) proteins prepared from transfected CV-1 cells with N- glycosidase F, as we did with the Sf9 cells expressing D5 with either the MBP-D5 fusion protein or MBP before im- receptors. Fig. 2C shows that N-glycosidase F digestion ap- munoblotting. All five proteins were detected with D5 anti- peared to alter only the mobility of the 50- to 66-kDa epitope- bodies, even in the presence of MBP (Fig. 4), whereas no tagged D5 receptors. Non-epitope-tagged D5 receptors, ex- proteins were detected when D5 antibodies were preincubated pressed in HEK293 cells transiently transfected with the pCB6 with MBP-D5 (data not shown). Taken together, these results eukaryotic cell expression vector containing D5 cDNA, also are consistent with the specificity of the D5 antibodies for migrated as a mixture of N-linked glycosylated polypeptides sequences found in the C terminus of the D5 receptor. ('50-66 kDa) and higher-molecular-mass forms (-66-200 We next localized the D5 receptor in the adult macaque PFC kDa) on SDS/PAGE (data not shown). These results indicate by light microscopy using affinity-purified D5 antibodies. Pre- that sizes of the D5 receptors detected on immunoblots are incubation of the antibodies with MBP-D5 fusion protein independent of the HA epitope, as well as the types of prevented labeling of these tissues. In the PFC, D5-receptor expression vectors and cultured cells used for transfections. antibodies prominently labeled pyramidal neuron cell bodies Similar behavior of other G protein-coupled receptors in and apical dendrites (Fig. SA and C). The cellular and laminar SDS/PAGE has been reported (22, 23). immunolabeling patterns closely resembled D5 receptor Next, we localized epitope-tagged D5 receptors in trans- mRNA expression in PFC using antisense cRNA probes (Fig. fected cells by confocal laser microscopy. As shown in Fig. 3, SB). In the caudate nucleus, D5 antibodies produced relatively confocal microscopic analysis of cells labeled with D5 antibod- light labeling of medium-sized neurons and large interneurons; ies showed bright staining at the cell margins, a result indicative D5 mRNA was also detected in similar cell populations of this of an integral plasma membrane protein. No signal was detected in untransfected CV-1 cells or in epitope-tagged D5 receptor-transfected cells when either the primary or second- ary antibody was omitted. Taken together, these results sup- 0. port the specificity of the D5 antibodies for the D5 dopamine receptor. .Q. X Z > > IC) Receptor in Primate Brain. of I O CO fO D Ds Expression Expression D5 ...... receptors was examined on immunoblots mem- 205- containing ...... :...:.:. brane fractions prepared from rhesus monkey brain and 19= peripheral tissues. Fig. 4 shows that D5 receptor antibodies 66- detected five polypeptide species of -49, 52, 70, 85, and 135 ';'.' 'u'.-,.'X,'.t r...... kDa in hippocampus, caudate, and substantia nigra. Proteins 45- of the same apparent molecular mass were also detected in D5-transfected CV-1 cells (Fig. 4) and in crude membrane preparations of the PFC (data not shown). Although the 50- 29- and 60-kDa polypeptides co-migrated with the more abundant glycosylated species in transfected cells, the 135-kDa polypep- FIG. 4. Comigration of D5 receptor antibody immunoreactive tide appeared the most abundant form of the receptor in proteins expressed in primate brain and in D5 receptor-transfected monkey brain. In contrast, D5 antibodies did not react with cells on SDS/PAGE. Immunoblot of membrane proteins prepared membrane proteins prepared from liver (Fig. 4) or spleen 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 (data not shown), suggesting that the D5 receptor polypeptides indicated. SN, substantia nigra. Proteins were separated on SDS/10% may be brain-specific. polyacrylamide gels and then electroblotted to poly(vinylidene di- To test whether the five reactive proteins shared D5 C- fluoride) filters. Positions of molecular mass markers are indicated at terminal sequence epitopes, D5 antibodies were preincubated left. Downloaded by guest on September 26, 2021 Neurobiology: Bergson et aL Proc. Natl. Acad. Sci. USA 92 (1995) 3471 noreactivity is specifically eliminated. It therefore seems rea- sonable to conclude that the antibodies we have produced are specific for the D5 dopamine receptor. In general, affinity-purified D5 antibodies and the HA- specific 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. Treat- ment 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 cul- ture (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 FIG. 5. Localization of D5 receptor protein and mRNA in the receptor in brain can be deglycosylated (C.B. and R.L., un- macaque PFC. Sections (30 gm thick) of comparable levels of Walker's published observations). area 9 of the PFC incubated with affinity-purified D5 antibodies (A) Light microscopic analysis of D5 receptor protein expression or D5 antisense RNA probes (B). Cells in layers III and V are the most indicates that the D5 receptor is relatively abundant in PFC prominently labeled in both sections. (X90) (C) Higher magnification pyramidal of a layer III pyramidal cell soma and apical dendrite shown in A. where D5 receptor antibodies prominently label (x360) wm, White matter. neurons. The D1 receptor shows a similar distribution in the primate PFC at this level of analysis. At the electron micro- nucleus (data not shown). The concordance of labeling pat- scopic level, the D1 receptor has been localized in numerous terns obtained with both antibody and cRNA probes further dendritic spines of pyramidal cells (8). The availability of supports the specificity of the antibodies for the D5 receptor. 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, DISCUSSION as well as in neuropathologies. For example, if the D5 receptor We have taken advantage of the sequence divergence in the C has a different distribution in pyramidal neurons of the PFC, terminus of the D1 and D5 dopamine receptors to generate it should be possible to determine the specific functional antibodies specific for the human D5 receptor. Antibody contributions of each receptor subtype to working memory (2, specificity was demonstrated by multiple criteria including (i) 25). It should also now be possible to investigate whether the reactivity with the D5 receptor and not with a variety of other D1 and/or D5 receptor subtypes exhibit alterations in abun- G protein-coupled receptors; (ii) reactivity and localization of dance or subcellular localization in cortical regions related to epitope-tagged D5 receptors in transfected cells; and (iii) lack diseases such as Parkinson disease or schizophrenia. of reactivity with brain tissue and immunoblots in the presence technical of D5 fusion proteins and not in the presence of nonspecific We thank M. Pappy and Y. Cao for their excellent assistance as well as Dr. H. Komuro for help with confocal imaging. fusion proteins. This research was supported by the National Alliance for Research on We observe antibody reactivity only with the D5 dopamine Schizophrenia and Depression (C.B.) and a National Institutes of receptor and not with other dopaminergic, adrenergic, sero- Mental Health Center Grant P50-MH44866-05. tonergic, and muscarinic receptors on immunoblots, suggesting that the antibodies are selective for the D5 receptor and are 1. Stoof, J. C. & Kebabian, J. W. (1984) Life Sci. 35, 2281-2296. unlikely to react with other members of the G protein-coupled 2. Sawaguchi, T. & Goldman-Rakic, P. S. (1991) Science 251, receptor superfamily. We affinity-purified antibodies from 947-950. serum using GST-D5 fusion protein to obtain 3. Grandy, D. K., Zhang, Q.-Y., Bouvier, C., Zhou, Q.-Y., Johnson, crude rabbit Civelli, for the D5 receptor portion of the MBP-D5 R. A., Allen, L., Buck, K., Bunzow, J. R., Salon, J. & 0. antibodies specific (1991) Proc. Natl. Acad. Sci. USA 88, 9175-9179. immunogen. These affinity-purified antibodies react with 4. Sunahara, R. K., Guan, H.-C., O'Dowd, B. F., Seeman, P., Lau- brain tissue and immunoblots containing membranes prepared rier, L. G., George, S. R., Torchia, J., Van Tol, H. M. & Niznik, from hippocampus, caudate, and substantia nigra in the pres- H. (1991) Nature (London) 350, 614-619. ence of other GST or MBP fusion proteins. However, in the 5. Lidow, M. S., Goldman-Rakic, P. S., Gallager, D. W. & Rakic, P. presence of GST-D5 or MBP-D5 fusion proteins, D5 immu- (1991) Neuroscience (Oxford) 40, 667-671. Downloaded by guest on September 26, 2021 3472 Neurobiology: Bergson et al Proc. Nati Acad Sci. USA 92 (1995)

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