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D3Dopamine Receptor Coupling to Adenylyl Cyclase
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
237, 394–399 (1997)
RC977146
Chimeric D2/D3 Dopamine Receptor Coupling to Adenylyl Cyclase Jean E. Lachowicz1 and David R. Sibley2 Molecular Neuropharmacology Section, Experimental Therapeutics Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892-1406
Received July 16, 1997
We have sought to determine which area of the D2 dopamine receptor’s third intracellular loop contributes to G-protein coupling by constructing reciprocal chimeric D2/D3 receptors with fusion points near the center of the third intracellular loop. Both receptor chimeras were expressed equally well in Chinese Hamster Ovary (CHO) cells and exhibited ligand binding properties similar to those of the wild type receptors. Surprisingly, both of the D2/D3 receptor chimeras were able to effectively inhibit adenylyl cyclase activity to almost the same extent as that seen with the D2 receptor whereas the D3 receptor was without effect. These results suggest that the D2 receptor possesses two redundant and independent domains for G-protein coupling and inhibition of adenylyl cyclase activity. q 1997 Academic Press
The neurotransmitter dopamine mediates numerous central nervous system functions and is associated with various neuronal disorders such as Parkinson’s Disease and schizophrenia (1,2). Dopamine exerts its action through the binding and activation of specific cell surface receptors which are members of the G protein-coupled receptor super-family. Dopamine receptors have generally been subclassified as ‘‘D1-like’’ or ‘‘D2-like’’ based on their functional and/or pharmacological profiles (3). Recently, molecular cloning techniques have been used to identify five distinct dopamine receptor proteins which are encoded for by separate genes (48). The deduced amino acid sequences of the dopamine receptors suggest that they contain seven alpha-helical transmembrane (TM) domains characteristic of recep1
Current address: Central Nervous System and Cardiovascular Department, Schering-Plough Research Institute, Kenilworth, NJ 07033. 2 Correspondence address: Experimental Therapeutics Branch, NINDS/NIH, Building 10, Room 5C108, 10 Center Drive, MSC 1406, Bethesda, MD 20892-1406. Fax: (301) 496-6609. E-mail: sibley@ helix.nih.gov. 0006-291X/97 $25.00
tors which couple to guanine nucleotide binding proteins (G-proteins) (9,10). Two of the cloned receptors, the D1 and D5 , have been characterized as belonging to the ‘‘D1-like’’ subfamily, whereas the D2 , D3 and D4 receptors belong to the ‘‘D2-like’’ subfamily. The D1 and D5 receptors couple to Gs to stimulate adenylyl cyclase activity while the D2 and D4 receptors mainly couple through Gi to inhibit adenylyl cyclase (reviewed in 11,12). Although D3 receptors belong to the ‘‘D2-like’’ subfamily, based on similar structure and pharmacology, they generally do not appear to be robustly coupled to adenylyl cyclase inhibition when expressed in mammalian cell lines (reviewed in 11,12). Structurally, the rat D2 and D3 receptors exhibit 52% overall amino acid homology, while their TM domains exhibit 75% homology (13). Since the TM regions of catecholamine receptors are believed to constitute the ligand binding sites (9,10,14,15), the high homology of these domains explains the relative lack of compounds with high selectivity between D2 and D3 receptors. Whereas the TM domains contain sites necessary for ligand binding, it is the third intracellular loop (IC3), especially its amino- and carboxyl-terminal regions, which is postulated to be mostly important for G-protein interactions and the initiation of signal transduction (14-17). In the IC3 domain, the D2 and D3 receptors are more divergent, exhibiting only about 25% structural homology (13). Chimeric receptor strategies have proven useful for evaluating the functional attributes of specific receptor domains (15-17). Since in most expression systems, the D3 receptor does not inhibit adenylyl cyclase activity, this receptor can serve as a null background with which to investigate regions of the D2 receptor which are important for Gi protein coupling. We have thus sought to determine which region of the D2 receptor’s IC3 contributes most to second messenger coupling by constructing chimeric D2 and D3 receptors with fusion points near the center of the third cytoplasmic loop. By leaving the corresponding transmembrane regions of each receptor intact, we have maintained the amino
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acid environment with which each region of the IC3 may crucially interact. One chimera, termed the D2/3 receptor, contains the D2 receptor TMs I-V and the amino-terminal portion of the IC3 fused to the D3 receptor carboxyl-terminal portion of the IC3 and TMs VI and VII. This chimera and its reciprocal, the D3/2 receptor, were stably expressed in CHO cells to examine their potential coupling to adenylyl cyclase. MATERIALS AND METHODS Chimera constructions. Rat D2L and D3 receptor cDNAs in the cloning vector pBluescript (Stratagene) were excised with the polylinker restriction enzymes Xho I (5*) and Xba I (3 *) (Boehringer Mannheim) and cleaved in approximately the center of the IC3 with Ecl 136 II (D2) or Eco 47 III (D3) (New England Biolabs). 5* and 3* fragments were separated by agarose gel electrophoresis and extracted from low melting agarose (GIBCO). The opposite 5* and 3* fragments were ligated together in the presence of Xho I/Xba I linearized pcDNA3 vector (Invitrogen). Because we have found that the expression levels differ depending on whether the 3 *-untranslated region of the receptor is that of the D2 or D3 receptor (unpublished observations), all wild type and chimeric constructs were designed to contain the D2 receptor 3*-untranslated region. The resulting plasmids were transformed into competent DH5-a cells (GIBCO). DNA was extracted from bacterial colonies and the inserts verified by restriction digests and Sanger DNA sequencing of the ligation junctions using Sequenase (Amersham). CHO-K1 cell culture and transfections. CHO-K1 cells were cultured in F12 nutrient media (GIBCO) containing 10% fetal bovine serum (FBS) and 3 ml/L gentamycin. Cells seeded into 150120 mm plates were transfected with 30 mg of construct-containing pcDNA3 plasmid DNA using the CaPO4 precipitation method described previously (18). Cells were split after 48 hr and selection in the form of 500 mg/ml G418 (geneticin, GIBCO) was applied 24 hr later. Individual colonies were isolated after about one week of selection and analyzed by cellular RNA dot blot hybridization assays using [32P]-labeled D2 and D3 receptor DNA probes. Further screening for receptor expression levels was performed by radioligand binding analysis (see below) using [3H]-methylspiperone ([3H]-MSP) (80-90 Ci/mmol, Amersham).
tion) containing 3 mM forskolin. cAMP generation was allowed to proceed for 5 minutes at 377C and the reaction was terminated at 1007C for 3 minutes. A competitive binding assay utilizing bovine heart protein kinase A regulatory subunit (Sigma) and [3H]-cAMP tracer (New England Nuclear) was used to quantify cAMP generated. Following a 2 hour 47C incubation, free [3H]-cAMP was removed by precipitation with charcoal/BSA and the remaining, bound cAMP was quantified by liquid scintillation spectroscopy using Hydroflour. The assay described was linear in the range of 0.5-25 pmoles cAMP/ assay tube. Basal cAMP values were approximately 0.6 pmol/250,000 cells whereas forskolin-stimulated values were about 15 pmol/ 250,000 cells.
RESULTS In order to create chimeric D2/D3 receptors with junctions in approximately the center of the IC3s, we took advantage of existing Ecl 136 II (D2 receptor) and Eco 47 III (D3 receptor) restriction sites which produce compatible cohesive ends (See Figure 1). Chimeric receptor D2/3 consists of the D2L receptor from its amino terminus to Arg 275 and the D3 receptor from Arg 301 to its carboxyl terminus. The reverse chimera, D3/2 , contains the D3 receptor from its amino terminus to Lys 300 and the D2L receptor from Arg 276 to its carboxyl terminus. The D2L receptor was used in this study rather than the D2S receptor because of its similarity in size to the D3 receptor in the IC3 region. In our hands, the D2L and D2S receptors have been found to exhibit similar
Radioligand binding assays. Cells were harvested for binding with 1 mM EDTA in Ca2//Mg 2/-free EDTA, washed 21 with Earle’s Balanced Salt Solution (EBSS) and binding buffer (50 mM Tris-HCl, pH 7.4 at 257C, 1 mM EDTA, 5 mM KCl, 1.5 mM CaCl2 , 4 mM MgCl2 , 120 mM NaCl), and Dounce homogenized. Crude membranes were collected by centrifugation at 34,0001g for 10 min and resuspended in binding buffer at about 0.3 mg protein/ml using a motor-driven teflon pestle. 100 ml of membrane suspension was added to assay tubes containing 0.03-1 nM [3H]-MSP in a final volume of 1 ml. 1mM (/)-butaclamol was used to define non-specific binding. Binding assays were incubated for 1 hour at 257C and were terminated by rapid filtration through GF/C filters pretreated with 0.3% polyethyleneimine, followed by washing with 514 ml ice-cold 50 mM TrisHCl, pH 7.4. Radioactivity bound to the filters was quantified by scintillation spectroscopy using Hydroflour (National Diagnostics) at a counting efficiency of 47%. Determination of cAMP production. The accumulation of adenosine 3*-, 5*-cyclic-monophosphate (cAMP) was measured in intact cells as follows. CHO cells were harvested, washed three times in EBSS, and resuspended in AC buffer (250 mM sucrose, 75 mM TrisHCl, pH 7.4 at 377C, 12.5 mM MgCl2 , 1.5 mM EDTA, 1 mM DTT, 200 mM sodium metabisulfite and 100 mM RO-20-1724 - a phosphodiesterase inhibitor). Cell suspensions (50 ml) containing 250,000 cells were added to a 10 ml solution of dopamine (0-100 mM final concentra-
FIG. 1. Schematic diagram of how the chimeric D2/3 and D3/2 receptors were constructed from the wild type D2 and D3 receptors. Solid lines represent D2 receptor sequences whereas the open lines represent D3 receptor sequences. The amino acid sequences of the D2 and D3 receptors within the IC3 regions containing the Ecl 136 II and Eco 47 III sites are shown at the bottom. The arrows indicate the positions of the restriction enzyme recognition sites.
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Summary of Results from Radioligand Binding and cAMP Accumulation Assays of Wild Type and Chimeric Dopamine Receptors Receptor
[3H]-MSP Bmax (pmol/mg protein)
D2 D2/3 D3/2 D3
2.65 2.25 2.47 2.85
[3H]-MSP Kd (M) 6.50 41.6 6.60 30.5
1 1 1 1
7-OH-DPAT Ki (M)
10011 10011 10011 10011
1.41 1.37 2.88 2.30
1 1 1 1
1006 1006 1008 1008
PD-128907 Ki (M) 7.47 4.54 2.70 5.94
1 1 1 1
1006 1007 1007 1009
Dopamine EC50 (M) 8.35 1 1008 1.27 1 1008 1.23 1 1008 —
The [3H]-MSP binding parameters were derived from saturation binding isotherms as described in the Material and Methods. The drug affinity (Ki) values were derived from competition binding assays as shown in Figs. 2 and 3. The EC50 values for cAMP accumulation were derived from experiments as shown in Fig. 4. All data represent average mean values from between three to seven separately performed experiments with SEM values of less than 20%.
ligand binding characteristics and efficacy at inhibiting adenylyl cyclase activity in CHO cells (18,19). Wild type and chimeric receptor constructs in pcDNA3 were stably transfected into CHO-K1 cells. CHO-K1 cells were used because they lack endogenous dopamine receptors and because D2 receptors transfected into CHO-K1 cells effectively inhibit adenylyl cyclase activity (18,19). Transfected cells were selected by G418 resistance, sequence-specific hybridization, and [3H]-MSP binding. Cell lines for each receptor were chosen so as to maximize receptor expression and minimize differences in expression levels among the four receptors. The mean expression level for the four cell lines was 2.56 pmol/mg protein with a standard deviation of less than 10% (Table 1). Receptor binding assays using [3H]-MSP were conducted not only to determine the level of receptor expression but also to test whether the binding properties of the chimeras differed from those of the wild type receptors. Notably, the D2 and D3/2 receptors bound the antagonist [3H]-MSP with higher affinity than did the D3 and D2/3 receptors (Table 1). Competition studies were also performed using the D3-selective agonist ligands, 7-hydroxydipropylaminotetralin (7-OH-DPAT) and PD-128907 (7,8). 7-OH-DPAT was more potent in displacing [3H]-MSP binding in the D3 and D3/2 receptor expressing cells than in the D2 and D2/3 receptor expressing cells (Figure 2 and Table 1). The affinities for the chimeras were somewhat intermediate to those of the wild type receptors. These results with [3H]-MSP binding and 7-OH-DPAT competition assays suggest that TMs VI and VII have a stronger influence on methylspiperone binding than TMs I-V, and that TMs I-V have a stronger influence on 7-OH-DPAT binding than TMs VI and VII. This premise is in agreement with previous reports demonstrating that, for catecholamine receptors, residues in TMs IV and V are critical for agonist binding and residues in TM VI and VII are critical for antagonist binding (9,10,14,15). Competition studies with PD-128907 yielded somewhat different results, however, than the studies with
7-OH-DPAT. PD-128907 competed for [3H]-MSP binding with about 1,000-fold greater potency at the D3 receptor than the D2 receptor (Figure 3, Table 1) in agreement with previous findings (7,8). In contrast with 7-OH-DPAT however, for which the affinity of each chimeric receptor correlated with the presence or absence of TMs I-V, PD-128907 displaced [3H]-MSP binding to the two chimeras with similar affinities which were intermediate to those of the wild type receptors (Figure 3, Table 1). This binding profile may suggest that PD-128907 interacts in a unique way with the receptors involving multiple TM contact sites. In any case, these preliminary binding studies indicate that the chimeric D2/D3 receptors are capable of being expressed in CHO cells and exhibit ligand binding parameters well within those exhibited by the wild type receptors. To determine whether the chimeric receptors would inhibit adenylyl cyclase activity in CHO cells like the D2 receptor or be uncoupled like the D3 receptor, the effects of dopamine on cAMP accumulation in intact stably transfected cells were investigated. Dopamine did not inhibit basal cAMP accumulation in either the wild-type or chimeric receptor expressing cells (data not shown), consistent with previous reports (18,19). When the cAMP accumulation was stimulated with 3 mM forskolin treatment, however, inhibition of this activity was detectable in the D2 , D2/3 , and D3/2 receptortransfected cells (Figure 4). No effects of dopamine on basal or forskolin-stimulated cAMP accumulation were observable in D3 receptor transfected cells, in agreement with previous studies (13). Dopamine activation of D2 receptors resulted in a maximal decrease in forskolin-stimulated cAMP production of about 65% with an EC50 of 84 nM (Figure 4, Table 1). For the D2/3 and D3/2 receptors, dopamine produced about a 50% and 45% decrease in forskolinstimulated cAMP production, respectively (Figure 4). The EC50 for dopamine at both receptors was about 12 nM (Table 1). This represents about a 7-fold increase in potency for dopamine in comparison to the wild type
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FIG. 2. Displacement of [3H]-MSP binding to wild type D2 and D3 and chimeric D2/3 and D3/2 receptors by 7-OH-DPAT in stably transfected CHO cells. Cell membranes were prepared and radioligand binding competition assays were performed using 0.1 nM [3H]-MSP as described in the Materials and Methods. A representative experiment is shown which was performed four times. Average affinity values are shown in Table 1.
D2 receptor expressing cells. Thus, both of the receptor chimeras were able to couple to adenylyl cyclase inhibition in the CHO cells, although the maximum degree of inhibition was 15-20% less than that seen with the wild type D2 receptor. DISCUSSION The mechanisms by which receptors selectively activate G-proteins have been difficult to elucidate. Con-
trary to initial expectations, it has not proven possible to predict the G protein specificity of any receptor based on its primary structure. Extensive investigations of catecholamine receptors using site-directed mutagenesis, receptor chimeras, peptides and antibodies, however, have implicated the third intracellular loop (IC3), especially its amino- and carboxyl-terminal regions, of the receptor as an important, but perhaps not exclusive, determinant of selective G-protein coupling (1417). This has also been suggested to be true for the D2
FIG. 3. Displacement of [3H]-MSP binding to wild type D2 and D3 and chimeric D2/3 and D3/2 receptors by PD-128907 in stably transfected CHO cells. Cell membranes were prepared and radioligand binding competition assays were performed using 0.1 nM [3H]-MSP as described in the Materials and Methods. A representative experiment is shown which was performed four times. Average affinity values are shown in Table 1. 397
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FIG. 4. Dopamine-mediated inhibition of forskolin-stimulated cAMP accumulation in CHO cells stably transfected with wild type D2 and chimeric D2/3 and D3/2 receptors. No inhibition of cAMP accumulation was observed in CHO cells stably transfected with D3 receptors (see text). cAMP accumulation assays were performed as described in the Materials and Methods. The data represent the % inhibition of the cAMP accumulation produced in response to forskolin. A representative experiment is shown which was performed a total of seven times. Average EC50 values are shown in Table 1.
dopamine receptor with respect to its ability to couple to Gi and inhibit adenylyl cyclase activity. Several studies have shown that antisera directed against sequences within the D2 receptor IC3 could disrupt Gprotein coupling in cell-free systems (20-23). In addition, synthetic peptides derived from the IC3 of the D2 receptor were shown to attenuate receptor-mediated inhibition of adenylyl cyclase in membrane preparations (24). On the other hand, Kozell et al. (25) using D1/D2 receptor chimeras found that the presence of both the second (IC2) and 3rd (IC3) intracellular loops of the D2 receptor were necessary to support receptormediated adenylyl cyclase inhibition. These results suggest that while the IC3 of the D2 receptor may be necessary for Gi coupling, it may not be sufficient. Several previous studies have used D3/D2 receptor chimeras to address the functional role of the D2 receptor IC3 in G-protein coupling. In an initial study, McAllister et al. (26) found that a chimera consisting of the D3 receptor with the IC3 of the D2 receptor was unable to couple to adenylyl cyclase inhibition. In contrast, two subsequent studies (27,28) have shown that similar D3/D2-IC3 receptor chimeras were, indeed, able to effectively couple to this signal transduction pathway. An important issue in the interpretation of these data is that the junctions of these chimeras (26-28) were either in or near the amino- and carboxyl-terminal regions of the IC3 - regions where G-protein coupling has been postulated to occur (14-17). Thus, it is possible that the existence of these junctions have perturbed some secondary structure of the receptor resulting in anomalous G-protein coupling properties. In
the present study, we have attempted to avoid this issue by placing the D2/D3 junctions in the middle of the IC3 thus leaving the environment of the aminoand carboxyl-terminal regions (as well the adjacent TM regions) of the IC3 intact. This approach has also allowed us to test the individual contributions of each half of the IC3 to G-protein coupling. Our present results suggest that D2/D3 receptor chimeras with mid-IC3 junctions can be effectively expressed without a major perturbation in their ligand binding characteristics. Although, the pharmacology of each chimera differed somewhat depending upon the ligand tested, each chimera exhibited ligand binding parameters well within those demonstrated by the wild type receptors. Of major significance, however, is that each chimera was able to support inhibition of adenylyl cyclase activity similarly and to almost the same extent as that observed with the wild type D2 receptor. While this may suggest that both the amino and carboxyl portions of the D2 receptor IC3 are required for maximal Gi coupling, it also implies that each region is capable of independently supporting functional coupling. This surprising finding suggests that the D2 receptor possesses redundant and mostly independent Gi coupling domains. A similar finding was recently reported for the a2adrenergic receptor which couples to multiple G-proteins, including Gi resulting in inhibition of adenylyl cyclase activity. Using a chimeric receptor approach, Eason et al. (17) found that either the amino- or carboxyl-terminal IC3 region of the a2-adrenergic receptor was capable of supporting Gi coupling. In addition,
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these investigators found that the IC2 of the a2-adrenergic receptor was mandatory for functional G-protein coupling to occur. This agrees well the D1/D2 receptor chimera study of Kozell et al. (25) which also implicated the necessity of the IC2 of the D2 receptor for inhibition of adenylyl cyclase. In the present study, it is interesting to note that only the D2/3 chimera possessed the IC2 of the D2 receptor whereas the D3/2 receptor possessed that of the D3 . This suggests that the IC2 of the D3 receptor (which is 50% homologous with that of the D2) is capable of supporting Gi coupling but not in conjunction with the D3 receptor’s IC3, in agreement with previous results (27,28). Given these findings for the D2 dopamine and a2-adrenergic receptors, it will be interesting to see if other Gi coupled receptors exhibit this phenomenon. In summary, we have provided evidence for the existence of two redundant and functionally independent Gi coupling domains in the D2 dopamine receptor. The rationale for the existence of two distinct G-protein coupling regions is unclear but may have to do with the fact that the D2 receptor (as are most Gi/o coupled receptors) is coupled to multiple signal transduction pathways (11,12). It will be interesting to investigate the coupling of these and related D2/D3 receptor chimeras to other D2 receptor-mediated signalling events as well as to use more specific mutagenesis techniques to further delineate the G-protein coupling domains of the D2 receptor. REFERENCES 1. Hornykiewicz, O. (1973) Fed. Proc. 32, 183–190. 2. Seeman, P. (1987) Synapse 1, 133–152. 3. Kebabian, J. W., and Calne, D. B. (1979) Nature 277, 93–96. 4. Sibley, D. R., and Monsma, F. J., Jr. (1992) Trends Pharmacol. Sci. 13, 61–69. 5. Civelli, O., Bunzow, J. R., and Grandy, D. K. (1993) Ann. Rev. Pharmacol. Tocxicol. 32, 281–307. 6. Gingrich, J. A., and Caron, M. G. (1993) Ann. Rev. Neuroci. 16, 299–321.
7. Sokoloff, P., and Schwartz, J.-C. (1995) Trends Pharmacol. Sci. 16, 270–275. 8. Neve, K. A., and Neve, R. L. (1997) in The Dopamine Receptors (Neve, K. A., and Neve, R. L., Eds.), pp. 77–104, Humana Press, Clifton, NJ. 9. Dohlman, H. G., Thorner, J., Caron, M. G., and Lefkowitz, R. J. (1991) Annu. Rev. Biochem. 60, 653–688. 10. Strader, C. D., Fong, T. M., Graziano, M. P., and Tota, M. R. (1995) FASEB J. 9, 745–754. 11. Robinson, S. W., and Caron, M. G. (1997) in The Dopamine Receptors (Neve, K. A., and Neve, R. L., Eds.), pp. 137–166, Humana Press, Clifton, NJ. 12. Huff, R. (1997) in The Dopamine Receptors (Neve, K. A., and Neve, R. L., Eds.), pp. 167–192, Humana Press, Clifton, NJ. 13. Sokoloff, P., Giros, B., Martres, M.-P., Bouthenet, M.-L., and Schwartz, J.-C. (1990) Nature 347, 146–151. 14. Strader, C. D., Fong, T. M., Tota, M. R., Underwood, D., and Dixon, R. A. F. (1994) Annu. Rev. Biochem. 63, 101–132. 15. Kobilka, B. K., Kobilka, T. S., Daniel, K., Regan, J. W., Caron, M. G., and Lefkowitz, R. J. (1988) Science 240, 1310–1316. 16. Wess, J. (1997) FASEB J. 11, 346–354. 17. Eason, M. G., and Liggett, S. B. (1996) J. Biol. Chem. 271, 12826–12832. 18. Zhang, L. J., Lachowicz, J. E., and Sibley, D. R. (1994) Molec. Pharmacol. 45, 878–889. 19. Rinaudo, M. S., Monsma, F. J., Jr., Black, L. E., Mahan, L. C., and Sibley, D. R. (1990) Soc. Neurosci. Abstr. 16, 209. 20. Plug, M. J., Dijk, J., Maassen, A., and Moller, W. (1992) Eur. J. Biochem. 206, 123–130. 21. Boundy, V. A., Luedtke, R. R., and Molinoff, P. B. (1993) J. Neurochem. 60, 2181–2191. 22. Boundy, V. A., Luedtke, R. R., Artmyshynm, R. P., Filtz, T. M., and Molinoff, P. B. (1993) J. Pharmacol. Exptl. Therap. 43, 666– 676. 23. Chazot, P. L., Doherty, A. J., and Strange, P. L. (1993) Biochem. J. 289, 789–794. 24. Malek, D., Munch, G., and Palm, D. (1993) FEBS Letts. 325, 215–219. 25. Kozell, L. B., Machida, C. A., Neve, R. L., and Neve, K. A. (1994) J. Biol. Chem. 269, 30299–30306. 26. McAllister, G. A., Knowles, M. R., Patel, S., Marwood, R., Emms, F., Seabrook, G. R., Graziano, M., Borkowski, D., Hey, P. J., and Freedman, S. B. (1993) FEBS Letts. 324, 81–86. 27. Van Leeuwen, D. H., Eisenstein, J., O’Malley, K., and MacKenzie, R. G. (1995) Molec. Pharmacol. 48, 344–351. 28. Robinson, S. R., and Caron, M. G. (1996) J. Neurochem. 67, 212– 219.
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Chimeric D2/D3 Dopamine Receptor Coupling to Adenylyl Cyclase Jean E. Lachowicz1 and David R. Sibley2 Molecular Neuropharmacology Section, Experimental Therapeutics Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892-1406
Received July 16, 1997
We have sought to determine which area of the D2 dopamine receptor’s third intracellular loop contributes to G-protein coupling by constructing reciprocal chimeric D2/D3 receptors with fusion points near the center of the third intracellular loop. Both receptor chimeras were expressed equally well in Chinese Hamster Ovary (CHO) cells and exhibited ligand binding properties similar to those of the wild type receptors. Surprisingly, both of the D2/D3 receptor chimeras were able to effectively inhibit adenylyl cyclase activity to almost the same extent as that seen with the D2 receptor whereas the D3 receptor was without effect. These results suggest that the D2 receptor possesses two redundant and independent domains for G-protein coupling and inhibition of adenylyl cyclase activity. q 1997 Academic Press
The neurotransmitter dopamine mediates numerous central nervous system functions and is associated with various neuronal disorders such as Parkinson’s Disease and schizophrenia (1,2). Dopamine exerts its action through the binding and activation of specific cell surface receptors which are members of the G protein-coupled receptor super-family. Dopamine receptors have generally been subclassified as ‘‘D1-like’’ or ‘‘D2-like’’ based on their functional and/or pharmacological profiles (3). Recently, molecular cloning techniques have been used to identify five distinct dopamine receptor proteins which are encoded for by separate genes (48). The deduced amino acid sequences of the dopamine receptors suggest that they contain seven alpha-helical transmembrane (TM) domains characteristic of recep1
Current address: Central Nervous System and Cardiovascular Department, Schering-Plough Research Institute, Kenilworth, NJ 07033. 2 Correspondence address: Experimental Therapeutics Branch, NINDS/NIH, Building 10, Room 5C108, 10 Center Drive, MSC 1406, Bethesda, MD 20892-1406. Fax: (301) 496-6609. E-mail: sibley@ helix.nih.gov. 0006-291X/97 $25.00
tors which couple to guanine nucleotide binding proteins (G-proteins) (9,10). Two of the cloned receptors, the D1 and D5 , have been characterized as belonging to the ‘‘D1-like’’ subfamily, whereas the D2 , D3 and D4 receptors belong to the ‘‘D2-like’’ subfamily. The D1 and D5 receptors couple to Gs to stimulate adenylyl cyclase activity while the D2 and D4 receptors mainly couple through Gi to inhibit adenylyl cyclase (reviewed in 11,12). Although D3 receptors belong to the ‘‘D2-like’’ subfamily, based on similar structure and pharmacology, they generally do not appear to be robustly coupled to adenylyl cyclase inhibition when expressed in mammalian cell lines (reviewed in 11,12). Structurally, the rat D2 and D3 receptors exhibit 52% overall amino acid homology, while their TM domains exhibit 75% homology (13). Since the TM regions of catecholamine receptors are believed to constitute the ligand binding sites (9,10,14,15), the high homology of these domains explains the relative lack of compounds with high selectivity between D2 and D3 receptors. Whereas the TM domains contain sites necessary for ligand binding, it is the third intracellular loop (IC3), especially its amino- and carboxyl-terminal regions, which is postulated to be mostly important for G-protein interactions and the initiation of signal transduction (14-17). In the IC3 domain, the D2 and D3 receptors are more divergent, exhibiting only about 25% structural homology (13). Chimeric receptor strategies have proven useful for evaluating the functional attributes of specific receptor domains (15-17). Since in most expression systems, the D3 receptor does not inhibit adenylyl cyclase activity, this receptor can serve as a null background with which to investigate regions of the D2 receptor which are important for Gi protein coupling. We have thus sought to determine which region of the D2 receptor’s IC3 contributes most to second messenger coupling by constructing chimeric D2 and D3 receptors with fusion points near the center of the third cytoplasmic loop. By leaving the corresponding transmembrane regions of each receptor intact, we have maintained the amino
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acid environment with which each region of the IC3 may crucially interact. One chimera, termed the D2/3 receptor, contains the D2 receptor TMs I-V and the amino-terminal portion of the IC3 fused to the D3 receptor carboxyl-terminal portion of the IC3 and TMs VI and VII. This chimera and its reciprocal, the D3/2 receptor, were stably expressed in CHO cells to examine their potential coupling to adenylyl cyclase. MATERIALS AND METHODS Chimera constructions. Rat D2L and D3 receptor cDNAs in the cloning vector pBluescript (Stratagene) were excised with the polylinker restriction enzymes Xho I (5*) and Xba I (3 *) (Boehringer Mannheim) and cleaved in approximately the center of the IC3 with Ecl 136 II (D2) or Eco 47 III (D3) (New England Biolabs). 5* and 3* fragments were separated by agarose gel electrophoresis and extracted from low melting agarose (GIBCO). The opposite 5* and 3* fragments were ligated together in the presence of Xho I/Xba I linearized pcDNA3 vector (Invitrogen). Because we have found that the expression levels differ depending on whether the 3 *-untranslated region of the receptor is that of the D2 or D3 receptor (unpublished observations), all wild type and chimeric constructs were designed to contain the D2 receptor 3*-untranslated region. The resulting plasmids were transformed into competent DH5-a cells (GIBCO). DNA was extracted from bacterial colonies and the inserts verified by restriction digests and Sanger DNA sequencing of the ligation junctions using Sequenase (Amersham). CHO-K1 cell culture and transfections. CHO-K1 cells were cultured in F12 nutrient media (GIBCO) containing 10% fetal bovine serum (FBS) and 3 ml/L gentamycin. Cells seeded into 150120 mm plates were transfected with 30 mg of construct-containing pcDNA3 plasmid DNA using the CaPO4 precipitation method described previously (18). Cells were split after 48 hr and selection in the form of 500 mg/ml G418 (geneticin, GIBCO) was applied 24 hr later. Individual colonies were isolated after about one week of selection and analyzed by cellular RNA dot blot hybridization assays using [32P]-labeled D2 and D3 receptor DNA probes. Further screening for receptor expression levels was performed by radioligand binding analysis (see below) using [3H]-methylspiperone ([3H]-MSP) (80-90 Ci/mmol, Amersham).
tion) containing 3 mM forskolin. cAMP generation was allowed to proceed for 5 minutes at 377C and the reaction was terminated at 1007C for 3 minutes. A competitive binding assay utilizing bovine heart protein kinase A regulatory subunit (Sigma) and [3H]-cAMP tracer (New England Nuclear) was used to quantify cAMP generated. Following a 2 hour 47C incubation, free [3H]-cAMP was removed by precipitation with charcoal/BSA and the remaining, bound cAMP was quantified by liquid scintillation spectroscopy using Hydroflour. The assay described was linear in the range of 0.5-25 pmoles cAMP/ assay tube. Basal cAMP values were approximately 0.6 pmol/250,000 cells whereas forskolin-stimulated values were about 15 pmol/ 250,000 cells.
RESULTS In order to create chimeric D2/D3 receptors with junctions in approximately the center of the IC3s, we took advantage of existing Ecl 136 II (D2 receptor) and Eco 47 III (D3 receptor) restriction sites which produce compatible cohesive ends (See Figure 1). Chimeric receptor D2/3 consists of the D2L receptor from its amino terminus to Arg 275 and the D3 receptor from Arg 301 to its carboxyl terminus. The reverse chimera, D3/2 , contains the D3 receptor from its amino terminus to Lys 300 and the D2L receptor from Arg 276 to its carboxyl terminus. The D2L receptor was used in this study rather than the D2S receptor because of its similarity in size to the D3 receptor in the IC3 region. In our hands, the D2L and D2S receptors have been found to exhibit similar
Radioligand binding assays. Cells were harvested for binding with 1 mM EDTA in Ca2//Mg 2/-free EDTA, washed 21 with Earle’s Balanced Salt Solution (EBSS) and binding buffer (50 mM Tris-HCl, pH 7.4 at 257C, 1 mM EDTA, 5 mM KCl, 1.5 mM CaCl2 , 4 mM MgCl2 , 120 mM NaCl), and Dounce homogenized. Crude membranes were collected by centrifugation at 34,0001g for 10 min and resuspended in binding buffer at about 0.3 mg protein/ml using a motor-driven teflon pestle. 100 ml of membrane suspension was added to assay tubes containing 0.03-1 nM [3H]-MSP in a final volume of 1 ml. 1mM (/)-butaclamol was used to define non-specific binding. Binding assays were incubated for 1 hour at 257C and were terminated by rapid filtration through GF/C filters pretreated with 0.3% polyethyleneimine, followed by washing with 514 ml ice-cold 50 mM TrisHCl, pH 7.4. Radioactivity bound to the filters was quantified by scintillation spectroscopy using Hydroflour (National Diagnostics) at a counting efficiency of 47%. Determination of cAMP production. The accumulation of adenosine 3*-, 5*-cyclic-monophosphate (cAMP) was measured in intact cells as follows. CHO cells were harvested, washed three times in EBSS, and resuspended in AC buffer (250 mM sucrose, 75 mM TrisHCl, pH 7.4 at 377C, 12.5 mM MgCl2 , 1.5 mM EDTA, 1 mM DTT, 200 mM sodium metabisulfite and 100 mM RO-20-1724 - a phosphodiesterase inhibitor). Cell suspensions (50 ml) containing 250,000 cells were added to a 10 ml solution of dopamine (0-100 mM final concentra-
FIG. 1. Schematic diagram of how the chimeric D2/3 and D3/2 receptors were constructed from the wild type D2 and D3 receptors. Solid lines represent D2 receptor sequences whereas the open lines represent D3 receptor sequences. The amino acid sequences of the D2 and D3 receptors within the IC3 regions containing the Ecl 136 II and Eco 47 III sites are shown at the bottom. The arrows indicate the positions of the restriction enzyme recognition sites.
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Summary of Results from Radioligand Binding and cAMP Accumulation Assays of Wild Type and Chimeric Dopamine Receptors Receptor
[3H]-MSP Bmax (pmol/mg protein)
D2 D2/3 D3/2 D3
2.65 2.25 2.47 2.85
[3H]-MSP Kd (M) 6.50 41.6 6.60 30.5
1 1 1 1
7-OH-DPAT Ki (M)
10011 10011 10011 10011
1.41 1.37 2.88 2.30
1 1 1 1
1006 1006 1008 1008
PD-128907 Ki (M) 7.47 4.54 2.70 5.94
1 1 1 1
1006 1007 1007 1009
Dopamine EC50 (M) 8.35 1 1008 1.27 1 1008 1.23 1 1008 —
The [3H]-MSP binding parameters were derived from saturation binding isotherms as described in the Material and Methods. The drug affinity (Ki) values were derived from competition binding assays as shown in Figs. 2 and 3. The EC50 values for cAMP accumulation were derived from experiments as shown in Fig. 4. All data represent average mean values from between three to seven separately performed experiments with SEM values of less than 20%.
ligand binding characteristics and efficacy at inhibiting adenylyl cyclase activity in CHO cells (18,19). Wild type and chimeric receptor constructs in pcDNA3 were stably transfected into CHO-K1 cells. CHO-K1 cells were used because they lack endogenous dopamine receptors and because D2 receptors transfected into CHO-K1 cells effectively inhibit adenylyl cyclase activity (18,19). Transfected cells were selected by G418 resistance, sequence-specific hybridization, and [3H]-MSP binding. Cell lines for each receptor were chosen so as to maximize receptor expression and minimize differences in expression levels among the four receptors. The mean expression level for the four cell lines was 2.56 pmol/mg protein with a standard deviation of less than 10% (Table 1). Receptor binding assays using [3H]-MSP were conducted not only to determine the level of receptor expression but also to test whether the binding properties of the chimeras differed from those of the wild type receptors. Notably, the D2 and D3/2 receptors bound the antagonist [3H]-MSP with higher affinity than did the D3 and D2/3 receptors (Table 1). Competition studies were also performed using the D3-selective agonist ligands, 7-hydroxydipropylaminotetralin (7-OH-DPAT) and PD-128907 (7,8). 7-OH-DPAT was more potent in displacing [3H]-MSP binding in the D3 and D3/2 receptor expressing cells than in the D2 and D2/3 receptor expressing cells (Figure 2 and Table 1). The affinities for the chimeras were somewhat intermediate to those of the wild type receptors. These results with [3H]-MSP binding and 7-OH-DPAT competition assays suggest that TMs VI and VII have a stronger influence on methylspiperone binding than TMs I-V, and that TMs I-V have a stronger influence on 7-OH-DPAT binding than TMs VI and VII. This premise is in agreement with previous reports demonstrating that, for catecholamine receptors, residues in TMs IV and V are critical for agonist binding and residues in TM VI and VII are critical for antagonist binding (9,10,14,15). Competition studies with PD-128907 yielded somewhat different results, however, than the studies with
7-OH-DPAT. PD-128907 competed for [3H]-MSP binding with about 1,000-fold greater potency at the D3 receptor than the D2 receptor (Figure 3, Table 1) in agreement with previous findings (7,8). In contrast with 7-OH-DPAT however, for which the affinity of each chimeric receptor correlated with the presence or absence of TMs I-V, PD-128907 displaced [3H]-MSP binding to the two chimeras with similar affinities which were intermediate to those of the wild type receptors (Figure 3, Table 1). This binding profile may suggest that PD-128907 interacts in a unique way with the receptors involving multiple TM contact sites. In any case, these preliminary binding studies indicate that the chimeric D2/D3 receptors are capable of being expressed in CHO cells and exhibit ligand binding parameters well within those exhibited by the wild type receptors. To determine whether the chimeric receptors would inhibit adenylyl cyclase activity in CHO cells like the D2 receptor or be uncoupled like the D3 receptor, the effects of dopamine on cAMP accumulation in intact stably transfected cells were investigated. Dopamine did not inhibit basal cAMP accumulation in either the wild-type or chimeric receptor expressing cells (data not shown), consistent with previous reports (18,19). When the cAMP accumulation was stimulated with 3 mM forskolin treatment, however, inhibition of this activity was detectable in the D2 , D2/3 , and D3/2 receptortransfected cells (Figure 4). No effects of dopamine on basal or forskolin-stimulated cAMP accumulation were observable in D3 receptor transfected cells, in agreement with previous studies (13). Dopamine activation of D2 receptors resulted in a maximal decrease in forskolin-stimulated cAMP production of about 65% with an EC50 of 84 nM (Figure 4, Table 1). For the D2/3 and D3/2 receptors, dopamine produced about a 50% and 45% decrease in forskolinstimulated cAMP production, respectively (Figure 4). The EC50 for dopamine at both receptors was about 12 nM (Table 1). This represents about a 7-fold increase in potency for dopamine in comparison to the wild type
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FIG. 2. Displacement of [3H]-MSP binding to wild type D2 and D3 and chimeric D2/3 and D3/2 receptors by 7-OH-DPAT in stably transfected CHO cells. Cell membranes were prepared and radioligand binding competition assays were performed using 0.1 nM [3H]-MSP as described in the Materials and Methods. A representative experiment is shown which was performed four times. Average affinity values are shown in Table 1.
D2 receptor expressing cells. Thus, both of the receptor chimeras were able to couple to adenylyl cyclase inhibition in the CHO cells, although the maximum degree of inhibition was 15-20% less than that seen with the wild type D2 receptor. DISCUSSION The mechanisms by which receptors selectively activate G-proteins have been difficult to elucidate. Con-
trary to initial expectations, it has not proven possible to predict the G protein specificity of any receptor based on its primary structure. Extensive investigations of catecholamine receptors using site-directed mutagenesis, receptor chimeras, peptides and antibodies, however, have implicated the third intracellular loop (IC3), especially its amino- and carboxyl-terminal regions, of the receptor as an important, but perhaps not exclusive, determinant of selective G-protein coupling (1417). This has also been suggested to be true for the D2
FIG. 3. Displacement of [3H]-MSP binding to wild type D2 and D3 and chimeric D2/3 and D3/2 receptors by PD-128907 in stably transfected CHO cells. Cell membranes were prepared and radioligand binding competition assays were performed using 0.1 nM [3H]-MSP as described in the Materials and Methods. A representative experiment is shown which was performed four times. Average affinity values are shown in Table 1. 397
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FIG. 4. Dopamine-mediated inhibition of forskolin-stimulated cAMP accumulation in CHO cells stably transfected with wild type D2 and chimeric D2/3 and D3/2 receptors. No inhibition of cAMP accumulation was observed in CHO cells stably transfected with D3 receptors (see text). cAMP accumulation assays were performed as described in the Materials and Methods. The data represent the % inhibition of the cAMP accumulation produced in response to forskolin. A representative experiment is shown which was performed a total of seven times. Average EC50 values are shown in Table 1.
dopamine receptor with respect to its ability to couple to Gi and inhibit adenylyl cyclase activity. Several studies have shown that antisera directed against sequences within the D2 receptor IC3 could disrupt Gprotein coupling in cell-free systems (20-23). In addition, synthetic peptides derived from the IC3 of the D2 receptor were shown to attenuate receptor-mediated inhibition of adenylyl cyclase in membrane preparations (24). On the other hand, Kozell et al. (25) using D1/D2 receptor chimeras found that the presence of both the second (IC2) and 3rd (IC3) intracellular loops of the D2 receptor were necessary to support receptormediated adenylyl cyclase inhibition. These results suggest that while the IC3 of the D2 receptor may be necessary for Gi coupling, it may not be sufficient. Several previous studies have used D3/D2 receptor chimeras to address the functional role of the D2 receptor IC3 in G-protein coupling. In an initial study, McAllister et al. (26) found that a chimera consisting of the D3 receptor with the IC3 of the D2 receptor was unable to couple to adenylyl cyclase inhibition. In contrast, two subsequent studies (27,28) have shown that similar D3/D2-IC3 receptor chimeras were, indeed, able to effectively couple to this signal transduction pathway. An important issue in the interpretation of these data is that the junctions of these chimeras (26-28) were either in or near the amino- and carboxyl-terminal regions of the IC3 - regions where G-protein coupling has been postulated to occur (14-17). Thus, it is possible that the existence of these junctions have perturbed some secondary structure of the receptor resulting in anomalous G-protein coupling properties. In
the present study, we have attempted to avoid this issue by placing the D2/D3 junctions in the middle of the IC3 thus leaving the environment of the aminoand carboxyl-terminal regions (as well the adjacent TM regions) of the IC3 intact. This approach has also allowed us to test the individual contributions of each half of the IC3 to G-protein coupling. Our present results suggest that D2/D3 receptor chimeras with mid-IC3 junctions can be effectively expressed without a major perturbation in their ligand binding characteristics. Although, the pharmacology of each chimera differed somewhat depending upon the ligand tested, each chimera exhibited ligand binding parameters well within those demonstrated by the wild type receptors. Of major significance, however, is that each chimera was able to support inhibition of adenylyl cyclase activity similarly and to almost the same extent as that observed with the wild type D2 receptor. While this may suggest that both the amino and carboxyl portions of the D2 receptor IC3 are required for maximal Gi coupling, it also implies that each region is capable of independently supporting functional coupling. This surprising finding suggests that the D2 receptor possesses redundant and mostly independent Gi coupling domains. A similar finding was recently reported for the a2adrenergic receptor which couples to multiple G-proteins, including Gi resulting in inhibition of adenylyl cyclase activity. Using a chimeric receptor approach, Eason et al. (17) found that either the amino- or carboxyl-terminal IC3 region of the a2-adrenergic receptor was capable of supporting Gi coupling. In addition,
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these investigators found that the IC2 of the a2-adrenergic receptor was mandatory for functional G-protein coupling to occur. This agrees well the D1/D2 receptor chimera study of Kozell et al. (25) which also implicated the necessity of the IC2 of the D2 receptor for inhibition of adenylyl cyclase. In the present study, it is interesting to note that only the D2/3 chimera possessed the IC2 of the D2 receptor whereas the D3/2 receptor possessed that of the D3 . This suggests that the IC2 of the D3 receptor (which is 50% homologous with that of the D2) is capable of supporting Gi coupling but not in conjunction with the D3 receptor’s IC3, in agreement with previous results (27,28). Given these findings for the D2 dopamine and a2-adrenergic receptors, it will be interesting to see if other Gi coupled receptors exhibit this phenomenon. In summary, we have provided evidence for the existence of two redundant and functionally independent Gi coupling domains in the D2 dopamine receptor. The rationale for the existence of two distinct G-protein coupling regions is unclear but may have to do with the fact that the D2 receptor (as are most Gi/o coupled receptors) is coupled to multiple signal transduction pathways (11,12). It will be interesting to investigate the coupling of these and related D2/D3 receptor chimeras to other D2 receptor-mediated signalling events as well as to use more specific mutagenesis techniques to further delineate the G-protein coupling domains of the D2 receptor. REFERENCES 1. Hornykiewicz, O. (1973) Fed. Proc. 32, 183–190. 2. Seeman, P. (1987) Synapse 1, 133–152. 3. Kebabian, J. W., and Calne, D. B. (1979) Nature 277, 93–96. 4. Sibley, D. R., and Monsma, F. J., Jr. (1992) Trends Pharmacol. Sci. 13, 61–69. 5. Civelli, O., Bunzow, J. R., and Grandy, D. K. (1993) Ann. Rev. Pharmacol. Tocxicol. 32, 281–307. 6. Gingrich, J. A., and Caron, M. G. (1993) Ann. Rev. Neuroci. 16, 299–321.
7. Sokoloff, P., and Schwartz, J.-C. (1995) Trends Pharmacol. Sci. 16, 270–275. 8. Neve, K. A., and Neve, R. L. (1997) in The Dopamine Receptors (Neve, K. A., and Neve, R. L., Eds.), pp. 77–104, Humana Press, Clifton, NJ. 9. Dohlman, H. G., Thorner, J., Caron, M. G., and Lefkowitz, R. J. (1991) Annu. Rev. Biochem. 60, 653–688. 10. Strader, C. D., Fong, T. M., Graziano, M. P., and Tota, M. R. (1995) FASEB J. 9, 745–754. 11. Robinson, S. W., and Caron, M. G. (1997) in The Dopamine Receptors (Neve, K. A., and Neve, R. L., Eds.), pp. 137–166, Humana Press, Clifton, NJ. 12. Huff, R. (1997) in The Dopamine Receptors (Neve, K. A., and Neve, R. L., Eds.), pp. 167–192, Humana Press, Clifton, NJ. 13. Sokoloff, P., Giros, B., Martres, M.-P., Bouthenet, M.-L., and Schwartz, J.-C. (1990) Nature 347, 146–151. 14. Strader, C. D., Fong, T. M., Tota, M. R., Underwood, D., and Dixon, R. A. F. (1994) Annu. Rev. Biochem. 63, 101–132. 15. Kobilka, B. K., Kobilka, T. S., Daniel, K., Regan, J. W., Caron, M. G., and Lefkowitz, R. J. (1988) Science 240, 1310–1316. 16. Wess, J. (1997) FASEB J. 11, 346–354. 17. Eason, M. G., and Liggett, S. B. (1996) J. Biol. Chem. 271, 12826–12832. 18. Zhang, L. J., Lachowicz, J. E., and Sibley, D. R. (1994) Molec. Pharmacol. 45, 878–889. 19. Rinaudo, M. S., Monsma, F. J., Jr., Black, L. E., Mahan, L. C., and Sibley, D. R. (1990) Soc. Neurosci. Abstr. 16, 209. 20. Plug, M. J., Dijk, J., Maassen, A., and Moller, W. (1992) Eur. J. Biochem. 206, 123–130. 21. Boundy, V. A., Luedtke, R. R., and Molinoff, P. B. (1993) J. Neurochem. 60, 2181–2191. 22. Boundy, V. A., Luedtke, R. R., Artmyshynm, R. P., Filtz, T. M., and Molinoff, P. B. (1993) J. Pharmacol. Exptl. Therap. 43, 666– 676. 23. Chazot, P. L., Doherty, A. J., and Strange, P. L. (1993) Biochem. J. 289, 789–794. 24. Malek, D., Munch, G., and Palm, D. (1993) FEBS Letts. 325, 215–219. 25. Kozell, L. B., Machida, C. A., Neve, R. L., and Neve, K. A. (1994) J. Biol. Chem. 269, 30299–30306. 26. McAllister, G. A., Knowles, M. R., Patel, S., Marwood, R., Emms, F., Seabrook, G. R., Graziano, M., Borkowski, D., Hey, P. J., and Freedman, S. B. (1993) FEBS Letts. 324, 81–86. 27. Van Leeuwen, D. H., Eisenstein, J., O’Malley, K., and MacKenzie, R. G. (1995) Molec. Pharmacol. 48, 344–351. 28. Robinson, S. R., and Caron, M. G. (1996) J. Neurochem. 67, 212– 219.
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